OX2 receptor homologs

专利摘要:

公开号:AU2008203172A1
申请号:U2008203172
申请日:2008-07-16
公开日:2008-08-07
发明作者:Neil A. Barclay；Marion H. Brown；Holly Cherwinski；Daniel M. Gorman；Robert M. Hoek；Lewis L. Lanier；Joseph H Phillips；Jonathan D. Sedgwick；Gavin J. Wright
申请人:Medical Research Council；Schering Corp；
IPC主号:C12N15-12

专利说明:
AUSTRALIA
Patents Act 1990 COMPLETE SPECIFICATION Standard Patent Applicant(s): MEDICAL RESEARCH COUNCIL and SCHERING CORPORATION Invention Title: OX2 RECEPTOR HOMOLOGS The following statement is a full description of this invention, including the best method for performing it known to me/us: P43750AU.2 Pa1_SI. Fbng Appoicaton 20W7.15.doc (S) OX2 RECEPTOR HOMOLOGS FIELD OF THE INVENTION The present invention relates to compositions and methods O 5 for affecting mammalian physiology, including immune system function. In particular, it provides reagents or methods which may regulate development and/or the immune system. Diagnostic and therapeutic uses of these materials are also described.
c- 10 BACKGROUND OF THE INVENTION 0O The OX2 antigen (OX2) is a cell surface protein identified Son a variety of cells including thymocytes, B lymphocytes, activated T lymphocytes, neurons, endothelial cells, and follicular dendritic cells. Barclay (1981) Immunoloav 44:727- 736. Sequence analysis indicates that it is a transmembrane protein containing two extracellular immunoglobulin-like (Iglike) domains and a short cytoplasmic domain. Clark, et al.
(1985) EMBO J. 4:113-118. This domain organization is common and found in many different leukocyte surface proteins. Barclay, et al. (1997) Leucocyte Antigens Factsbook (2d. ed.) Academic Press, London. These types of proteins often interact with other proteins on the surfaces of other cells, also having Ig-like domains.
The distribution of the OX2 antigen is consistent with a hypothesis that OX2 relays a signal through a binding partner, the OX2 receptor (OX2R), to cells within the leukocyte lineage including macrophages, which express the receptor (Preston, et al. (1997) Eur. J. Immunol. 27:1911-1918) and possibly other cells of the monocyte-macrophage lineage. Also, the OX2 has been implicated in regulation of various functions of macrophages. In this scenario, for instance, expression of OX2 on neurons could establish a direct means of communication to the resident macrophages of the brain called microglia that might express OX2R, since they originate from the monocyte-macrophage lineage. Perry and Gordon (1988) Trends Neurosci. 11:273-277.
Generally, defective or exaggerated activation of macrophages contributes to pathogenesis of a wide range of 00 S immunological and other diseases. See, McGee, et al. (eds.
1992) Oxford Textbook of Patholoav Oxford University Press, Oxford; Lewis and McGee (eds. 1992) The Macrophaae IRL Press, NO Oxford; and Bock and Goode (eds. 1997) The Molecular Basis of Cellular Defence Mechanisms Wiley Sons.
Also, identification of the OX2 interacting proteins, e.g., the OX2R for the OX2 antigen, is difficult because the affinities c of the interactions are often very low. This means that the binding of recombinant forms of cell surface proteins, OX2, to their binding partners, the interacting proteins, OX2R, is insufficiently stable to allow detection by normal methods.
Thus, the interaction between CD48 and CD2, of which both partners contain two Ig-like domains in their extracellular regions, has a half-life of a fraction of a second. See Van der Merwe, et al. (1993) Biochem. Soc. Trans. 21:340S; and Van der Merwe and Barclay (1994) Trends Biochem. Sci. 19:354-358.
Recombinant forms of cell surface proteins such as OX2 can be made multivalent by a number of methods and used to detect novel proteins. An OX2 has been engineered to include a tag of two Ig-like domains from CD4. The recombinant soluble proteins are expressed by conventional expression methods in eukaryotic cells. In an earlier study, an interaction was observed between the multivalent recombinant OX2 protein on fluorescent beads and mouse macrophages. Preston, et al. (1997) Eur. J. Immunol.
27:1911-1918.
Despite the above, attempts to identify OX2R on mouse macrophages through use of a blocking antibody OX89 were not successful. Preston, et al. (1997) Eur. J. Immunol. 27:1911- 1918.
From the foregoing, it is evident that the discovery, identification, and understanding of novel receptors for OX2-like molecules would be highly advantageous. The present invention provides new receptor homologs for OX2 ligands and related compounds, and methods for their use.
I
00 0 SUMMARY OF THE INVENTION The present invention is directed to novel receptor homologs, for the ligand designated OX2, rodent and primate O embodiments. These have been designated generically OX2 receptor homologs (OX2RH), with embodiments from various rodent and primate species. Two have been established as actually binding to the respective species OX2. In particular, it provides c description of homologs designated OX2RH1, OX2RH2, OX2RH3, and C OX2RH4. It includes nucleic acids encoding the polypeptides 0 10 themselves and methods for their production and use. The nucleic Sacids of the invention are characterized, in part, by their homology to cloned complementary DNA (cDNA) sequences enclosed herein.
The present inventors have produced a new monoclonal antibody (mAb), designated OX102, for OX2R on rat macrophages which blocks the interaction between OX2 and OX2R. They have also isolated and characterised the rat OX2R gene and polypeptide. Sequences for the rat OX2R nucleic acid molecule and'polypeptide (predicted amino acid sequence) are provided herein. By analogy with similar proteins, the inventors teach that the nucleotide and amino acid sequences of human OX2R will be at least 50% homologous with the corresponding rat OX2R sequences. The availability of the rat OX2R cDNA and a predicted OX2R polypeptide sequence enable identification of the equivalent human OX2R sequences, either through screening of known human sequences or the isolation of human nucleic acids by hybridisation or PCR technology.
The presence of a large cytoplasmic sequence in the OX2R polypeptide indicates that OX2R has a role in macrophage function, either in signalling or through interactions with components of the cytoplasm. Thus, the present invention provides for reagents based on OX2R that either mimic or recognise OX2R polypeptide or nucleic acid sequences, small molecular entities designed to react with OX2R binding sites, mAbs raised against OX2R or antisense sequences, which reagents constitute therapeutically useful compounds for modifying the 00 O function of cells carrying OX2 and/or OX2R cell surface proteins the function of cells such as macrophages, activated lymphocytes, neurons, endothelial cells, dendritic cells, thymocytes and B lymphocytes), either by enhancing or inhibiting cell activity. Thus, reagents based on OX2R that either mimic or ci recognise OX2R polypeptide or nucleic acid sequences have potential applications for controlling the wide range of m functions of macrophages, including responses to bacterial 00 CA infections, autoimmune diseases, etc.
Since the extracellular domain of OX2R is believed to be S responsible for interacting with OX2, the present inventors provide a means of screening candidate compounds for an ability to affect (positively or negatively) binding between OX2 and OX2R. Thus OX2R as provided by the present invention can, e.g., be used to detect compounds which inhibit the interaction between OX2 and OX2R, and hence which are likely to affect the interaction between macrophages and other cells of the immune system, such as lymphocytes or follicular dendritic cells.
The nucleic acid and amino acid sequences for rat OX2R are shown in Table 1. Various aspects of the invention are stated below. Other aspects are clear from the detailed description.
Hence, in a first aspect, the present invention provides a substance comprising a polypeptide having the amino acid sequence set out in Table 1.
In a further aspect, the present invention provides a substance comprising a polypeptide having at least 50% amino acid sequence identity with the amino acid sequence set out in Table 1.
In a further aspect, the present invention provides a polypeptide which is a mutant, variant, derivative or allele of an above polypeptide and which has a characteristic property of full-length OX2R, an ability to bind with an OX2 or with an antibody for full-length OX2R.
In a further aspect, the present invention provides a substance which is a fragment of an above polypeptide a fragment of a polypeptide having the amino acid sequence set out.
00 0 C in Table which fragment exhibits a characteristic property of Sfull-length OX2R protein. For example, the fragment may bind with an OX2 protein or with an antibody for full-length OX2R protein. In one embodiment, the fragment includes part or all of the cytoplasmic domain of OX2R or an active portion of that CI domain. In another embodiment, the fragment includes part or all of the extracellular domain of OX2R or an active portion of that Sdomain. Since the extracellular domain is believed to be C responsible for interacting with OX2, such fragments according to 0 10 the present invention including part or all of the extracellular CI domain, can be used to screen candidate compounds for an ability to interfere with the binding between OX2 and OX2R.
Accordingly, the present invention provides methods and materials for screening candidate compounds likely to have the ability to interfere with the OX2/OX2R interaction between macrophages and other cells, including thymocytes, B lymphocytes, activated T lymphocytes, neurons, endothelial cells and follicular dendritic cells.
Polypeptides and fragments as above may be recombinant and/or isolated polypeptides.
In a further aspect, the present invention provides a substance comprising a nucleic acid having the nucleotide sequence of Table 1. The present invention also provides a substance which comprises a nucleic acid molecule encoding an above polypeptide or fragment. Thus, Table 1 shows the cDNA sequence of an exemplary nucleic acid molecule coding for an OX2R polypeptide. The nucleic acid molecule may have at least sequence homology with the nucleic acid sequence of Table 1.
The invention also provides a substance comprising a nucleic acid molecule having part of a coding nucleotide sequence of Table 1. Where the substance comprises a part of a coding nucleotide sequence of Table 1, it will be a part which is characteristic of an OX2R gene. Thus, the part may encode a polypeptide fragment as stated above, which binds with OX2 or an antibody for full-length OX2R. Alternatively, the part may comprise at least 4 to 7 contiguous codons, often at least 7 to 9 00 contiguous codons, typically at least 9 to 13 contiguous codons and, most preferably, at least 20 to 30 contiguous codons of a nucleotide sequence of Table 1. Alternatively, the part may encode at least 4 to 7 contiguous amino acids, often at least 7 to 9 contiguous amino acids, typically at least about 9 to 13 C contiguous amino acids and, most preferably, at least about 20 to 30 contiguous amino acids of a polypeptide sequence of Table 1.
SNucleic acid molecules as above may be recombinant and/or 00 isolated.
o 10 In further aspects, the present invention provides vectors C comprising an OX2R nucleic acid as herein provided, e.g., expression vectors in which an OX2R nucleic acid sequence is operably linked to control sequences to direct its expression.
Also provided are host cells transformed with such vectors. The present invention further includes a method of producing OX2R polypeptides, comprising culturing such host cells and isolating OX2R polypeptide produced.
In a further aspect, the present invention provides a method of expressing OX2R in host cells, the method including the steps of inserting a nucleic acid molecule as above into a host cell and providing conditions for expression of said nucleic acid molecule in the host cell. The method may employ an expression vector.
In a further aspect, the present invention provides a composition comprising a soluble form of an OX2R polypeptide or fragment as above, the composition also optionally including an adjuvant, pharmaceutical carrier, or excipient. The composition can be used, to generate an antibody response to an OX2R polypeptide.
In further aspects, the present invention provides above OX2R polypeptides and nucleic acid molecules for use in screening candidate compounds likely to be useful as therapeutics. The present invention provides the use of an OX2R polypeptide or fragment as above in the screening for substances likely to be useful for the treatment of bacterial infections, autoimmune diseases, and the like.
00 The present invention also provides the.use of OX2R polypeptides, polypeptide fragments, and nucleic acids for the identification of ligands for OX2R other than OX2. The present O invention also provides the use of OX2R polypeptides, polypeptide fragments, and nucleic acids for the design of mimetics of OX2.
In a further aspect, the present invention provides antibodies capable of specifically binding to OX2R polypeptides, polypeptide fragments, and nucleic acids as above, and 0 compositions comprising such antibodies. These antibodies can be 00 10 used in assays to detect and quantify the presence of OX2R, as Swell as in methods of purifying OX2R. The antibodies may be polyclonal. Preferably, the antibodies are IgG antibodies, more preferably monoclonal IgG antibodies.
In a further aspect, the present invention provides the use of OX2R polypeptides, polypeptide fragments, and nucleic acid molecules as provided herein to produce binding molecules, such as substances with one or more antibody domains, which can block the interaction between OX2 and OX2R. These may be included in a composition likely to be useful in the preparation of medicaments for the treatment of bacterial infections, autoimmune diseases, and the like. Where the binding molecules are antibodies, they may be IgG antibodies, preferably monoclonal IgG antibodies.
In a further aspect, the present invention provides the use of OX2R nucleic acids as defined above in the design of antisense oligonucleotides to restrict OX2R expression in a population of macrophage cells, phosphorothiolated or cholesterol-linked oligonucleotides which can facilitate internalization and stabilization of the oligonucleotides.
In a further aspect, the present invention provides a method of amplifying a nucleic acid test sample, which comprises priming a nucleic acid polymerase reaction with primer oligonucleotides obtainable from the sequence information provided herein. The nucleic acid test sample may be of a human, such that nucleic acid coding for a human OX2R is amplified using such a method.
In a further aspect, the present invention provides a method of obtaining a nucleic acid molecule coding for part or all of an 00 0 I OX2R from a species other than rat, human OX2R, which Scomprises probing a nucleic acid test sample from the species of IN interest with a nucleic acid probe obtainable from the sequence information provided herein.
In a further aspect, the present invention provides a method of obtaining an OX2R polypeptide sequence from a species other Sthan rat, a human OX2R, which comprises searching databases 8 for polypeptide sequences at least 50% homologous with an OX2R 00 amino acid sequence as provided herein (Table Similarly, OX2R nucleic acid sequences from species other than rat, e.g., r human OX2R, can be obtained by searching databases for nucleotide sequences at least 50% homologous with an OX2R nucleotide sequence as provided herein.
The present invention also provides the use of the nucleic acid sequence information provided herein in the search for mutations in OX2R genes, using techniques such as single stranded conformation polymorphism
(SSCP).
The present invention provides a composition of matter selected from: a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 2; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 2; a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 2; a fusion polypeptide comprising rat OX2RH1 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 4; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 4; a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 4; a fusion polypeptide comprising human OX2RH1 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of 00 0 SEQ ID NO: 6; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 6; a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 6; a fusion polypeptide comprising mouse OX2RH1 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four m amino acids identical to segments of SEQ ID NO: 8; a C substantially pure or recombinant polypeptide comprising at least 00 two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 8; a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 8; a fusion polypeptide comprising human OX2RH2 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 10; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 10; a natural sequence rodent OX2RH2 polypeptide comprising mature SEQ ID NO: 10; a fusion polypeptide comprising mouse OX2RH2 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 12; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 12; a natural sequence rodent OX2RH3 comprising mature SEQ ID NO: 12; a fusion polypeptide comprising mouse OX2RH3 sequence; a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 20; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 20; a natural sequence primate OX2RH1.2 polypeptide comprising mature SEQ ID NO: 20; a fusion polypeptide comprising primate OX2RH1.2 sequence; a 00 S substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino 'n acids identical to segments of SEQ ID NO: 23; a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to C segments of SEQ ID NO: 23; a natural sequence rodent OX2RH4 polypeptide comprising mature SEQ ID NO: 23; or a fusion n polypeptide comprising mouse OX2RH4 sequence. Some preferred C embodiments include wherein the distinct nonoverlapping segments 00 C 10 of identity: include one of at least eight amino acids; include C one of at least four amino acids and a second of at least five amino acids; include at least three segments of at least four, five, and six amino acids, or include one of at least twelve amino acids. Other preferred embodiment include those wherein the: a) OX2RH1 polypeptide: comprises a mature sequence of Tables 1 or 2; is an unglycosylated form of OX2RH polypeptide; is from a primate, such as a human; is from a rodent, such as a rat or mouse; comprises at least seventeen amino acids of SEQ ID NO: 2, 4, 6, or 20; exhibits at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 2, 4, 6, or 20; is a natural allelic variant of OX2RH1; has a length at least about amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate or rodent OX2RH1; is glycosylated; has a molecular weight of at least 30 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is 5-fold or less substituted from natural sequence; or is a deletion or insertion variant from a natural sequence; b) OX2RH2 polypeptide: comprises a mature sequence of Table 2; is an unglycosylated form of OX2RH2 polypeptide; is from a primate, such as a human; is from a rodent, such as a mouse; comprises at least seventeen amino acids of SEQ ID NO: 8 or 10; exhibits at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 8 or 10; is a natural allelic variant of OX2RH2; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a primate or rodent OX2RH2; is 00 glycosylated; has a molecular weight of at least 30.kD with c natural glycosylation; is a synthetic polypeptide; is attached to Sa solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence; c) OX2RH3 polypeptide: comprises a mature sequence of Table 3; is an unglycosylated form of OX2RH3; is from a rodent, such as a mouse; comprises at least seventeen amino acids of SEQ ID NO: 12; Sexhibits at least four nonoverlapping segments of at least seven 00 10 amino acids of SEQ ID NO: 12; is a natural allelic variant of SOX2RH3; has a length at least about 30 amino acids; exhibits at least two non-overlapping epitopes which are specific for a rodent OX2RH3; is glycosylated; has a molecular weight of at least 30 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is 5-fold or less substituted from natural sequence; or is a deletion or insertion variant from a natural sequence; or d) OX2RH4 polypeptide: comprises a mature sequence of Table 2; is an unglycosylated form of OX2RH4; is from a rodent, such as a mouse; comprises at least seventeen amino acids of SEQ ID NO: 23; exhibits at least four nonoverlapping segments of at least seven amino acids of SEQ ID NO: 23; is a natural allelic variant of OX2RH4; has a length at least about amino acids; exhibits at least two non-overlapping epitopes which are specific for a rodent OX2RH4; is glycosylated; has a molecular weight of at least 30 kD with natural glycosylation; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is 5-fold or less substituted from natural sequence; or is a deletion or insertion variant from a natural sequence. In yet other embodiments, the invention provides a composition comprising: al) a substantially pure OX2RH1 and another Ig superfamily member; a2) a substantially pure OX2RH2 and: another Ig superfamily member, DAP12, or DAP10; a3) a substantially pure OX2RH3 and: another Ig superfamily member, DAP12, or DAP10; a4) a substantially pure OX2RH4 and: another Ig superfamily member, DAP12, or DAP10; or a 00 §l sterile OX2RH1 polypeptide; a sterile OX2RH2 polypeptide; a sterile OX2RH3 polypeptide; a sterile OX2RH4 polypeptide; the SOX2RH1, OX2RH2, OX2RH3, or OX2RH4 polypeptide and a carrier, S wherein the carrier is an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration.
Fusion polypeptides are also provided, comprising: Smature protein sequence of Tables 1-3; a detection or 0 purification tag, including a FLAG, His6, or Ig sequence; or 00 10 sequence of another Ig superfamily protein. Kits are also Sprovided, comprising an OX2RH polypeptide and: a C compartment comprising the protein or polypeptide; or instructions for use or disposal of reagents in the kit.
The invention also embraces various antibody like reagents, including antibodies derived from different species. It provides, a binding compound comprising an antigen binding site from an antibody, which specifically binds to a natural OX2RH polypeptide, OX2RH1, OX2RH2, OX2RH3, and/or OX2RH4, wherein: the binding compound is in a container; the OX2RH polypeptide is from a rodent or primate; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; or the antibody: is raised against a peptide sequence of a mature polypeptide of Tables 1-3; is raised against a mature OX2RH; is raised to a purified mammalian OX2RH; is immunoselected; is a polyclonal antibody; binds to a denatured OX2RH; exhibits a Kd to antigen of at least 30 pM; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label. Kits are thereby provided, comprising such binding compounds and: a compartment comprising the binding compound; or instructions for use or disposal of reagents in the kit. Methods are also provided, producing an antigen:binding compound or antigen:antibody complex, comprising contacting under appropriate conditions a mammalian OX2RH polypeptide with an antibody, thereby allowing the complex to form. Preferably, in this method: the complex is 00 O purified from other cytokine or Ig superfamily receptors; the complex is purified from other antibody; the contacting is with a Ssample comprising a mammalian OX2; the contacting allows IND quantitative detection of the antigen; the contacting is with a sample comprising the antibody; or the contacting allows quantitative detection of the antibody. Related compositions are
C
made available, comprising: a sterile binding compound, or the binding compound and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or 00 10 formulated for oral, rectal, nasal, topical, or parenteral administration.
C
The present invention further provides nucleic acids, e.g., an isolated or recombinant nucleic acid encoding a OX2RH polypeptide wherein the: OX2RH is from a mammal; or the nucleic acid: encodes an antigenic peptide sequence of Tables 1-3; encodes a plurality of antigenic peptide sequences of Tables 1-3; exhibits identity over at least thirteen nucleotides to a natural cDNA encoding the segment; is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence; is less than 6 kb, preferably less than 3 kb; is from a primate or rodent; comprises a natural full length coding sequence; is a hybridization probe for a gene encoding the OX2RH; further encodes DAP12 or DAP10; or is a PCR primer, PCR product, or mutagenesis primer. Cells comprising the recombinant nucleic acid are also provided, wherein the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell. Kits comprising the nucleic acid are provided, e.g., with a compartment comprising the nucleic acid; with a compartment further comprising a mammalian OX2RH polypeptide; or with instructions for use or disposal of reagents in the kit.
Alternatively, the invention provides a nucleic acid which: hybridizes under wash conditions of 30 minutes at 40° C and less than 2M salt to the coding portion of SEQ ID NO: 1, 3, 5, 7, 9, 11, 19, or 22; or exhibits identity over a stretch of at least 14 00 D about 30 nucleotides to a primate or rodent OX2RH cDNA.
S Preferably, the wash conditions are at: 50° C and/or 500 mM salt; n or 60* C and/or 150 mM salt; the stretch is at least 0 nucleotides or 75 nucleotides; or the nucleic acid further encodes a DAP12 or DAP10 peptide.
Other methods are further embraced, a method of modulating physiology or development of a cell or tissue culture M c cells comprising contacting the cell with an agonist or c- antagonist of a mammalian OX2RH. Often, the cell is transformed 00 10 with a nucleic acid encoding an OX2RH.
00 C DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
SOUTLINE
I. General O 5 II. Activities III. Nucleic acids A. encoding fragments, sequence, probes C- B. mutations, chimeras, fusions C. making nucleic acids
T
10 D. vectors, cells comprising IV. Proteins, Peptides 0 C A. fragments, sequence, immunogens, antigens
O
SB. muteins SC. agonists/antagonists, functional equivalents D. making proteins V. Making nucleic acids, proteins A. synthetic B. recombinant C. natural sources VI. Antibodies A. polyclonals B. monoclonal C. fragments; Kd D. anti-idiotypic antibodies E. hybridoma cell lines VII. Kits and Methods to quantify OX2RHs A. ELISA B. assay mRNA encoding C. qualitative/quantitative D. kits VIII. Therapeutic compositions, methods A. combination compositions B. unit dose C. administration IX. Screening X. Ligands I. General The present invention provides amino acid sequences and DNA sequences of mammalian, herein primate and rodent, receptor-like subunit molecules, these designated OX2 receptor homologs (OX2RH). These genes have particular defined properties, either or both structural and biological. Various cDNAs encoding these molecules were obtained from mammal, human and rodent, cDNA 00 S sequence libraries. Other mammalian, primate, rodent, or other, counterparts would also be desired.
The OX2 antigen was first characterized in rat, using a O monoclonal antibody (mAb) MRC OX2. See, McMaster and Williams (1979) Eur. J. Immunol. 9:426-433; Barclay (1981) Immunoloav 44:727-736; Barclay (1981) Immunology 42:593-600; Bukovsky, et al. (1984) Immunology 52:631-640; and Webb and Barclay (1984) J. Neurochem. 43:1061-1067. Using this antibody 0 in immunohistochemical (IHC) staining of tissue sections or cell 00 10 suspensions for flow cytometry revealed that the OX2 antigen was Sexpressed by a wide variety of cells, e.g. neurons, vascular
C
endothelium, B cells, activated T cells, follicular dendritic cells, smooth muscle cells and trophoblasts. Furthermore, human OX2 is known to be expressed in normal brain and by B cells.
McCaughan, et al. (1987) Immunogenetics 25:329-335.
Characterization of the rat protein recognized by MRC OX2 (Clark, et al. (1985) EMBO J. 4:113-118) revealed that OX2 consists of about 248 amino acids comprising two extracellular immunoglobulin (Ig) domains, a transmembrane domain and a short C-terminal cytoplasmic tail. The molecule is glycosylated through 6 Nlinked glycosylation sites, three of which are present in the Nterminal V-like Ig domain and the others reside in the membrane proximal C2-like Ig domain. This places OX2 in the Ig superfamily (IgSF), forming a sub-group of small IgSF molecules with molecules like CD2, CD48, CD58, CD80, CD86, CD90, and CD147, which are characterized structurally, by the existence of the immunoglobulin-like domains corresponding to Ig variable and constant domains, a transmembrane segment, an intracellular domain, and characteristic cysteine and tryptophan residue spacings. See, Campbell, et al. (1979) Nature 282:341-342.
Interestingly, CD90 is also highly expressed by neurons.
Williams, et al. (1977) Cold SDrina Harb. SvmD. Quant. Biol. 41 Pt 1:51-61. Furthermore, it was shown that 0X2 was a structural homologue of CD80 and CD86 (Borriello, et al. (1997) J. Immunol.
158:4548-4554) and that the OX2 gene was closely linked to those coding for CD80 and CD86 on chromosome 16 in the mouse.
00 Borriello, et al. (1998) Mamm. Genome 9:114-118. Both CD80 and CD86 serve as ligands in a process known as co-stimulation, and Stherefore it is likely that OX2 would act as a ligand as well.
The OX2 antigen will be referred hereafter as the OX2 protein or ligand OX2. The binding partner will be referred to as the OX2 receptor.
To identify the receptor for OX2 (OX2R), a multivalent reagent was prepared using rat OX2-rat CD4 fusion protein bound Sto fluorescent beads. This reagent was shown to bind to mouse 00 10 and rat peritoneal macrophages, and this binding could be blocked Sby the mAb MRC OX88. Preston, et al. (1997) Eur. J. Immunol.
C 27:1911-1918. This mAb was shown to bind to macrophages isolated from both peritoneum and spleen and in IHC on spleen sections staining was found in areas known to contain high proportions of macrophages.
A second monoclonal antibody raised by the Barclay group, designated OX102, was shown to bind macrophages in the rat species and also to prevent specifically the binding of the OX2 molecule to rat peritoneal macrophages. Isolation of material binding to the OX102 molecule and N-terminal sequencing showed the putative OX2 receptor (OX2R) to be a novel molecule. This was cloned as described herein. That the protein recognized by the OX102 antibody was indeed the receptor was supported by the demonstration of a longer cytoplasmic tail on this molecule relative to the OX2 molecule itself (the ligand). Clark, et al.
(1985) EMBO J. 4:113-118. Preliminary analysis of the OX2R did not reveal obvious motifs consistent with known signaling molecules although this does not exclude the potential role of this molecule in mediating OX2-delivered signals.
Then, a mouse homolog was identified, designated OX2RH1.
Because the terminology OX2R should be reserved for those proteins which have been verified to actually bind to the OX2, the initial designation applied is a receptor homolog of the group 1. The nucleotide and amino acid sequences of this molecule are described herein.
00 0 Further analysis of available sequence databases revealed CM the presence of another distinct form of OX2RH, a molecule that Sshowed significant homology in the putative extracellular Ig- 4 domain structures with OX2RH1 but with a different transmembrane S 5 and cytoplasmic sequence. These forms have herein been designated OX2RH2, and both human and mouse embodiments have been CN identified. Of particular note is the presence of a lysine (K) moiety at positions 224 (human) and 170 (nouse) that lies within Sthe transmembrane portion of the molecule. Such a residue OO 10 suggests that this molecule will associate with molecular Spartners such as DAP12 known to express motifs capable of 0 C signaling for cellular activation. See, Lanier, et al.
(1998) Nature 391:703-707; Colonna(1998) Nature 391:642-3; Campbell, et al. (1999) Int. J. Biochem. Cell. Biol. 31:631-636; and Lopez-Botet, et al. (1999) Curr. OQin. Immunol. 11:301-307.
Moreover, such suggests various signaling pathways and associated biochemistry. See, Lanier, et al. (1998) Immunity 8:693- 701; Smith, et al. (1998) J. Immunol. 161:7-10; Gosselin, et al.
(1999) J. Leukoc. Biol. 66:165-171; Tomasello, et al.(1998) J.
Biol. Chem. 273:34115-34119; and McVicar, et al. (1998) J. Biol.
Chem. 273:32934-32942. But, a full length mouse or human OX2RH2 form is yet to be isolated.
There is high homology between the mouse and rat extracellular regions of the OX2RH1 molecule, both of which have been confirmed to bind to their respective species OX2. Thus, the rat and mouse OX2RH1 embodiments are properly also referred to functionally as OX2R. Both contain typical extracellular, transmembrane, and intracellular domain structures. Human OX2RH1 embodiments were discovered. Additionally, soluble forms of the rat and mouse OX2RH1 may exist.
Related homologs, designated OX2RH2 and OX2RH4, have also been described, various embodiments originating in mouse and human. OX2RH2, H3, and H4 embodiments exhibit a charged lysine residue in the transmembrane segment. The human OX2RH2 embodiment lacks a signal sequence and shows some genomic sequence earmarks, suggesting that the functional form of the 00 c- natural human OX2RH2 should be closely related but slightly different from the sequence provided. The functional C- relationship of the mouse and human homologs 2 and 4 remain to be confirmed.
A further OX2R homolog was also found in the mouse.
Although its homology is much more divergent, it exhibits some similarities in sequence. In particular, it has a lysine residue in the transmembrane region. Thus, like the other OX2RH2, H3, and H4 molecules exhibiting this feature, it would be expected to signal via an associating molecule such as DAP12. This embodiment is herein designated OX2RH3 from rodent, mouse.
Ongoing analysis of the expression patterns of the OX2RH1 indicates that in rat, mouse, and human leukocytes, OX2RH1 (as determined by flow cytometric staining with the OX102 antibody and/or analysis of mRNA expression by PCR techniques) is expressed most strongly by monocytes, granulocytes, and mast cells, marginally by B cells, and weakly by T cells. This is consistent with the preferential binding of the ligand OX2 to macrophages in earlier studies. In the normal rat central nervous system, a proportion of resident macrophage (or microglial cells) also express the OX2R, but at a low level.
Some applicable standard methods are described or referenced, in Maniatis, et al. (1982) Molecular Cloning,
A
Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular Cloning:
A
Laboratory Manual, (2d vols. 1-3, CSH Press, NY; Ausubel, et al., Bioloy, Greene Publishing Associates, Brooklyn, NY; or Ausubel, et al. (1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York; each of which is incorporated herein by reference.
Nucleotide (SEQ ID NO: 1) and corresponding amino acid sequence (SEQ ID NO: 2) of a rodent, rat, OX2 receptor homolog 1 (OX2RH1) coding segment is shown in Table 1.
Similarly, further embodiments, primate, human, and rodent, mouse, are described, designated OX2RH1, 1.2, 2, and 4.
The nucleic acid sequences are SEQ ID NO: 3, 19, 5, 7, 9, and 22;.
00 O the corresponding amino acid sequences are SEQ ID NO: 4, 20, 6, 8, 10, and 23 which are presented in Table 2. Table 3 provides 3 the sequence of other rodent, mouse, OX2RH3 (SEQ ID NO: 11 ND and 12).
Reverse translation nucleic acid sequences are provided in Table 4 (SEQ ID NO: 13-14, 21, 15-17, 24, and 18). Table provides alignment and numeric comparison of polypeptide sequences.
00 21 00 Table 1: Nucleotide and polypeptide sequences of rodent 0X2R (hornolog rat OX2RH1 (SEQ ID NO: 1 and 2): S 5 agcggaggga tcctggtcat ggtcaccgct gctcccctac ctgtgaagag aaagagcacc gagtgagccg ctgaaaacca gaaaaccgaa atg ctC tgc ttt tgg aga act tct 114 Met Leu Cys Phe Trp Arg Thr Set cac gia gca gta ctc ttg atc tgg ggg gtc ttc gcg gct gag tca agt 162 His Val Ala Val Leu Leu Ile Trp, Gly Val Phe Ala Ala Giu. Ser Ser -10 -5 -1 00 tgt cct gat aag aat caa aca atg cag aac aat tca tca act atg aca 210 Cys Pro Asp Lys A-sn Gin Thr Met Gin A-sn A-sn Ser Ser Thr Met Thr 1 5 10 gaa gtt aac act aca gtg ttt gta cag atg ggt aaa aag gct ctg ctc 258 Glu Vai A-sn Thr Thr Val Phe Val Gin Met Gly Lys Lys Ala Leu Leu 25 tgc tgc cct tct. att tca ctg aca aaa gta ata tta ata aca tgg aca 306 Cys Cys Pro Ser Ile Set Leu Thr Lys Val Ile Leu Ile Thr Trp Thr 40 ata acc ctc aga gga cag cct tcc tgc ata ata tcc tac aaa gca gac 354 Ile Thr Leu Arg Gly Gin Pro Ser Cys Ile Ile Set Tyr Lys Ala Asp 50 55 aca agg gag acc cat gaa agc aac tgc tcg gac aga agc atc acc tgg 402 Thr Arg Giu Thr His Giu Ser A-sn Cys Ser Asp Arg Ser Ile Thr Trp 70 75 gcc tcc aca cct gac ctc gct cct gac ctt cag atc agt gca gtg gcc 450 Ala Ser Thr Pro Asp Leu Ala Pro Asp Leu. Gin Ile Set Ala Val. Ala 90 ctc cag cat gaa ggg cgt tac tca tgt gat ata gca gta cct gac ggg 498 Leu Gin His Glu Gly Arg Tyr Set Cys Asp Ile Ala Val Pro Asp Giy 100 105 110 aat ttc caa aac atc tat gac ctc caa gtg ctg gtg ccc cct gaa gta 546 Asn Phe Gln A-sn Ile Tyr Asp Leu Gin Val Leu Val. Pro Pro Glu Val 115 120 125 acc cac ttt cca ggg gaa aat aga act gca gtt tgt gag gcg att gca 594 Thr His Phe Pro Gly Giu Asn Arg Thr Ala Val Cys Giu Ala Ile Ala 130 135 140 ggc aaa cct gct. gcg cag atc tct tgg acg cca gat ggg gat tgt gtc 642 Gly Lys Pro Ala Ala Gin Ile Ser Trp Thr Pro Asp Gly Asp Cys Vai 145 150 155 160 gct aag aat gaa tca cac agc aat ggc acc gtg act gtc cgg agc aca 690 Ala Lys A-sn Giu. Set His Set Asn Gly Thr Val. Thr Val Arg Set Thr 165 170 175 tgc cac tgg gag cag agc cac gig tci gtc gig tic igt gtt gic tct Cys His Trp Giu Gin Ser His Val Ser Val Val Phe Cys Val Val Ser 180 155 190 cac ttg aca act ggt aac cag tci ctg tct His Leu Thr Thr Gly Asn Gin Ser Leu Ser ata gaa ctg Ile Giu Leu 205 atc Ile ggt aga ggg Gly Axrg Gly cca ict ati Pro Ser Ile ggt gac Gly Asp 210 att ati Ile Ile caa tta tia gga Gin Leu Leu Giy t ca S er 215 tac ait caa tac aic Tyr Ile Gin Tyr Ile 220 ttg atc aic Leu Ile Ile 225 tgc Cys ata Ile 230 itg Leu gga igc ati igt Gly Cys Ile Cys cii Leu 235 g ci Al a tig aaa atc agi Leu Lys Ile Ser ggC Gi y 240 786 834 882 930 978 1026 aga aaa igi Arg Lys Cys cca aaa icg Pro Lys Ser gga Gi y 250 act cca. gai Thr Pro Asp att gag Ile Giu 255 gag gat gaa Glu Asp Giu ctc tai gat Leu Tyr Asp 275 atg cag Met Gin 260 aci gig Thr Val ccg tat gci Pro Tyr Ala agc Ser 265 iac aca Tyr Thr gag aag agc aat cca Giu Lys Ser Asn Pro 270 cca gcg tea caa ggc Pro Ala Ser Gin Giy 285 acc acg acg gag gca cac Thr Thr Thr 280 Giu Ala His aaa gic aai ggc aca gac igt cti act tig ica Lys Val Asn Gly Thr Asp Cys Leu Thr Leu Ser 290 295 iagaaccaag gaaaagaagt caagagaeat eaiaattact ctccaatgga gggaaaitag ctcttcigaa gitcitagaa ttigcctita agttcticta tcaiiggaag tttggaaici taggaagaac tgatttaaii atiacaaaga aagcacaitg gcc aig gga ate Ala Met Giy Ile 300 gettitt agcacaaatg tigctgctac tiaiggtaaa acigcagaag cccigigtai gtatatatgc atttaigaaa gtgcaataca aigatgaaaa taciaaagca tttcatgiga tatggtatgt gtgtgcctt ttatcaagac aictcigica tgagtaitgg agaigtttt ctgagticc gitcitccaa atatgtigi aaaactcaci ata tcaagaaata aa a aga a aat ttacaaaigi ggcgttccag ctiaaactt iictaatgga cigttaattc atatcaaat gaacaaicai acgacaigai acacaiciga gciaataaag 1071 1131 1191 1251 1311 1371 1431 1491 1551 1574 MLCFWRTSWVAVLLIWGVFAAES SCPDKNQTMQNNS STN'TEVNTTVFVQMGKKALLCCPS ISLTKVI LITWTITLR GQPSCIIs ATElENSPSTASPLPLIAAQERSCIVDNQIDQLPE THFP GEN RTAVCEAIAGKPAAQI
SWTPDGDCVANESHSNGTVTVRSTCHWEQSHVSWVFCWSHLTTGNQSLSIE
LGGDLGYQI S IIGICLISCKKPSATDEDMPASTKNLDVT
EAHPASQGKVNGTDCLTLSAMGI
23 Table 2: Nucleotide and polypeptide sequences of additional OX2R hono logs.
primate, human, OX2RH1 (SEQ ID NO: 3 and 4): cagagaaaag cttctgttcg tccaagttac taaccaggct aaaccacata gacgtgaagg aaggggctag aaggaaggga gtgccccact gttgatgggg taagaggatc ctgtactgag aagttgacca gagagggtct caccatgcgc acagttcctt ct gtaccagt gtggaggaaa agtactgagt gaagggcaga aaaagagaaa acagaa atg ctc tgc cct tgg aga Met Leu Cys Pro Trp Arg act Thr gaa Giu agc Ser a aa Lys tca Ser atc Tle gct gCg Ala aag Lys cag Gin 30 tgg Trp gca Al a aac Asn gag Giu gag Glu att Ile cct Pro tta cta Leu ggt Gly -1 aat Asn aca Thr gta Val aga ggg Gly g ct Al a 1 cat His cag Gin aag Lys aat cta Leu -15 gct Al a gct Ala aac As n atg Met 50 ttg cig ttg Leu Leu caa cca Gin Pro tta gct Leu Al1a 20 tac tcg Tyr Ser 35 gct aca Ala Thr aic ata att ttg Ile Leu aac aac Asn Asn 5 tca agc Ser Ser aaa gta Lys Val aat gct Asn Ala ata aca act Thr -10 t ca Ser agt Ser ct C Leu gtg Val 55 tgg atc Ile tta Leu tta Leu g ca Ala ctt Leu gaa ttc Phe atg Met tgt Cys gaa Glu tgt Cys ata Ile tta Le u ctg Le u atg Met gtt Val tg c Cys at c Ile gtg Val caa Gin gat Asp aac As n cct Pro ctg Leu gcc Al a act Thr gaa Glu act Thr cet Pro a ga Arg 120 180 234 282 330 378 426 474 522 570 618 666 714 762 Leu Arg Asn Leu Ile Ile Ile Thr Trp Glu ggc Gi y aag Lys gat Asp ggg Gi y 125 gga Gi y cag Gin gaa Glu cag Gin 110 tat Tyr tat Tyr cct Pro acc Thr aat As n tac Tyr ca C His tcc Ser aac As n t cg Ser aga Arg ct C Leu tgC Cys tgi Cys ga c Asp tgc Cys caa Gin 145 aca Th r act Thr ct t Leu ata Ile 130 gtg Val aaa Lys gat As p cag Gin 115 atg Met tta Leu g cc Al.a gag Gi u 100 att Ile gta Val gtt Val tac Tyr 85 aga Arg cgt Ar g aca Th r aca Th r aag aaa Lys Lys ata acc Ile Thr acc gtg Thr Val cct gat Pro Asp 135 cct gaa Pro Glu 150 gaa Glu tgg T rp gcc Al a 120 ggg Gi y gtg Val1 aca Thr gtc Val 105 atc Ile aat As n a cc Thr aat As n tcc Ser act Thr ttc Phe ctg Leu gag Glu a ga Arg cat His cat His ttt Ph e 155 acc Thr cct Pro gac Asp cgt Arg 140 ca a Gln aac agg aat aga. act Asn Arg Asn gcg cat atc Ala His Ile 175 tac tgg agc Tyr Trp Ser AMg Thr 160 tcc tgg Ser Trp gca gta Ala Val atc cca Ile Pro tgc aag gca gtt gca Cys Lys Ala Val Ala ggg aag cca gct Gly Lys Pro Ala gag Glu 180 gat tgt gcc Asp Cys Ala act Thr 185 tgc Cys 170 aag caa gaa Lys Gin Glu cac tgg gag His Trp Glu aat ggc aca Asn Gly Thr 190 cac His gtg Val 195 gtg Val act gtt aag agt Thr Val Lys Ser aca Thr 200 tcc Ser gt C Val 205 aac Asn aat gtg tct Asn Val Ser acc tgc cac Thr Cys His gtC Val 215 cat ttg act His Leu Thr 810 858 906 954 1002 1050 1098 aag agt ctg Lys Ser Leu ta c Tyr 225 ata Ile gag cta ctt Giu Leu Leu gtt cca ggt gcc Val Pro Gly Ala aaa aaa Lys Lys 235 atc agc aaa Ile Ser Lys att Ile 240 tat tcc ata Tyr Ser Ile cct. tac tat Pro Tyr Tyr tat tat tta Tyr Tyr Leu 250 ctg cag aaa Leu Gin Lys gac cat cgt ggg att cat ttg gtt Asp His Arg Gly Ile His Leu Val 255 260 gaa agt caa. tgg Glu Ser Gin Trp 265 ata taaattgaat aaaacagaat ctactccagt tgttgaggag gatgaaatgc Ile agccctatgc cagctacaca gagaagaaca atcctctcta. tgatactaca aacaaggtga aggcatctga ggcattacaa agtgaagttg acacagacct ccatacttta taagttgttg gactctagta ccaagaaaca acaacaaacg agatacatta taattactgt ctgattttct tacagttcta gaatgaagac ttatattgaa attaggtttt ccaaggttct tagaagacat tttaatggat tctcattcat acccttgtat aattggaatt tttgattctt agctgctacc agctagttct ctgaagaact gatgttatta caaagaaaat acatgcccat gaccaaatat tcaaattgtg caggacagta aataatgaaa accaaatttc ctcaagaaat aactgaagaa ggagcaagtg tgaacagttt cttgtgtatc ctt 1151 1211 1271 1331 1391 1451 1511 1571 1604 MIJCPWRTANLGLLLI LTI FLVAF.AEGAAQPNNS LMLQTS KENHALAS SS LCMDEKQITQNYSKVLAEVNTSWPVKM
ATNAVLCCPPIAL.PNLIIITWEIILRGQPSCTKAYKKETNETKETNCTDERITWNSRPDQNSDLQIRTVAITHDGY
YRCIMVTPDGNFHRGYHLQVLVT PEVTLFQNPNRTAVCKAVAGKPAAHI SWI PEGDCATKQEYWSNGTVTVKSTCH WEVHNVSTVTCHVS HLTGNS LY IELL PVPGAKKI SKI I YS I YH PYYYYLDHRGI HLVVES QWLQKI ID NO: 19 and primate, human, OX2RH1.2 (SEQ 00 atg ctc tgc cct tgg aga act gct aac cta ggg cta ctg ttg att ttg Met Leu Cys Pro Trp Arg Thr Ala Asn act Thr tca Se r agt Ser ct C Leu gt g Val 55 tgg Trp aaa Lys aCC Thr gtg Val gat Asp 135 gaa Glu gtt Val tgt Cys atc Ile tta Leu tta Leu gca Al a ctt Leu gaa Glu gaa Glu tgg T rp gcc Al a 120 ggg Gly gtg Val gca Ala g cc Al a ttc Phe aig Met tgt Cys gaa Gi u tgt Cys a ta Ile aca Thr gtc Val 105 atc Ile aat As n acc Thr 9gg Gi y act Thr 185 tta Leu ctg Leu 10 atg Met gtt Val tgC Cys atc Ile aat Asn t cc Ser act Thr ttc Phe Ctg Leu aag Lys 170 aag Lys -20 gaa Gi u agc Ser aaa Lys tca Ser 45 atc Ile Gi y aag Lys gat Asp ggg Gi y 125 gga Gi y aac As n gcg Al a tac Tyr gcg Al a aag Lys cag Gin 30 tgg Trp gca Al a ca g Gin ga a Glu ca g Gin 110 tat Tyr tat Tyr agg Arg ca g Gin tgg Trp 190 Leu ggt Gi y -1I aat As n a ca Thr gta Val1 aga Arg tcc Ser aac Asnr tcg Ser aga Ar g ct C Leu aga Arg 160 tcc Ser aat As n Gi y gct Al a 1 cat His cag Gin aag Lys aat As n 65 tgc Cys tgt Cys gac Asp tgC Cys caa Gin 145 act Thr tgg Trp ggc Gi y Leu Leu Leu gct caa cca Ala Gin Pro gCt tta gct Ala Leu Ala aac tac tcg Asn Tyr Ser atg gct aca Met Ala Thr ttg atc ata Leu Ile Ile aca aaa gCC Thr Lys Ala act gat gag Thr Asp Giu 100 ctt cag att Leu Gin Ile 115 ata atg gta Ile Met Val 130 gtg tta gtt Val Leu Val gca gta tgc Ala Val Cys atc cca gag Ile Pro Giu 180 aca gtg act Thr Vai Thr 195 Ile aac As n
S
tca Ser aaa Lys aat As n ata Ile tac Tyr aga Arg cgt Arg a ca Thr aca Thr aag Lys 165 ggc Gi y gtt Val Leu aac As n agc Ser gta Val gct Al a aca Thr agg A.rg ata Ile Pro cct Pro C Ct Pro 150 gca Al1 a gat Asp aag Lys 48 96 144 192 240 288 336 384 432 480 528 576 624 672 agt aca Ser Thr 200 tgc cac tgg gag Cys His Trp Glu gtc Val1 205 cac aat gtg tct His Asn Val Ser acc Thr 210 gtg acc tgc cac Val Thr Cys His Val1 215 tcc cat ttg act Ser His Leu Thr ggc Gi y 220 aac aag agt ctg Asn Lys Ser Leu tac Tyr 225 ata gag cta ctt Ile Glu Leu Leu cct Pro 230 gtt cca ggt gcc Val Pro Gly Ala aaa Lys 235 aaa tca gca aaa Lys Ser Ala Lys tat att cca tat Tyr Ile Pro Tyr atc atc Ile Ile 245 ctt act att Leu Thr Ile gtc aat ggc Val Asn Gly 265 att Ile 250 att ttg acc atc Ile Leu Thr Ile gtg Val 255 gga ttc ait tgg Gly Phe Ile Trp ttg ttg aaa Leu Leu Lys 260 tct act cca Sez Thr Pro 720 768 816 864 912 960 1008 1047 tgc aga aaa tat Cys Arg Lys Tyr aaa Lys 270 ttg aat aaa aca Leu Asn Lys Thr gaa Giu 275 gtt gtt Val. Val 280 gag gag gat gaa Glu Giu Asp Giu atg Met 285 cag ccc tat gcc Gin Pro Tyr Ala agc Ser 290 tac aca gag aag Tyr Thr Glu Lys aac As n 295 aat cct ctc tat Asn Pro Leu Tyr gat Asp 300 act aca aac aag Thr Thr Asn Lys aag gca tct cag gca Lys Ala Ser Gin Ala 310 tta caa agt gaa Leu Gln Ser Glu gtt Val 315 gac aca gac ctc Asp Thr Asp Leu cat act tta taa His Thr Leu 320 MLCPWRTANLGLLLI LT I FLVAEAEGAAQ PNN S LMLQTS KEN HALAS S S LCMDEKQI TQNYS KVLAEVNT SWPVKM
ATNAVLCCPPIAIJRNLIIITWEIILRGQPSCTKAYRKETNETKETNC:TDERITWVSRPDQNSDLQIRPVAITHDGY
YRCIMVTPDGNFHRGYHLQVLVT PEVTLFQNRNRTAVCKAVAGKPAAQ ISWI PEGDCATKQEYWSNGTVTVKSTCH WEVHNVSTVTCHVS HLTGNKS LYI ELLPVP GAKKSAKLYI PYI I LT I I I LT IVGFIWLLKVN GC RXYKLNKT EST P VVEEDEM4QPYASYTEKNN PLYDTTNKVKA.SQALQSEVDTDLHTLZ 27 rodent, mouse, 0X2P.H1 (SEQ ID NO: 5 and 6): aaaaccgaa atg ttt tgc tit tgg aga act tct gcc cta gca gtg ctc tta 51 Met Phe Cys Phe Trp Azg Thr Ser Ala Leu Ala Val Leu Leu 1 5 ata tgg ggg gtc ttt gtg gct ggg tca agt tgt act gat aag aat caa 99 Ile a ca Thr gtg Val c ca Pro ctg Leu acc Thr agt Ser tac Tyr gac Asp aac As n atc Ile 175 agc Set Trp aca Thr tct Ser ctg Leu cca Pro 80 agc Se r cat Pro aca Thr ci c Leu aga Arg 160 t 5cr aat As n Giy Val cag aac Gin Asn gta cag Val GIn aca aaa Thr Lys tcc tgc Ser Cys tgc tig Cys Leu gaa ct Giu Leu tgi gag Cys Giu 130 caa gtg Gin Val 145 tct gca Set Ala tgg tct Trp Ser ggc act Gly Thr Phe aac As n ata Ile gca Al a a ca Thr gg C Gly ca g Gin 115 aca Thr ctg Leu gt c Val cca Pro gig Val1 195 Val Ala 20 agt tca Set Set ggt aca Gly Thr gia tia Val Leu ata gca Ile Ala 85 agg aac Arg Asn 100 atc agt Ile Ser gta aca Val Thr gig ccc Val Pro tgt gag Cys Glu 165 gat ggg Asp Giy 180 act gic Thr Val Gly t ci Ser aag Lys aic Ile 70 ia c Tyr aic Ile gca Al a acti Pro cci Pro 150 gca Al a gac Asp agg Arg Ser cci Pro gct Al a 55 aca Th r aaa Lys acc Thr gig Val gaa Giu 135 gaa Giu aig Met igi Cys agc Ser Set Cys 25 cig aca Leu Thr 40 cig cic Leu Leu tgg ata Trp Ile gia gat Val Asp igg gcc Trp Ala 105 acc ctc Thr Leu 120 ggg aai Gly Asn gia acc Val Thr gca ggc Al a Gly gic act Val Thr 185 aca igc Thr Cys 200 Thr Asp caa gig Gin Vai igc tgc Cys Cys ata aag Ile Lys aca aag Thr Lys icc aca Ser Thr cag cat Gin His iii gaa Phe Giu tac tt Tyr Phe 155 aag cci Lys Pro 170 acg agi Thr Set cac igg His Trp Lys aac As n iii Phe ci c Leu acc Thr act Pro gag Gi u aaa Lys 140 cca Prto g ci Al a gaa Glu gag Glu Asn Gin act aca Thr Thr tct ati Set Ile aga ggc Arg Gly aat gaa Asn Glu gac cac Asp His 110 ggg act Gly Thr 125 aac tat As n Tyt gag aaa Giu Lys gca cag Ala Gin ica cac Set His 190 cag aac Gin Asn 205 147 195 243 291 339 387 435 483 531 579 627 aai gtg Asn Val tct gat gig Ser Asp Vai 210 icc tgc ati Ser Cys Ile gi c Val1 ict cat tig act Ser His Leu Thr ggi aac caa Gly Asn Gin 220 tct ctg tcc ata, gaa ctg agt aga ggt ggt aac caa tca 'tta. cga cca Ser Leu Ser Ile Giu Leu Sex Arg Gly Giy A.5n Gin Ser Leu Arg Pro 225 230 235 tat att cca tac Tyr Ile-Pro Tyr 240 tgc att tgt ctt 0 Cvs Ile Cys Leu atc ata cca Ile Ilie Pro 245 tca. ati atc att ttg atc atc ata gga.
Ser Ile Ile Ilie Leu Ile Ile Ile Gly 250
I
ttg a Leu Lvs 00 2552 aaa Lys tta gaa gct.
Leu Giu Aia act Thr 275 260 tca Ser atc agt ggc ttc aga aaa Ile Ser Giy Phe Azg Lys 265 gct att. gag gag gat gaa Ala Ile Giu Giu Asp Giu 280 agc aat cca ctc tat gat Ser Asn Pro Leu Tyr Asp 295 tca caa ggc gaa gtc aat Ser Gin Giy Giu Val Asn 310 tgc aaa ttg cca Cys Lys Leu Pro 27 0 atg cag cct tat Met Gin Pro Tyr 285 act gtg act aag Thr Val Thr Lys 300 ggc aca gac tgc Gly Thr Asp Cys 315 771 819 867 915 963 gct agc tat Ala Ser Tyr gtg gag gca Val Giu Ala aca. gag aag Thr Giu Lys 290 ttt cca gta Phe Pro Val ctt act ttg tcg gcc att gga atc tagaaccaag aaaaaagaag tcaagagaca Leu Thr Leu Ser Ala Ile Giy Ile S320 325 tcataattac tgctttgctt tctttaaaat tcgacaatgg aaggactact. tggaaatta.
ctcttccaaa gctattaaaa agcacaaatg ttctaatgaa attgc-attta aattctatc ttggaagttt ggaatctctg ctgctacctg ttaattttag gaagaactga tttaattat acaaagaaag cacatggtta tggtgaaata tcaagttgtg caataaagta tgatgaaaa tgagtttcct caagaaataa ctgcaggagg aacaatcatc actaaagaat ttcatgtga ttcttacaaa aaaattccta. tgtatacatg actatggtat gtgtgtccaa. ttacatgtt atttacaaat gtgtatatat gcacacattt gcttttcagg acatctcctt gtaaaaaac cactggagtt ttggatttat. aaaagcttat aaagtgagca tiggagatat ttt a t c g 1017 1077 1137 1197 1257 1317 1377 1437 1490 MFCFWRT SALAVLLIWGVFVAG S S CTDKN QTTQNN 5S SSPLTQWNTTVSVOI GT KALLCCFS I P LTKAVLI TWI IKL RGLP SCT IAYKVDTKTNETSCLG RN ITWAST PDH S PELQ I SAVTLQHEGTYTCETVT PEGN FEKNYDLQV LVPEV TYFP EKN RSAVCEAMAGKPAAQI SW S P DGDCVTT SES HSN GTVTVRS TCHWEQNNVS DVS CIVSHiLTGNQSLS I EL S GNSLPIPIIPSIIIL CICLISGFKKPKF7S. EEQYSY KNPYTTV
AFPVSQGEVNGTDCLTLSAIGI
29 primate, human, OX2RI{2 (SEQ ID NO: 7 and 8): atg ggt gga aag cag aig aca cag aac tat ica aca att tt' Met Gly Gly Lys Gin Met Thr Gin Asn Tyr Ser Thr Ile Phi 1 5 10 ggt aac att tca cag cct gta ctg atg gat ata. aat gct gt Gly Asn Ile Ser Gin Pro Val Leu Met Asp Ile Asn Aia Va 25 3 tgc cct cct att gca tta aga aat ttg atc ata ata aca tg Cys Pro Pro Ile A-1a Leu Arg Asn Leu Ile Ile Ile Thr Tx 40 atc ctg aga ggc cag cct tcc tgc aca aaa gcc tac aag a Ile Leu Arg Giy Gin Pro Ser Cys Thr Lys Ala Tyr Lys L 55 60 aai gag acc aag gaa acc aac tgt act gtt gag aga ata ac Asn Giu Thr Lys Giu Thr Asn Cys Thr Val Giu Arg Ile T~ 70 75 tct aga cct gat cag aat tcg gac ctt cag att cgt ccg g Ser A~rg Pro Asp Gin Asn Ser Asp Leu Gin Ile Arg Pro V.
85 90 act cat gac ggg tat tac aga ggc ata gtg gta aca cct g Thr His Asp Giy Tyr Tyr Arg Gly Ile Val Vai Thr Pro A 100 105 1 ttc cat cgt gga tat cac ctc caa gtg tta gtt aca ccc g Phe His Arg Giy Tyr His Leu Gin Val Leu Vai Thr Pro G 115 120 125 cta tit caa agc agg aat ata act gca gia tgc aag gcag Leu Phe Gin Ser Axg Asn Ile Thr Ala Val Cys Lys Ala N 130 135 140 aag cca gct gcc cag atc tcc tgg atc cca gag gga ict Lys Pro Ala Ala Gin Ile Ser Trp Ile Pro Giu Giy Ser 145 150 155 act aag caa gaa tac tgg ggc aat ggc aca gtg acg gtt Thr Lys Gin Giu Tyr Trp Giy Asn Gly Thr Val Thr Val 165 170 tgc ccc tgg gag ggc cac aag tct act gtg acc tgc cat Cys Pro Trp Giu Giy His Lys Ser Thr Vai Thr Cys His 180 185 ttg act ggc aac aag agt ctg tcc gta aag ttg aat tca Leu Thr Giy Asn Lys Ser Leu Ser Val Lys Leu Asn Ser 195 200 205 acc tca gga tct cca gcg ttg icc tta ctg atc att ct Thr Ser Giy Ser Pro Ala Leu Ser Leu Leu Ile Ile Leu 210 215 220 g 1 0 g
P
La Lg al at 11 t aa at T1 gca gaa Ala Giu ctt tgt Leu Cys gaa ata Giu Ile gaa aca Giu Thr tgg gtc Trp Val gac acc Asp Thr ggg aat Giy Asn gtg aac Vai Asn t aca ggg 1 Thr Gly t ctt gcc e Leu Ala 160 .g agt aca ~s Ser Thr 17 :c tcc cat il Ser His gt ctc aga ly Leu Arg at gtg aaa yr Vai Lys 48 96 144 192 240 288 336 384 432 480 528 57 6 624 672 etc tet ctt ttt gtg gtc att ctg gic acc aca gga ttt gtt ttc ttc Leu Ser Leu Phe Val Val Ile Leu Val. Thr Thr Giy Phe Val. Phe Phe 225 230 235 240 cag agg ata aat cat gtc aga aaa gtt ctt taaagaagaa ggaagggtct Gin Arg Ile Asn His Val. Arg Lys Vai Leu 245 250 720 7*7 0 tcttttgctt ctcctacttg tctctggact gcaacattgg tgagatgagt gatggtccag 830 cagtgaatt gggecatgga tgatgttaag gatagaagcc actcagtagg atagaagaaa 890 agaaagatgg aagaaggatc ctgggcttga tgaccatgaa gtttecctat aaaccctcaa 950 ccacctattc attgacttct tttgtgttag agtgaataaa attttgttca tgccagtgtt 1010 MGGKQMTQNYST IFAEGNI SQPLMDI NAVLCCP P IALLI IITWEI I LRGQ PS CT KAYKKETNET
KETNCVR
ITWVSRPDQNSDLQIRPVDTTHDGYYRGIVVT'PDGN FHRGYHiLQVLW PEVt'LFQS RNITAVCKAVTGKPAAQI
SW
I PEGS I AKEWNGVVSCP GKTTHSHTNSLVL GLRT SGS PALS LLI I LYVKL5 LF
VILVTTGFFFQRINH{VP.KV
rodent, mouse, OX2RH2 (SEQ ID NO: 9 and aga ggc Arg Gly 1 cag act tcc tgc ata atg gcc Gin Pro Ser Cys Ile Met Aia 5 tac aaa gta gaa aca Tyr Lys Val Giu Thr 10 Lys Giu acc aat gaa Thr Asn Giu gac cac ati Asp His Ile -i S 35 ace Th r tgc ttg ggc agg Cys Leu Gly Arg ate ace tgg gc Ile Thr Trp Ala tee aca cct Ser Thr Pro eag cat gag Gin His Giu cat gae ctt cag Pro Asp Leu Gin ate Ile 40 agt gcg gtg gee Ser Aia Val Aia etc Leu ggg aat Giy Asn s0 tac tta tgt gag ata aca aca ect gaa Tyr Leu Cys Giu Ile Thr Thr Pro Glu ggg eat ttc cat Gly Asn Phe His gta ace tac ttt Val Thr Tyr Phe aaa Lys etc Leu s0 192 240 288 tat gac etc caa Tyr Asp Leu Gin ctg gtg ccc ect Leu Val Pro Pro ggg gaa aat aga Gly Giu Asn Arg gea cag ate tet Ala Gin Ile Ser gca gtt tgt gag Ala Vai Cys Giu gca Al a 90 atg gea ggc aeg Met Ala Gly Lys ect get Pro Ala tgg act cca gat Trp Th~r Pro Asp gac tgt gte act Asp Cys Vai Thr aag agt gag Lys Ser Giu 110 eec tgg gag His Trp Glu tca eac age Ser His Ser 115 aat ggc act gtg Asn Gly Thr Va).
act Thr 120 gte egg age act Val Arg Ser Thr eag eec Gin Asn 130 aat gtg tct get Asn Val Ser Ala gtg Val 135 tee tgc att gte Ser Cys Ile Val cat tcg act ggt His Ser Thr Gly 432 00 00 aai cag ict cig tcc ata gaa ctg agt aga ggt acc ace agc aec acc Asn Gin Ser Leu Se Ile Giu Leu Ser Arg Gly Thr Thr Ser Thr Thr 145 150 155 160 cct icc tig cig acc ait ctc tac gtg aaa atg gtc cit tig ggg at Pro Ser Leu Leu Thr Ile Leu Tyr Val Lys Met Vai Leu Leu Gly Ile 165 170 175 att ctt cit aaa gig gga iii get tic tte cag aag aga aat gtt ace Ile Leu Leu Lys Val Gly Phe Ala Phe Phe Gin Lys Arg Asn Val Thr 180 185 190 aga aca igaatatcca gatitctgga ageteattag tctgatgaea caiaceagaa Mrg Thr aacageatii giaatcaact tetcaitgg aatceagcti acecgtecct gcigiciica tgtgttag acacteacci ccaaatictt aactgagaag ggciectgtc iaaaggaaat atggggacaa atigiggage aiagaccaaa agaaaggcca tccagagact gcccaccia aggacccate ecatatacag acaccaaaee cagacaciac tgaagatgct gcgaagegii tgctgacagg agcigitat agctgtctcc igagaggete agccagagcc tgacaaatac ataggiagat gciigcagcc aacaactgga ctgagcaaaa aatetccatt ggaggagtta gagaaaggac tgaagagggi gaaagggtit geagcecat aggaagaaca acaataicaa ccaaccagat cicccagagc teccagggac iaa 480 528 576 632 692 752 812 872 932 992 1052 1085 RGQPSCIMAYKVETKETNETCLGRNITWASTPDHI PDLQISAVALQHEGNYLCEITTPEGNFHKVYDLQVLVPPEV TYFLGEINRTAVCEAMAGKPAAQI SWTPDGDCV'rKSESHSNGTV'VRSTCHWEQNNVSAVSCIVSHSTGNQSLSIEL SRGTTSTTPSLLTILYVKMVLLGI ILLKVGFAFFQKRNVTRT Rodent, mouse, OX2RH-4 aig cat get cig ggg agg ati Met His Ala Leu Giy Axg Ile -20 aat ait iii gig tet ggg tea Asn Ilie Phe Val Ser Gly Ser cag aat gac agi tea tet tet Gin Asn Asp Ser Ser Ser Ser (SEQ ID ceg act Pro Thr agi igi Ser Cys -1 cig aca Leu Thr 15 NO: 22 and 23): ttg act tig ctg Leu Thr Leu Leu -1i5 act gat gag aat Thr Asp Giu Asn 1 eaa git aae act Gin Val Asn Thr ate te ate Ile Phe Ile eaa aca ata Gin Thr Ile aca atg tet Thr Met Ser agt eca ctg Ser Pro Leu cac etg cct His Leu Pro gta cag Vai Gin atg gat aaa aag get Met Asp Lys Lys Ala 30 ctg ete tge tge ttt tet Leu Leu Cys Cys Phe Ser 192 240 ata aat gea gta tia ate aea tgg ata Ile Asn Ala Vai Leu Ile Thr Trp Ile ata aaa cac aga Ile Lys His Azg icc tgc aca ata Ser Cys Thr Ile tgc ttg ggc agg Cys Leu Gly Arg g ca Al1 a tac aac cia gat Tyr Asn Leu Asp aaa aag acc: aat gaa acc agc Lys Lys Thr Asn Glu Thr Ser 65 icc aca cci gac cac agi cct Ser Thr Pro Asp His Ser Pro aac: atc acc igg Asn Ile Thr Trp gaa cit cag atc Glu Leu Gin Ile agt. gca gig Ser Ala Val gcc: Al a 95 ctc cag cat gag Leu Gin His Glu ggg Gi y 100 act tac aca Thr Tyr Thr 288 336 384 432 480 igt gag Cys Glu 105 caa gtg Gin Val 120 ata gte ace. cci Ile Val Th~r Pro gaa Glu 110 ggg aai tia gaa Gly Asn Leu Glu aaa Lys 115 gtc tat gac cic Val Tyr Asp Leu cig gtg ccc Leu Val Pro Cct Pro 125 gag gta acc tac Glu Val Thr Tyr cca ggg aaa aac Pro Gly Lys Asn ega Arg 135 act gca gtc tgt Thr Ala Val Cys gag Glu 140 gca aig gca ggc Ala Met Ala Gly cct gct gca cag Pro Ala Ala Gin etc ici Ile Ser 150 igg act cca Trp, Thr Pro ggg gac tgt gic Gly Asp Cys Val act Thr 160 aag agi gag ice Lys Ser Glu Ser cac agc aet His Ser Asn 165 eac: aat gig Asn Asn Val 528 576 624 672 ggc act gtg Gly Thr Val 170 ict. gti gig Ser Val Val 185 act gic egg egc Thr Val Arg Ser tgc cac tgg gag Cys His Trp Glu icc igc tia 5cr Cys Leu ict cat tcg act Ser His 5cr Thr ggt Gly 195 aat cag ici cig Asn Gin Sex Leu icc Se r 200 ata gea cig agi Ile Glu Leu Ser ca Gin 205 ggi aca aig acc Gly Thr Met Thr ccc: cgi icc ttg Pro Arg Ser Leu ci g Leu 215 acc ati cic tat Thr Ile Leu Tyr gig Val 220 eaa aig gcc cit tig gig at att cii cit aac Lys Met Ala Leu Leu Val Ile Ile Leu Leu Asn 225 230 gte gge iii gci tic: tic cag aag aga aat iii gcc age aca tga Val Gly Phe Ala Phe Phe Gin Lys Arg Asn Phe Al1a Arg Thr 235 240 245 MALGRI PTLTLLI FIN IFVSGS SCTDENQT IQNDS S SSLTQVNTTMSVQKALCCFS
SPLINALIT
WI IKHRHLPSCTIAYNLDKKTNETSCLGRNITWASTPDHSPELQI
SAVALQHEGTYTCEIVTPEGNLEKVY
DLQVLVPPEVTYFPGKNRTAVCEAMAGKPAAQI SWTPDGDCVrKSESHSNGTVTVRSTCHWEQNl4VSWVSC LVS HSTGNQS LS IELS QGTMTT PRS LLT ILYVO4ALLVI I LLNVGFAFFQKRN FART 33 Table 3: Rodent, mouse, OX2RH3 (SEQ ID N~O: 11 and 12): ggcacgagtt acgatttgtg cttaaeetga ctecactcca g atg cat gat ttg ggg 56 Met His Ala Leu Gay agg act ctg gat ttg atg tta etc atc ttc atc act att ttg gtg cet 104 AMg Thr Leu Ala Leu Met Leu Leu Ile Phe Ile Thr Ile Leu Val Pro 15 -10 gag tca agt tgt tea gtg aaa gga egg gag gag atc cca ecg gat gat 152 Giu Ser Ser Cys Ser Val Lys Gly Arg Giu Glu Ile Pro Pro Asp Asp -1 1 5 tca ttt act ttt tea gat gat aat ate tte ect gat gga gtg ggc gtc 200 Ser Phe Pro Phe Ser Asp Asp Asn Ilie Phe Pro Asp Gly Val. Gly Val 20 ace atg gag ati gag att ate act cca gtg tet gta cag ata ggt atc 248 Thr Met Giu Ile Giu Ile Ile Thr Pro Val Ser Val Gin Ile Gly Ile 35 aag get cag ett ttc tgt cat act agt cca tca aaa gaa gca aca ctt 296 Lys Ala Gin Leu Phe Cys His Pro Ser Pro Ser Lys Giu Ala Thr Leu 45 50 55 aga ata tgg gaaata act ccc aga gac tgg cct tcc tgc aga eta ccc 344 Arg Ile Trp Glu Ile Thr Pro Mrg Asp Trp Pro Ser Cys Arg Leu Pro '70 tac aga gca gag ttg eag cag ate agt aaa aaa ate tgt act gag aga 392 Tyr Mrg Ala Giu Leu Gin Gin Ile Ser Lys Lys Ile Cys. Thr Giu Mzg 85 gga ace act agg gte act gca eat cac cag agt tet gac ctt eec ate 440 Gly Thr Thr AMg Val Pro Ala His His Gin Ser Ser Asp Leu Pro Ile 100 105 aaa tea atg gee etc aag cat gat ggg cat tac tea tgt egg ata gaa 488 Lys Ser Met Ala Leu Lys His Asp Gly His Tyr Sex Cys Arg Ile Glu 110 115 120 aca aca gat ggg att ttc caa gag aga cat age ate eaa gtg eca ggg 536 Thr Thr Asp Gly Ile Phe Gin Giu Arg His Ser Ile Gin Val pro Giy 125 130 135 140 gaa aat aga act gta gtt tgt gag gea att gca age aag act get atg 584 Giu Asn Azg Thr Val Val Cys Giu Ala Ile Ala Ser Lys Pro Ala Met 145 150 155 eag ate ttg tgg act eca gat gag gac tgt gte act aag agt aaa tea 632 Gin Ile Leu Trp Thr Pro Asp Giu Asp Cys Val. Thr Lys Ser Lys Ser 160 165 170 cac aat gac ace atg att gte agg age aag tgc cac agg gag aaa aac 680 His Asn Asp Thr Met Ile Val Arg Ser Lys Cys His Mrg Giu Lys Asn 175 180 185 aat ggc cac agt gtg ttc tgc tit atc tcc cat ttg act gat aac igg Asn Gly His Ser Val Phe Cys Phe Ile Ser His Leu Thr Asp Asn Trp 190 195 200 att ctc tcc atg gaa cag aat cga ggt aca ace agc ate ctg cct tcc Ile Leu Ser Met Glu Gin Asn Arg Gly Thr Thr Ser Ile Leu Pro Ser 205 210 215 220 ttg ctg agc att etc tat gtg aaa ctg gct gta act git ctc atc gta Leu Leu Ser Ile Leu Tyr Val Lys Leu Ala Val Thr Val Leu Ile Val 225 230 235 gga ttt gct ttt ttc cag aag aga aat tat ttc aga gtg eca gaa ggc Gly Phe Ala Phe Phe Gin Lys Arg Asn Tyr Phe Arg Val Pro Giu Gly 240 245 250 tee tgaggagagt ggtctgtggt taagatgaga tttaccacca tctgaaagac Ser 728 776 824 872 925 atcttgteta ccgcgcagcg tgctgagatt ccgagaagca gccaeagaae ctactaggaa 985 gacaaatctg atgtggttgt caatcctttc aatggacctg agtacttcta. taaacccgag 1045 tgaggttgtg ctggacccag gagccaggct aggtcatata tgttgatttt tgctgcaaga 1105 ectcatggtt tatctacaaa tcctaaattc tttcacttcc agttttaaaa cttttggccc 1165 aagcatttta tccacagcat aacaccttta aagaaactct cccacggaaa ctgctggttc 1225 catggaatgg aaaattgcaa catggtttac aagacagtgc aaaccaagca gcattceaag 1285 atatgaqctt cagaaagtta caggaaetgt cttgggacga'gaaagaagga ttaaatagtt 1345 cccagtcec 1354 MHALGRTLALMLLI FITI LVESSCSVKGREEI PPDDSFP FSDDI FPDGG IEI ITPVSVQI GI KQLFCH PSPSKEATLRIWEIT PPDWPSCRLPYRAELQQI SKKI CTERGTTRVPAI*IQS
SDLPIKSMALKHDGHYSCRIETTD
GI FQEP.HS I QVP GEN RTVVCFAIAS K PAM~QI LWT PDEDCVT KS KSIINDTMI VRSKCHREKNNGH SVFC F1 SHLTDN WILSMEQNRGTTS ILPSLLSILYVKLAVTVLIVGFAFFQKRNYFRVPEGS Table 4: Reverse translations of OX2R homologs: rodent, e.g., rat, OX2RH1 (SEQ ID NO: 13): atgytntgyt gcngazwsnw gargtnaaya athwsnyina tgyathathw wsnathacnt ytncarcayg athtaygayy acngcngtnt ggngaytgyg tgycaytggg ggnaaycarw athcartaya aarathwsng gargaygara gtnacnacna acnytnwsng tytggmgnac sntgyccnga cra cngtntt cnaargtnat sntayaargc gggcnwsnac arggnnignta tncargtflyt gygargcnat tngcnaaraa arcarwsnca snytnwsnat thathccnws gntgymgnaa tgcarccnta cngargcnca cnatgggnat nwsncaygtl ya ara a ycar ygtncaratg hytnathacn ngayacnmgi nccngayytn ywsntgygay ngtnccnccn hgcnggnaar yga rws ncay ygtnwsngtn hgarytnggn nathathath rtgyaarytl ygcnwsntay yccngcnwsn h gcngtnytny acflatgcara ggriaaraarg tggacnatha garacncayg gcnccngayy athgcngtnc gargtflacnc ccngcngcnc wsnaayggna gtnttytgyg rngnggnggng ytnathatha ccnaarwsng acngaraarw carggnaarg tnathtgggg ayaaywsnws cnytnytntg cnytnmgngg arwsnaaytg tncarathws cngayggnaa ayttyccngg arathwsntg cngtna cngt tngtnwsnca aycarytnyt thggntgyat gngcna cnc snaayccnyt tnaayggnac ngtnttygcn nacnatgacn ytgyccnwsn ncarccnwsn yws nga ymgn ng cngtngcn yttycaraay ngaraayngn gacnccngay nmgnwsna cn yytnacnacn nggnwsntay htgyytnytn ngayathgar ntaygayacn ngaytgyytn 120 180 240 300 360 420 480 540 600 660 '72 0 780 840 900 960 981 primate, human, OX2RH1 (SEQ ID NO: 14): atgytntgyc gtngcngarg garaaycayg taywsnaarg gtnytntgyt ytrngnggnc acna aytgya carathmgna gayggnaayt ttycaraaym athwsntgga gtna cngtna gtnwsncayy aaraarathw mgnggnathc cntggmgnac cnga rggng c cnytngcnws tnytngcnga gyccnccnat arccnwsntg cngayga rig cngtngcnat tycayngngg gnaaymgnac thccngargg arwsnacntg tna cnggnaa snaarathat ngcnaayytfl ngcncarccn nwsnwsnytn rgtnaayacn hgcnytngn yacnaargcn nathacntgg hacncaygay ntaycayytn ngcngtntgy ngaytgygcn ycaytgggar yaarwsnytn htaywsnath ggnytnytny a aya aywsnfy tgyatggayg wsntggccng aayytnatha tayaaraa rg gtnwsnngnc ggntaytaym ca rgtnytng aargcngtng acnaarcarg gtncayaayg tayathgary taycayccnt tnathytnac tnatgytnca araarcarat tnaaratggc thathacntg aracnaayga cngaycaraa gntgyathat tnacnccnga cnggnaar cc artaytggws tnwsnacngt tnytnccngt aytaytayta arath nathttyytn racnwsnaar hacncaraay nacnaaygcn ggazathath racnaargar ywsngayytn ggtnacnccn rgtna cnytn ngcngcncay naayggnacn nacntgycay nccnggngcn yytngaycay 120 180 240 300 360 420 480 540 600 660 720 780 840 885 ayytngtngt ngarwsncar tggytncara 00 primate, human, OX2RI{1.2 (SEQ ID NO: 21): ngcnaayytn ggnytnytny tnathytnac nathttyytn atgytntgyc cntggmgnac gtngcngarg cngarggngc garaaycayg cnytngcnws taywsnaarg tnytngcflga gtnytfltgyt gyccnccflat ytnimgnggflc arccnwsfltg acnaaytgya cngaygarmg carathmgnc cngtngcnat gayggnaayt tycaymgngg ttycaraaym gnaaymgflac aihwsntgga thccngargg gtnacflgtfla arwsnacfltg gtnwsncayy tnacnggnaa aaraarwsflg cnaarytlt~a gtnggnttya thtggytnyt garwsnacflc cngtngtflga aayaayccfly tntaygayac gtngayacng ayytncayacaayaaywsfly ngcncarccfl nwsnwsnytn rgtlaayacfl hg cnytnrngn yacnaargcfl nathacntgg hacncaygay ntaycayytfl ngcngtfltgy ngaytgygcfl ycaytgggar yaarwsnytfl yathccntay naargtnaay rgargaygar nacnaayaar nytn tgyatggayg wsniggcclg aayytnatha tayrngnaarg gtnwsflfgrlc ggntaytaym cargtlytflg aargclgtflg acflaarcarg gincayaayg tayathgary athathytna g gftgymgfla atgcarccnt araarcarat tnaaratggc thatha cntg aracna ayga cngaycaraa gntgyathat tnacnocnga cnggnaarcc artaytggjws inwsnacngt tnytrnccngt cnathathat artayaaryt aygcnwsflta hacncaraay nacnaaygcfl ggarathath racnaargar ywsngayytl g gtn acn ccn rgtflacfytfl ngcngcrlcar naaygglacf nacntgycay nccnggngcn hytnacnath naayaaracfl yacngaraar.
ncarwsngar 180 240 300 360 420 480 540 600 660 '720 780 840 900 960 1020 1044 gtnaargCflw sncargcnyt rodent, mouse, 0X2PJ{1 (SEQ ID NO: atgttytgyt tytggmgnac gcnggnwsnw sntgyacnga acncargtna ayacnacrigt wsnathccny tnacnaargc wsntgyacna thgcntayaa aayathacnt gggcnwsnac ytncarcayg arggnacflta aaytaygayy tncargtnyt wsngcngtlt gygargcflat ggngaytgyg tnacnacnws tgycaytggg arcaraayaa aaycarwsny tnwsnathga ccntayatha thccnwsnat athwsnggnt tymgnaartg gaygaratgc arccntaygc acnaargtng argcnttycc ytnwsngcna thggnath nwsngclytl yaa ra a ycar nwsngtncar ngtnytnath rgtngaya cn nccngaycay yacntgygar ngtnccflccf ggcnggflaar ngarwsncay ygtnwsflgay rytnwsramgl hathathytn yaarytnccn nwsntayacn ngtnwsncar gcngtlytfly acnacflcara athggnacna .acntggatha aaracflaayg wsnccngary acngtlacflc ga rgtflacft ccngcngcnc wsnaayggna gtnwsntgya ggnggflaayc athathathg aarytflgarg garaarwsfla tnathtgggg ayaayw.sflws argcnytnyt thaar ytnmg aracnwsfltg tncar athws cngargglaa ayttyccflga a rathwsntg cngtnaclgt thgtrxwsnca a rwsnytnimg gnt gyathtg cnacnwsrigc ayccnytnta ngtnttygtl nwSflccfytl 120 ntgytgytty 180 nggnytlccf 240 yytngglmgl 300 ngcngtnacn 360 yttygaraar 420 raaraaymgn 480 gwsnccngay 540 rimgnwsnacl 600 yytnacnggl 660 nccntayath 720 yyinytnaar 780 nathgargar 840 ygayacngtfl 900 ytgyytnacn 960 978 ggngargtla ayggriacnga primate, human, OX2RH2 (SEQ ID NO: 16): arcaratgac ncaraaytay wsnacnatht tygcngargg naayathwsn atgggnggna carc-cngtny ytnathatha aaraargara wsrngnccng taytaymgng gtnytngtla gcngtnaclg acnaarcarg SS ggncayaarw gtnaarytna ytntaygtna ca rtgn ath a tnatggayat thacntggga cnaaygarac aycaraayws griathgtng t cnccnga rgt gnaarcrflgc artaytgggg snacngtlac aywsnggflyt arytnwsflyt aycaygtflmg haaygcngtn rathathytn naargaracfl ngayytncar nacnccflgay naayytfltty ngcncarath naaygglacf ntgycaygtfl nmgna cnwsn nttygtlgtfl naargtnytfl ytntgytgyc mgn ggn car C aaytgyaclg athxngnccng ggnaayttyc carwsflmgfla st gga thc gtna cngtna wsncayytna ggnwsnccng athytngtna cnccnathgc cnwsntgyac inga rmgnat tngayacnac a yxgnggnta ayathacngc cngarggnws arwsnacfltg cnggnaayaa criyinws nyt cnacnggntt naargcfltay hacntgggtl ncaygayggl ycayytnca r ngtntgyaa r nathytngcn yccntggga r rwsnytlwsf nytnathath ygtnttytty 120 180 240 300 360 420 480 540 600 660 720 750 rodent, mouse, OX2RH2 (SEQ ID NO: 17): mgnggncarc tgyytnggnm wsngcngtng aayttycaya ggngaraaym tggacnccng gtnmgnwsna caywsna cng ccnwsnytny gtnggnttyg cnwsntgyat gnaayathac cnytncarca argtntayga gnacngcflgt ayggngaytg cntgycaytg gnaayca rws tna cnathyt cnttyttyca hatggcntay aargtngara cnaargarac naaygaracn ntgggcnwsl ygarggnaay yytncargtn ntgygargcl ygtnacnaar ggarcaraay nytnwsnath ntaygtnaar raarmgnaay acnccngayc tayytntgyg ytngtlccflc atggclggfla wsngarwsnc aaygtlwsfg garytflwsnm atggtlytfly gtna crungna ayathccnga arathacnac cngazgtnac arccngcngc aywsnaaygg cngtnwsntg gnggnacnac tnggnathat cn yytncarath nccngarggn ntayttyytn ncarathwsn nacngtna cn yathgtnwsn nwsnacnacn hytnytnaar 120 180 240 300 360 420 480 540 582 rodent, mouse OX2R1i4 (SEQ ID NO: 24): atgcaygany wsnggnwsnw acncargtna wsnwsnccny wsntgyacna aayathacnt ytncarcayg gtntaygayy acngcngtnt ggngaytgyg tgycaytggg aaycarwsny acnathytnt ttyttycara tnggnmgnat.
sntgyacnga ayacnacnat tnathaaygc thgcntayaa gggcnwsnac arggnacrita tncargtnyt gygarganat tnacnaarws arcaraayaa tnws nath ga aygtnaarat armgnaaytt hccnacnytn ygaraaycar gwsngtncar ngtnytnath yytngayaar nccngaycay yacntgygar ngtnnccnf ggcnggnaar nga rws ncay ygtnwsngtl rytnwsncar ggcnytnytl yg cnxgna cn a cnytnytna acnathcara atggayaara a cntggatha aaracnaayg wsnccngary athgtnacnc gargtnacnt ccngcngcnc wsnaayggna gtnwsntgyy ggnacnatga gtnathathy thttyathaa aygaywsnws argcnytnyt thaa rca ymg aracrlwsntg tncarathws cngarggnaa ayttyccngg arathwsntg cngtnacngt tngtnwsnca cnacnccrng tnytnaaygt yathttygtn nwsnwsnytn ntgytgytty ncayytnccn yytnggnmgn ngcngtngcn yytngaraar naaraaymgn gacnccngay nmgnwsnacn ywsnacnggn nws nytnytn nggnttygcn 120 180 240 300 360 420 480 540 600 660 720 780 810 rodent, mouse, OX2RH3 (SEQ ID NO: 18): atgcaygcny ccngarwsnw ttywsngayg acnccngtnw aargargcna ccntaymgng mgnigtn ccn g gayggncayt athcargtnc atgcarathy acnatgathg ttyathwsnc wsnathytnc gtnggnttyg tnggnxngna c sntgywsngt ayaayathtt sngtncarat cnytrngnat cngarytnca cncaycayca ayws ntgymg cnggngaraa tntggacncc tnimgnwsnaa ayytna cnga cnws nytn yt cnttyttyca n ytngcnytfl naarggnlmnf yccngayggn hggnathaar htgggarath rcarathwsn rwsnwsngay nathgaracn ymgnacngtl ngaygargay rt gyca ymgn yaaytggath nwsnathytn raarmgnaay atgytnytna gargarathc gtnggngtria g cncar ytn t acnccimgng aaraaratht ytnccnatha a cngayggna gtntgygarg tgygtnacna garaaraaya ytnwsnatgg taygtnaary Sa yttymgn g thttyatha c cnccngayga cnatggarat tytgycaycc aytggccnws gyacngarmg arwsnatggc thttycarga cna thgcnws arwsnaarws ayggncayws arcaraaymg tng cngtnac tnacngargg nathytngtn ywsnttyccn hgarathath nwsnccnwsn ntgymgnytn nggnacnacn nytnaarcay rmgn ca yws n naarccngcn ncayaaygay ngtnttytgy nggnacnacn ngtnytnath nwsn 120 180 240 300 360 420 480 540 600 660 720 780 834 Table 5: A-lignment of various species OX2R homologs 1 and 2: OX2RHl_MU OX2RHI_RT OX2R2MU OX2RHIHU OX2RH2_MU OX2RJUMU OX2RHIRT OX2RH2_MU OX2RI~iHU OX2RH2_MU ox2RH1_MU oX2RHlRT OX2RH2_MU OX2RHiHU OX2P.H2_MU OX2RH1_MU OX2RHlRT OX21RH2_MU OX2RH1_MiU OX2M~2_HU OX2RH1_MU OX2RH1_RT OX2RH2_MU OX2RHiHU) OX2RH2_Mt 0X2RPHI Mx OX2RHlR' OX2RH2_M OX2RHiHi OX2RH2_H
CTDKNQTTQN
CPDKNQTMQN
MLC PWRTANLGLLLI LTI FLVAEAEGAAQPNN SLMLQTS KENHALA.SS
SLCMDEKQITQN
MGGKQMTQN
NSS SPLTQVNTTVSVQI GTKALLCCFSI
PLTKALITWIIKLRGLPSCTIAYVT-TN
NSST-MTENTTFQMGKKALLCCPSI SLTKVILITWTITLRGQPSCII
SYI(ADTRETH
RGQPSCIMAYKVETKETN
YSVLENSWVNTA
CPI.RLIITWEIILRGQPSCTKAYKKETNETK
YSTI-FAEGNISQPMDINALCCPPIALRLI 1ITWEIILRGQPSCTKAYKKETNETK ETSCLGPNITWASTPDHSPELQISATQETTCT
ENENYLVVPV
ESNCSDRSITWASTPDLAPDLQI
SAVALQHEGRYSCDIAVPDGNFQNIYDLQVLVPPEVT'
ET-CLGRNITWASTPDHIPDLQI
SAVALQHEGNYLCEITTPEGNFVDLQVLVPPEVT
ETNCTDERITWVSRPDQNSDLQI RTVAITHDGYYRCIMTPDGNERGYHLQL~PE~r ETNCTVERITWVS RPDQNS DLQI RPVDTTHDGYYRGI VVT PDGNFHRGYHLQVLVTPEVN YFPEKNPRSAVCEAMAGKPAAQISWS
PDG-DCVTTSESHSNGTVTRSTCWEQNNVSDVS
HFPGENRTAVCEAIAGKPAAQI SWTPDG-DCVA@IES
MSNGTVTVRSTCMWEQSHVSWVF
YFLGENRTAVCEAMAGKAAQI SWTPDG-DCVTKS ES HSNGTVrVRSTCHWEQNNVSAVS LFQNRNRTAVCKAVAGKPAAHI SWI PE-DAKEWS
T~VSCWVNSV
LFQSRNITAVCKAV'rGKPAAQI SWI PEGS ILATKQEYWGNGTTVKSTCPWEG-KSV CIVSHLT-GNQSLSIELSRGGNQSLRPYI PYI I PS IILI 11GCI CLLKI SGFRKCK
CVVSHLTTGNQSLSIELGRGGDQLLGSYIQYIIPSIIILIIIGCICLLKISGCRKLP
CHVSHLTGNKSLYIEL-LPVPGAKI SKI IYSI YHPYYLDHRG-IHL CHVSHLT -GNKS LSVKLN SGLRT SG S PALS LLI I LYVKLSLF--VVI LVTTG- FVFFQR 1SGATPDI EEDEMQPYASYTEKSNPLYDTTTEHPAQGKVJNGTDCLTLSAMGI J PNVTRT ~J SQWLQKI UT INHVRKVI OX2R homolog polypeptide relationships human Hl human H2 mouse I rat Hi Ig domain 54 52 72 TM/cyt 0 84 mouse H3 Ig domain 33' 29 39 TM/cyt 7 46 0 mouse H12 Ig domain 60 51 82 TM/cyt "7 49 0 mouse Hi Ig domain 53 47 TM/cyt 7 0 human H12 Ig domain 79 TM! cyt 7 mouse H12 73 0 mouse H3 32 sequence unavailable; "10"1 no significant matching Comparison of primate and rodent H2 with rodent H4 polypeptides; note similarity between the rodent Hi2 anid H{4: pOX2RH2 rOX2PRi2 rOX2RH~4 pOX2RH2 rOX2RH2 rOX2RH4 pOX2RH2 rOX2 RH2 rOX2RH4 pOX2RH2 rOX2RH2 rOX2RH4 pOX2RH2 rOX2PJ{2 r=XRH4 MGGK QMTQN-YSTIFAEGNISQPVL 1 MHLGRIPTLTLLIFINIFVSGSSCTDENQTIQNDSSSSLTQNTTMVQ 25 MfINAVLCCPPIALRNLIIITWEIILRGQPSCTKAYKKTNETKETNCTV 74 1 RGQPSCIMAYKVETKETNET -CLG 23 51 MDKKALLCCFSSPLINAVLITWIIKHRHLPSCTIAYN-LDKKTNETSCLG 99 75 ERTVRDNDQRVTHGYRIVPGFRYLVV 124 24 RNITWASTPDHIPDLQISAVALQHEGNYLCEITTPEGNFHKVYDLQVLVP 73 100 RNITWASTPDHS PELQI SAVALQHEGTYTCEIVTPEG3NLEKVYDLQVLVP 149 125 PEVNLFQSRNI TAVCKAVTGKPAAQI SWIPEGS ILATKQEYWGWGTVTVK 174 74 PEVTYFLGENRTAVCEAI1AGKPAAQISWTPDGDCVTKSESHSNGTTVR 122 150 PEVTYFPGKNRTAVCEAMAGKPAAQI SWTPDG DCKSESHSNGTV1'VR 198 175 STCPWEG-HKSTVTCHVSHLTGNKSLSVKLNSGLRTSGSPALSLLIILYV 223 123 STCWEQNNVSAVSCIVSHSTGNQSLSIELSRGTTST-TP--SLLTILYV 169 199 STCHWEQNNVSVVSCLVS}{STGNQSLSIELSQGTMTT--PR--SLLTILYV 245 pOX2RH2 rOX2RH2 rOX2RH4 224 KLSLFVVILVTTGFVFFQRINHVRKVL 170 KMVLLGIILLKVGFAFFQKR1NVTRT 246 1a4ALLVIILLNVGFAFFQKRNFART 250 194 270 00 o The OX2RH1 and 2 embodiments show particular similarity to 0 one another, see, Table 5. Particular regions or Spositions of interest are, for the rat HI: boundaries adjacent S to (before, at, or after) cys2, leu33, cys35, ile46, trp48, arg53, pro56, cys58, tyr62, cys 7 4 thr80, trp81, leu91, ile93, hisl00, gly10 2 tyrl0 4 glyll 3 phell5, leu122, vall123, prol2 7 asnl3 6 ala13 9 vall40, cysl41, ala143, lysl4 7 prol48, ala149, ile152, trpl54, prol56, asnl6 9 thrl71, vall 7 4, serl76, cysl78, 8 glul81, serl86, vall88, cysl90, serl93, hisl94, thrl96, asnl98, 00 10 leu202, gly215, tyr21 7 leu23 7 lys238, and ile304. Many of the Sresidues are conserved across the H1 and H2 classes. Likewise 0 C with H2 and H4. See Table 5. Particular domains of interest in rat OX2RH1 are the C2 domain from about cys2 to prol2 7 the C2 domain from about glul28-gly215, the TM segment from about tyr217-leu2 37 and the intracellular domain from about lys238ile304. Corresponding segments in mouse HI are about ser24glul51-gly 2 3 1, tyr239-leu2 5 9 and lys260-ile 3 2 6 In the mouse H2, the segments correspond, in available sequence, from about argl-pro 7 4 glu75-glyl55, prol61-glyl8 2 and phel83thrl94. For human H2, the transmembrane segment is about ala214-va1 2 3 3 and thr234-leu250, and in mouse H3, about proll9gly2 3 7 with the intramembrane lys228, and phe238-gly2 5 2 Table also indicates alignment of the H2 snd H4 embodiments.
Additional positions of interest, as boundaries for fragments, will be those conserved across homolog groups with the rat OX2RH1 or various subsets of the family members.
Functionally, the rat and mouse HI have been shown to bind to the OX2. This has not yet been confirmed for the human HI, but can be easily tested. Ligand matching for the H2, H4, and H3 groups is described below.
The rodent H3 has been shown to associate with DAP12, as predicted. Recombinantly expressed epitope tagged DAP12 is not membrane associated in the absence of coexpression of a chaparone partner. see, Bakker, et al. (1999) Proc. Nat'l Acad. Sci.
USA 96:9792-9796. Mouse H3 can serve as the chaparone partner.
However, the signal pathway through DAP12 requires binding to the O H3 ligand, which has not yet been identified, but can be found C using appropriate screening strategies, biochemical or Sphysical methods. Sequence similarity of H2 and rodent H4 suggest a similar association with either the DAP12, or possibly the The mouse H2 and H4 and human H2 are likely also to possess C such properties, association with DAP12 (activating) or The signaling pathways have been determined with some of Sthe related receptors on NK cells. See, Lanier, et al.
OO 10 (1998) Immunity 8:693-701; Smith, et al. (1998) J. Immunol.
S161:7-10; Gosselin, et al. (1999) J. Leukoc. Biol. 66:165-171; Tomasello, et al.(1998) J. Biol. Chem. 273:34115-34119; and McVicar, et al. (1998) J. Biol. Chem. 273:32934-32942. Because of the similarity of the extracellular domains with the H1 members, OX2-like genes, particularly OX2, are likely ligands.
As used herein, the term OX2RH1, OX2RH2, or OX2RH4 shall be used to describe a protein comprising amino acid sequences shown, in Tables 1-2. In many cases, a substantial fragment thereof will be functionally or structurally equivalent, 'including, an extracellular or intracellular domain. The invention also includes protein variants of the respective OX2RH alleles whose sequences are provided, muteins or soluble extracellular constructs. Typically, such agonists or antagonists will exhibit less than about 10% sequence differences, and thus will often have between 1 and 11 residue substitutions, 2, 3, 5, 7, and others. It also encompasses allelic and other variants, natural polymorphic, of the proteins described. Typically, the receptor will bind to a corresponding biological ligand with high affinity, at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. The term shall also be used herein to refer to related naturally occurring forms, alleles, polymorphic variants, and metabolic variants of the mammalian proteins. Preferred forms of the receptor complexes will bind the appropriate ligand 00 0 with an affinity and selectivity appropriate for a ligand- CI receptor interaction.
Z This invention also encompasses combinations of proteins or peptides having substantial amino acid sequence identity with the amino acid sequences in Tables 1-3. It will include sequence variants with relatively few substitutions, preferably less (C than about A substantial polypeptide "fragment", or "segment", is a stretch of amino acid residues of at least about 8 amino acids, 00 10 generally at least 10 amino acids, more generally at least 12 Samino acids, often at least 14 amino acids, more often at least CI 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids. Sequences of segments of different proteins can be compared to one another over appropriate length stretches. In many situations, fragments may exhibit functional properties of the intact subunits, the extracellular domain of the transmembrane receptor may retain the ligand binding features, and may be used to prepare a soluble receptor-like complex.
Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches. In some comparisons, gaps may be introduced, as required. See, Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al. (1983) chapter one in Time Warps, String Edits, and Macromolecules: The Theory and Practice of Seauence Comparison, Addison-Wesley, Reading, MA; and software packages from IntelliGenetics, Mountain View, CA; and the University of Wisconsin Genetics Computer Group (GCG), Madison, WI; each of which is incorporated herein by reference. This changes when considering conservative substitutions as matches. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; 00 O and phenylalanine, tyrosine. Homologous amino acid sequences are C intended to include natural allelic and interspecies variations in the receptor homolog sequence. Typical homologous proteins or ID peptides will have from 50-100% homology (if gaps can be introduced), to 60-100% homology (if conservative substitutions are included) with an amino acid sequence segment of Tables 1-3.
1 Homology measures will be at least about 70%, generally at least S76%, more generally at least 81%, often at least 85%, more often at least 88%, typically at least 90%, more typically at least 00 10 92%, usually at least 94%, more usually at least 95%, preferably Sat least 96%, and more preferably at least 97%, and in particularly preferred embodiments, at least 98% or more. The degree of homology will vary with the length of the compared segments. Homologous proteins or peptides, such as the allelic variants, will share most biological activities with the embodiments described in Tables 1-3.
As used herein, the term "biological activity" is used to describe, without limitation, effects on inflammatory responses, innate immunity, and/or morphogenic development by receptor-like proteins. For example, these receptors are likely to mediate :their effects through phosphatase or phosphorylase activities, which activities are easily measured by standard procedures.
See, Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al.
(1991) Meth. Enzvmol. 200:38-62; Hunter, et al. (1992) Cell 70:375-388; Lewin (1990) Cell 61:743-752; Pines, et al. (1991) Cold SDrina Harbor Svm. Ouant. Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738. The receptor homologs, or portions thereof, may be useful as phosphate labeling enzymes to label general or specific substrates. The subunits may also be functional immunogens to elicit recognizing antibodies, or antigens capable of binding antibodies.
The terms ligand, agonist, antagonist, and analog of, e.g., an OX2RH, include molecules that modulate the characteristic cellular responses to binding of 0X2 proteins, as well as molecules possessing the more standard structural binding 00 compeition features of ligand-receptor interations, where the antagonist is a soluble extracellular domain of a receptor homolog or an antibody. The cellular responses likely are typically mediated through receptor tyrosine kinase pathways.
Also, a receptor homolog may be a molecule which serves either as a natural receptor to which said ligand, or an analog thereof, binds, or a molecule which is a functional analog of the Snatural receptor. The functional analog may be a receptor 0 homolog with structural modifications, or may be a wholly O 10 unrelated molecule which has a molecular shape which interacts C with the appropriate ligand binding determinants. The ligands may serve as agonists or antagonists, see, Goodman, et al.
(eds. 1990) Goodman iln's The Pharmacoloical Bass of Therapeutics, Pergamon Press, New York.
Rational drug design may also be based upon structural studies of the molecular shapes of a receptor homolog, antibody, or other effectors or receptor homolog associated entities. See, Herz, et al. (1997) J. Recent. Signal Transduct. Res.
17:671-776; and haiken, t al. (1996) rends Biotechnl14:369- 375. Effectors may be other proteins which mediate other functions in response to ligand binding, or other proteins which normally interact with the receptor homolog. One means for determining which sites interact with specific other proteins is a physical structure determination, x-ray crystallography or 2 dimensional NMR techniques. These will provide guidance as to which amino acid residues form molecular contact regions. For a detailed description of protein structural determination, see, Blundell and Johnson (1976) Protein Crvstalloarachy, Academic Press, New York, which is hereby incorporated herein by reference.
II. Activities The receptor-like proteins will have a number of different biological activities, modulating cell proliferation, or in phosphate metabolism, being added to or removed from specific substrates, typically proteins. Such will generally result in
I
00 8 modulation of an inflammatory function, other innate immunity response, or a morphological effect. The receptor homolog will probably have a specific low affinity binding to the ligand, as O described.
The OX2RH1 has motifs suggestive of a receptor signaling through a receptor tyrosine kinase pathway. See, Ihle, et al. (1997).Stm Cells 15(suppl. 1):105-111; Silvennoinen, et al.
(1997) APMTS 105:497-509; Levy (1997) Cvtokine Growth Factor SReview 8:81-90; Winston and Hunter (1996) Current Biol. 6:668- 00 10 671; Barrett (1996) Baillieres Clin. Gastroenterol. 10:1-15; and SBriscoe, et al. (1996) Philos. Trans. R. Soc. Lond. B. Biol. Sci.
351:167-171.
The biological activities of the OX2R homologs will likely be related to addition or removal of phosphate moieties to substrates, typically in a specific manner, but occasionally in a non specific manner. Substrates may be identified, or conditions for enzymatic activity may be assayed by standard methods, e.g., as described in Hardie, et al. (eds. 1995) The Protein Kinase FactBook vols. I and II, Academic Press, San Diego, CA; Hanks, et al. (1991) Meth. Enzvmol. 200:38-62; Hunter, et al. (1992) £Cll 70:375-388; Lewin (1990) ECl. 61:743-752; Pines, et al. (1991) Cold SDrina Harbor SvmD. Ouant. Biol. 56:449-463; and Parker, et al. (1993) Nature 363:736-738.
A receptor homolog may combine with one or more other proteins to form functional complexes, which may be useful for binding ligand or preparing antibodies. These will have substantial diagnostic uses, including detection or quantitation.
III. Nucleic Acids This invention contemplates use of isolated nucleic acid or fragments, which encode these or closely related proteins, or fragments thereof, to encode a corresponding polypeptide, preferably one which is biologically active. In addition, this invention covers isolated or recombinant DNAs which encode combinations of such proteins or polypeptides having characteristic sequences, of the homologs. Typically, the nucleic acid is capable of hybridizing, under appropriate conditions, with a nucleic acid sequence segment shown in Tables 1-3, but preferably not with a corresponding segment of other known Ig superfamily receptors. Said biologically active protein or polypeptide can be a full length protein, or fragment, and will typically have a segment of amino acid sequence highly -homologous, exhibiting significant stretches of identity, M to one shown in Tables 1-3. Further, this invention covers the use of isolated or recombinant nucleic acid, or fragments 00 10 thereof, which encode proteins having fragments which are equivalent to the OX2RH proteins. The isolated nucleic acids can have the respective regulatory sequences in the 5' and 3' flanks, promoters, enhancers, poly-A addition signals, and others from the natural gene.
An "isolated" nucleic acid is a nucleic acid, an RNA, DNA, or a mixed polymer, which is substantially pure, e.g., separated from other components which naturally accompany a native sequence, such as ribosomes, polymerases, and flanking genomic sequences from the originating species. The term embraces a nucleic acid sequence which has been removed from its naturally occurring environment, and includes recombinant or cloned DNA isolates, which are thereby distinguishable from naturally occurring compositions, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems. A substantially pure molecule includes isolated forms of the molecule, either completely or substantially pure.
An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain heterogeneity, preferably minor. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
A "recombinant" nucleic acid is typically defined either by its method of production or its structure. In reference to its method of production, a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence.
00 Typically this intervention involves in vitro manipulation, although under certain circumstances it may involve more classical animal breeding techniques. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of S two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, naturally CI occurring mutants as found in their natural state. Thus, for example, products made by transforming cells with an unnaturally Soccurring vector is encompassed, as are nucleic acids comprising 00 10 sequence derived using most any synthetic oligonucleotide Sprocess. Such a process is often done to replace a codon with a I redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a restriction enzyme sequence recognition site. Alternatively, the process is performed to join together nucleic acid segments of desired functions to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms, encoding a fusion protein.
Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design. A similar concept is intended for a recombinant, e.g., fusion, polypeptide. This will include a dimeric repeat.
Specifically included are synthetic nucleic acids which, by genetic code redundancy, encode equivalent polypeptides to fragments of OX2 receptor homologs and fusions of sequences from various different related molecules, other Ig superfamily members.
A "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least 21 nucleotides, more generally at least 25 nucleotides, ordinarily at least 30 nucleotides, more ordinarily at least 35 nucleotides, often at least 39 nucleotides, more often at least nucleotides, typically at least 50 nucleotides, more typically at least 55 nucleotides, usually at least 60 nucleotides, more S usually at least 66 nucleotides, preferably.at least 72 nucleotides, more preferably at least 79 nucleotides, and in particularlY preferred embodiments will be at least 85 or more Snucleotides. Typically, fragments of different genetic sequences can be compared to one another over appropriate length stretches, particularly defined segments such as the domains described below.
A nucleic acid which codes for OX2R homologs will be particularly useful to identify genes, mRNA, and cDNA species 00 10 which code for itself or closely related proteins, as well as DNAs which code for polymorphic, allelic, or other genetic variants, from different individuals or related species.
Preferred probes for such screens are those regions of the receptor homolog which are conserved between different polymorphic variants or which contain nucleotides which lack specificity, and will preferably be full length or nearly so. In other situations, polymorphic variant specific sequences will be more useful. Quantitation or specific sequence analysis may be useful as markers for disease or medical conditions, or in selecting particular therapeutic treatments.
This invention further covers recombinant nucleic acid molecules and fragments having a nucleic acid sequence identical to or highly homologous to the isolated DNA set forth herein. In particular, the sequences will often be operably linked to DNA segments which control transcription, translation, and/or DNA replication. These additional segments typically assist in expression of the desired nucleic acid segment.
Homologous, or highly identical, nucleic acid sequences, when compared to one another, OX2RH sequences, exhibit significant similarity. The standards for homology in nucleic acids are either measures for homology generally used in the art by sequence comparison or based upon hybridization conditions.
Comparative hybridization conditions are described in greater detail below.
Substantial identity in the nucleic acid sequence comparison context means either that the segments, or their complementary 00 o strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 60% of the nucleotides, generally at least 66%, ordinarily Sat least 71%, often at least 76%, more often at least usually at least 84%, more usually at least 88%, typically at least 91%, more typically at least about 93%, preferably at least about 95%, more preferably at least about 96 to 98% or more, and in particular embodiments, as high at about 99% or more of the nucleotides, including, segments encoding structural 00 10 domains such as the segments described below. Alternatively, 8substantial identity will exist when the segments will hybridize under selective hybridization conditions, to a strand or its complement, typically using a sequence derived from Tables 1-3.
Typically, selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 14 nucleotides, more typically at least about 65%, preferably at least about 75%, and more preferably at least about 90%. See, Kanehisa (1984) Nucl. Acids Res. 12:203-213, which is incorporated herein by reference. The length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, generally at least about 20 nucleotides, ordinarily at least about 24 nucleotides, usually at least about 28 nucleotides, typically at least about 32 nucleotides, more typically at least about 40 nucleotides, preferably at least about 50 nucleotides, and more preferably at least about 75 to 100 or more nucleotides. This includes, 125, 150, 175, 200, 225, 246, 273, 300, 325, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, and other lengths.
Stringent conditions, in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters typically controlled in hybridization reactions. Stringent temperature conditions will usually include temperatures in excess of about 30" C, more usually in excess of about 37- C, typically in excess of about 45' C, more typically in excess of 00 about 50" C, 55' C or 60" C, preferably in excess of about c 65' C, and more preferably in excess of about 70" C. Stringent Ssalt conditions will ordinarily be less than about 1 M, more IO ordinarily less than about 500 mM, usually less than about 400 mM, more usually less than about 300 mM, typically less than about 200 mM, preferably less than about 100 mM, and more preferably less than about 80 mM, less than 50 mM, even down to less than about 20 mM. However, the combination of Sparameters is much more important than the measure of any single 00 10 parameter. See, Wetmur and Davidson (1968) J. Mol. Biol.
S31:349-370, which is hereby incorporated herein by reference.
C The isolated DNA can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications generally result in novel DNA sequences which encode this protein or its derivatives. These modified sequences can be used to produce mutant proteins (muteins) or to enhance the expression of variant species. Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms. Such mutant OX2R homolog derivatives include predetermined or site-specific mutations of the protein or its fragments, including silent mutations using genetic code degeneracy. "Mutant OX2 receptor homolog" as used herein encompasses a polypeptide otherwise falling within the definition of the OX2 receptor homologs as set forth above, but having an amino acid sequence which differs from that of other Ig superfamily as found in nature, whether by way of deletion, substitution, or insertion. In particular, "site specific mutant OX2 receptor homolog" encompasses a protein having substantial sequence identity with a protein of Tables 1-3, and typically shares some or most of the biological activities or effects, immunogenicity, of the forms disclosed herein.
Although site specific mutation sites are predetermined, mutants need not be site specific. Mammalian OX2 receptor homolog mutagenesis can be achieved by making amino acid insertions or deletions in the gene, coupled with expression.
00 0 Substitutions, deletions, insertions, or many combinations may be (c generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be N conducted at a target codon and the expressed mammalian OX2RH mutants can then be screened for the desired activity, providing some aspect of a structure-activity relationship. Methods for making substitution mutations at predetermined sites in DNA having a known sequence are well known in the art, by M13 primer mutagenesis. See also Sambrook, et al. (1989) and 00 10 Ausubel, et al. (1987 and periodic Supplements).
SThe mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
The phosphoramidite method described by Beaucage and Carruthers (1981) Tetra. Letts. 22:1859-1862, will produce suitable synthetic DNA fragments. A double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Polymerase chain reaction (PCR) techniques can often be applied in mutagenesis. Alternatively, mutagenesis primers are commonly used methods for generating defined mutations at predetermined sites. See, Innis, et al. (eds. 1990) PCR Protocols: A Guide to Methods and ADDlications Academic Press, San Diego, CA; and Dieffenbach and Dveksler (1995; eds.) PCR Primer: A Laboratory Manual Cold Spring Harbor Press, CSH, NY.
Certain embodiments of the invention are directed to combination compositions comprising the receptor homolog or ligand sequences described. In other embodiments, functional portions of the sequences may be joined to encode fusion proteins. In other forms, variants of the described sequences may be substituted.
00 0 IV. Proteins, Peptides As described above, the present invention encompasses mammalian OX2RH polypeptides, whose sequences are disclosed in Tables 1-3, and described above. Allelic and other variant S 5 polypeptides are also contemplated, including, fusion proteins combining portions of such sequences with others, ;C including, epitope tags, functional domains, and DAP12 or sequences.
r The present invention also provides recombinant proteins, S 10 heterologous fusion proteins using segments from these 0 primate or rodent proteins. A heterologous fusion protein is a CI fusion of proteins or segments which are naturally not normally fused in the same manner. Thus, the fusion product of two OX2RHs is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties, sequence or antigenicity, derived from each source peptide. A similar concept applies to heterologous nucleic acid sequences.
Combinations of various designated proteins into complexes are also provided.
In addition, new constructs may be made from combining similar functional or structural domains from other related proteins, other ITIM, ITAM, or YxxM motif containing receptors, including species variants. For example, ligandbinding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, Cunningham, et al.
(1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol.
Chem. 263:15985-15992, each of which is incorporated herein by reference. Thus, new chimeric polypeptides exhibiting new combinations of specificities will result from the functional linkage of receptor-binding specificities. For example, the ligand binding domains from other related receptor homolog molecules may be added or substituted for other domains of this or related proteins. The resulting protein will often have hybrid function and properties. For example, a fusion protein may include a targeting domain which may serve to provide 00 O sequestering of the fusion protein to a particular subcellular CI organelle.
Candidate fusion partners and sequences can be selected from various sequence data bases, GenBank, c/o IntelliGenetics, Mountain View, CA; and BCG, University of Wisconsin Biotechnology Computing Group, Madison, WI, which are each incorporated herein C by reference. In particular, combinations of polypeptide sequences provided in Tables 1-3 are particularly preferred.
SVariant forms of the proteins may be substituted in the described OO 10 combinations.
O The present invention particularly provides muteins which CI bind OX2-like ligands, and/or which are affected in signal transduction. Structural alignment of various members of the 0X2 receptor homolog family show conserved features/residues. See, Table 5. Alignment of the OX2R homolog sequences indicates various structural and functionally shared features. See also, Bazan, et al. (1996) Nature 379:591; Lodi, et al. (1994) Science 263:1762-1766; Sayle and Milner-White (1995) TIBS 20:374-376; and Gronenberg, et al. (1991) Protein Enaineering 4:263-269.
Substitutions with either mouse sequences or human sequences are particularly preferred. Conversely, conservative substitutions away from the ligand binding interaction regions will probably preserve most signaling activities; and conservative substitutions away from the intracellular domains will probably preserve most ligand binding properties.
"Derivatives" of a mammalian OX2RH include amino acid sequence mutants, glycosylation variants, metabolic derivatives and covalent or aggregative conjugates with other chemical moieties. Covalent derivatives can be prepared by linkage of functionalities to groups which are found in the OX2RH amino acid side chains or at the N- or C- termini, by means which are well known in the art. These derivatives can include, without limitation, aliphatic esters or amides of the carboxyl terminus, or of residues containing carboxyl side chains, O-acyl derivatives of hydroxyl group-containing residues, and N-acyl derivatives of the amino terminal amino acid or amino-group 00 S containing residues, lysine or arginine. Acyl groups are C selected from the group of alkyl-moieties, including C3 to C18 normal alkyl, thereby forming alkanoyl aroyl species.
In particular, glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. Particularly preferred means for accomplishing this are Sby exposing the polypeptide to glycosylating enzymes derived from 8 cells which normally provide such processing, mammalian 0 10 glycosylation enzymes. Deglycosylation enzymes are also 0 contemplated. Also embraced are versions of the same primary q amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine.
A major group of derivatives are covalent conjugates of the receptor homologs or fragments thereof with other proteins of polypeptides. These derivatives can be synthesized in recombinant culture such as N- or C-terminal fusions or by the use of agents known in the art for their usefulness in cross-linking proteins through reactive side groups. Preferred derivatization sites with cross-linking agents are at free amino groups, carbohydrate moieties, and cysteine residues.
Fusion polypeptides between the receptor homologs and other homologous or heterologous proteins are also provided.
Homologous polypeptides may be fusions between different receptors, resulting in, for instance, a hybrid protein exhibiting binding specificity for multiple different OX2 related ligands, or a receptor which may have broadened or weakened specificity of substrate effect. Likewise, heterologous fusions may be constructed which would exhibit a combination of properties or activities of the derivative proteins. Typical examples are fusions of a reporter polypeptide, luciferase, with a segment or domain of a receptor, a ligand-binding segment, so that the presence or location of a desired ligand may be easily determined. See, Dull, et al., U.S. Patent No.
4,859,609, which is hereby incorporated herein by reference.
00 0 Other gene fusion partners include glutathione-S-transferase C, (GST), bacterial E-galactosidase, trpE, Protein A, B-lactamase, alpha amylase, alcohol dehydrogenase, and yeast alpha mating t factor. See, Godowski, et al. (1988) Science 241:812-816.
NO
5 Labeled proteins will often be substituted in the described combinations of proteins.
C- The phosphoramidite method described by Beaucage and SCarruthers (1981) Tetra. Letts, 22:1859-1862, will produce Ssuitable synthetic DNA fragments. A double stranded fragment C 10 will often be obtained either by synthesizing the complementary Sstrand and annealing the strand together under appropriate C-q conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
Such polypeptides may also have amino acid residues which have been chemically modified by phosphorylation, sulfonation, biotinylation, or the addition or removal of other moieties, particularly those which have molecular shapes similar to phosphate groups. In some embodiments, the modifications will be useful labeling reagents, or serve as purification targets, e.g., affinity ligands.
Fusion proteins will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods.
Techniques for nucleic acid manipulation and expression are described generally, for example, in Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual (2d Vols. 1-3, Cold Spring Harbor Laboratory, and Ausubel, et al. (eds. 1987 and periodic supplements) Current Protocols in Molecular Biology, Greene/Wiley, New York, which are each incorporated herein by reference. Techniques for synthesis of polypeptides are described, for example, in Merrifield (1963) J. Amer. Chem. Soc.
85:2149-2156; Merrifield (1986) Science 232:341-347; and Atherton, et al. (1989) Solid Phase Peotide Synthesis: A Practical Approach, IRL Press, Oxford; each of which is incorporated herein by reference. See also Dawson, et al. (1994) Science 266:776-779 for methods to make larger polypeptides.
This invention also contemplates the use of derivatives of 00 O an OX2RH other than variations in amino acid sequence or C glycosylation. Such derivatives may involve covalent or Saggregative association with chemical moieties. These Io derivatives generally fall into three classes: salts, (2) side chain and terminal residue covalent modifications, and (3) adsorption complexes, with cell membranes. Such covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity Spurification of a receptor or other binding molecule, an 00 10 antibody. For example, an OX2 ligand can be immobilized by covalent bonding to a solid support such as cyanogen C bromide-activated Sepharose, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of an OX2RH, antibodies, or other similar molecules.
The ligand can also be labeled with a detectable group, for example radioiodinated by the chloramine T procedure, covalently bound to rare earth chelates, or conjugated to another fluorescent moiety for use in diagnostic assays.
A combination, including an OX2RH, of this invention can be used as an immunogen for the production of antisera or antibodies specific, capable of distinguishing between other 0X2 receptor homologs, or for desired combination specificity. The OX2RHs can be used to screen monoclonal antibodies or antigen-binding fragments prepared by immunization with various forms of impure preparations containing the protein.
In particular, the term "antibodies" also encompasses antigen binding fragments of natural antibodies, Fab, Fab2, Fv, etc. The purified OX2RH can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of expression, or immunological disorders which lead to antibody production to the endogenous receptor homolog.
Additionally, OX2RH fragments may also serve as immunogens to produce the antibodies of the present invention, as described immediately below. For example, this invention contemplates antibodies having binding affinity to or being raised against the 00 amino acid sequences shown in Tables 1-3, fragments thereof, or C' various homologous peptides or subsets. In particular, this invention contemplates antibodies having binding affinity to, or having been raised against, specific fragments which are
O
predicted to be, or actually are, exposed at the exterior protein surface of the native OX2RH. Complexes of combinations of CI proteins will also be useful, and antibody preparations thereto can be made.
The blocking of physiological response to the receptor 1' 0 ligands may result from the inhibition of binding of the ligand 00 to the receptor, likely through competitive inhibition, or CI perhaps down-regulation of receptor expression due to antibody binding. Thus, in vitro assays of the present invention will often use antibodies or antigen binding segments of these antibodies, or fragments attached to solid phase substrates.
These assays will also allow for the diagnostic determination of the effects of either ligand binding region mutations and modifications, or other mutations and modifications, which affect signaling or enzymatic function.
This invention also contemplates the use of competitive drug screening assays, where neutralizing antibodies to the receptor complexes or fragments compete with a test compound for binding to a ligand or other antibody. In this manner, the neutralizing antibodies or fragments can be used to detect the presence of a polypeptide which shares one or more binding sites to a receptor and can also be used to occupy binding sites on a receptor that might otherwise bind a ligand.
V. Making Nucleic Acids and Protein DNA which encodes the protein or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or by screening genomic libraries prepared from a wide variety of cell lines or tissue samples. Natural sequences can be isolated using standard methods and the sequences provided herein, in Tables 1-3. Reverse translation sequences are provided in Table 4. Other species counterparts can be identified by hybridization 00 techniques, or by various PCR techniques, combined with or by searching in sequence databases, GenBank. Antibodies may Sbe used in expression cloning efforts on species counterparts.
NO This DNA can be expressed in a wide variety of host cells 'for the synthesis of a full-length receptor or fragments which can, be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified ligand binding or kinase/phosphatase domains; and for structure/function studies. Variants or fragments can be 00 10 expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent. The protein, or portions thereof, may be expressed as fusions with other proteins. Combinations of the des-cribed proteins, or nucleic acids encoding them, are particularly interesting.
Expression vectors are typically self-replicating DNA or RNA constructs containing the desired receptor homolog gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The multiple genes may be coordinately expressed, and may be on a polycistronic message. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used. Generally, the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation. Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
00 0 The vectors of this invention include those which contain CI DNA which encodes a combination of proteins, as described, or a biologically active equivalent polypeptide. The DNA can be under the control of a viral promoter and can encode a selection
NO
marker. This invention further contemplates use of such expression vectors which are capable of expressing eukaryotic Cl cDNAs coding for such proteins in a prokaryotic or eukaryotic host, where the vector is compatible with the host and where the 3eukaryotic cDNAs are inserted into the vector such that growth of 00 10 the host containing the vector expresses the cDNAs in question.
SUsually, expression vectors are designed for stable replication OC in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, it is possible to effect transient expression of the protein or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of the protein encoding portions into the host DNA by recombination.
Vectors, as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. Expression vectors are specialized vectors which contain genetic control elements that effect expression of operably linked genes. Plasmids are the most commonly used form of vector but all other forms of vectors which serve an equivalent function and which are, or become, known in the art are suitable for use herein. See, Pouwels, et al. (1985 and Supplements) Cloning Vectors: A Laboratory Manual, Elsevier, and Rodriguez, et al. (eds. 1988) Vectors: A Survey of Molecular Clonino Vectors and Their Uses, Buttersworth, Boston, which are incorporated herein by reference.
Transformed cells are cells, preferably mammalian, that have been transformed or transfected with vectors constructed using recombinant DNA techniques. Transformed host cells usually 8 express the desired proteins, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the Ssubject proteins. This invention further contemplates culturing Stransformed cells in a nutrient medium, thus permitting the proteins to accumulate. The proteins can be recovered, either from the culture or, in certain instances, from the culture medium.
For purposes of this invention, nucleic acid sequences are 0 operably linked when they are functionally related to each other.
00 10 For example, DNA for a presequence or secretory leader is C operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide. A promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide; a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation. Usually, operably linked means contiguous and in reading frame, however, certain genetic elements such as repressor genes are not contiguously linked but still bind to operator sequences that in turn control expression.
Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include both gram negative and gram positive organisms, E. coli and B. subtilis.
Lower eukaryotes include yeasts, S. cerevisiae and Pichia, and species of the genus Dictyostelium. Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, insect cells, and birds, and of mammalian origin, human, primates, and rodents.
Prokaryotic host-vector systems include a wide variety of vectors for many different species. E. coli and its vectors will be described, but equivalent vectors and hosts can generally be substituted. A representative vector for amplifying DNA is pBR322 or many of its derivatives. Vectors that can be used to express the receptor homolog or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipp promoter (the 00 O pIN-series); lambda-pP or pR promoters (pOTS); or hybrid C' promoters such as ptac (pDR540). See Brosius, et al. (1988) S"Expression Vectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters", in Vectors: A Survey of Molecular Cloning Vectors and Their Uses, (eds. Rodriguez and Denhardt), Buttersworth, Boston, Chapter 10, pp. 205-236, which is C incorporated herein by reference.
Lower eukaryotes, yeasts and Dictyostelium, may be transformed with OX2RH sequence containing vectors. The most 00 10 popular lower eukaryotic host is the baker's yeast, Saccharomyces Scerevisiae. It will be used to exemplify lower eukaryotes, CI though many other strains and species are also available. Yeast vectors typically consist of a replication origin (unless of the integrating type), a selection gene, a promoter, DNA encoding the receptor homolog or its fragments, and sequences for translation termination, polyadenylation, and transcription termination.
Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the; alcohol dehydrogenase 2 promoter or metallothionine promoter.
Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series), self-replicating high copy number (such as the YEp-series); integrating types (such as the YIp-series), or mini-chromosomes (such as the YCp-series).
Higher eukaryotic tissue culture cells are normally the preferred host cells for expression of functionally active OX2RH proteins. In principle, many higher eukaryotic tissue culture cell lines are workable, insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
However, mammalian cells are preferred. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines. Expression vectors for such cell lines usually 00 0 include an origin of replication, a promoter, a translation CI initiation site, RNA splice sites (if genomic DNA is used), a Spolyadenylation site, and a transcription termination site.
These vectors also usually contain a selection gene or
IO
amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, from CI such sources as from adenovirus, SV40, parvoviruses, vaccinia virus, or cytomegalovirus. Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama, et al.
S 10 (1985) Mol. Cell Biol. 5:1136-1142; pMClneo PolyA, see Thomas, et 00 Sal. (1987) Cell 51:503-512; and a baculovirus vector such as pAC Cl 373 or pAC 610.
For secreted proteins and some membrane proteins, an open reading frame usually encodes a polypeptide that consists of a mature or secreted product covalently linked at its N-terminus to a signal peptide. The signal peptide is cleaved prior to secretion of the mature, or active, polypeptide. The cleavage site can be predicted with a high degree of accuracy from empirical rules, von-Heijne (1986) Nucleic Acids Research 14:4683-4690, and Nielsen, et al. (1997) Protein Ena. 10:1-12.
And the precise amino acid composition of the signal peptide often does not appear to be critical to its function, e.g., Randall, et al. (1989) Science 243:1156-1159; Kaiser, et al.
(1987) Science 235:312-317. The mature proteins of the invention can be readily determined using standard methods.
It will often be desired to express these polypeptides in a system which provides a specific or defined glycosylation pattern. In this case, the usual pattern will be that provided naturally by the expression system. However, the pattern will be modifiable by exposing the polypeptide, an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system. For example, the OX2RH gene may be co-transformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable in prokaryote or other cells. Expression in prokaryote cells will typically 00 lead to unglycosylated forms of protein.
C The source of OX2RH can be a eukaryotic or prokaryotic host Sexpressing recombinant polypeptide, such as is described above.
o The source can also be a cell line, but other mammalian cell lines are also contemplated by this invention, with the preferred cell line being from the human species.
Now that the sequences are known, the primate OX2RH, Sfragments, or derivatives thereof can be prepared by conventional Sprocesses for synthesizing peptides. These include processes 00 10 such as are described in Stewart and Young (1984) Solid Phase Peotide Synthesis, Pierce Chemical Co., Rockford, IL; Bodanszky C and Bodanszky (1984) The Practice of Peptide Synthesis, Springer-Verlag, New York; and Bodanszky (1984) The Principles of Peptide Synthesis, Springer-Verlag, New York; all of each which are incorporated herein by reference. For example, an azide process, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD)/additive process can be used. Solid phase and solution phase syntheses are both applicable to the foregoing processes. Similar techniques can be used with partial OX2RH sequences.
The OX2RH proteins, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid. Amino groups that are not being used in the coupling reaction typically must be protected to prevent coupling at an incorrect location.
If a solid phase synthesis is adopted, the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group. The insoluble carrier should have a binding capability to a reactive carboxyl group, halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl 00 resins, phenol resins, tert-alkyloxycarbonylhydrazidated resins, and the like.
An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide. This solid-phase approach is C generally described by Merrifield, et al. (1963) in J. Am. Chem.
Soc 85:2149-2156, which is incorporated herein by reference.
SThe prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, by extraction, precipitation, electrophoresis, various forms of chromatography, and the like. The receptor homologs of this invention can be obtained in varying degrees of purity depending upon desired uses. Purification can be accomplished using standard protein purification techniques or the antibodies herein described in immunoabsorbant affinity chromatography methods. Typically, affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate cells, lysates of other cells expressing the OX2RH, or lysates or supernatants of cells producing the protein as a result of DNA techniques, see below.
Generally, the purified protein will be at least about pure, ordinarily at least about 50% pure, usually at least about pure, typically at least about 70% pure, more typically at least about 80% pure, preferable at least about 90% pure and more preferably at least about 95% pure, and in particular embodiments, 97%-99% or more. Purity will usually be on a weight basis, but can also be on a molar basis. Different assays will be applied as appropriate. Individual proteins may be purified and thereafter combined.
VI. Antibodies Antibodies can be raised to the various mammalian, e.g., 00 primate, OX2RH proteins and fragments thereof, both in naturally occurring native forms and in their denatured forms. Antibodies raised to native OX2RH are more likely to recognize epitopes which are only present in the native conformations. Denatured antigen detection can also be useful in, Western analysis.
Anti-idiotypic antibodies are also contemplated, which would be c-i useful, diagnostic reagents.
Antibodies, including binding fragments and single chain M versions, against predetermined fragments of the protein can be N-~2 10 raised by immnization of animals with conjugates of the 00 fragments with immunogenic proteins. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective protein, or screened for agonistic or antagonistic activity.
These monoclonal antibodies will usually bind with at least a KD of about 1 mM, more usually at least about 300 pM', typically at least about lO0iiM, more typically at least about 30 11M, preferably at least about 10 pM, and more preferably at least about 3 -pM or better.
The antibodies, including antigen binding fragments, of this invention can have significant diagnostic or therapeutic value.
They can be potent antagonists that bind to a receptor homolog and inhibit binding to ligand or inhibit the ability of the receptor homolog to elicit a biological response, act on its substrate. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides to bind OX2RH producing cells. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly, by means of a linker.
The antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they might bind to the OX2RH without inhibiting ligand or substrate binding. As neutralizing antibodies, they can be useful in competitive binding assays. They will also be useful in detecting or quantifying ligand. They may be used as reagents for Western blot analysis, or for iinmunoprecipitation or 8 immunopurification of the respective protein. Likewise, nucleic acids and proteins may be immobilized to solid substrates for Saffinity purification or detection methods. The substrates may MO be, solid resin beads, sheets of plastic, or derivatized glass.
Protein fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined M polypeptides, to be used as immunogens. Mammalian OX2RH 0 polypeptides and fragments may be fused or covalently linked to a 0 10 variety of immunogens, such as keyhole limpet hemocyanin, bovine Sserum albumin, tetanus toxoid, etc. See Microbioloav, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) STecificitv of Seroloaical Reactions, Dover Publications, New York; and Williams, et al. (1967) Methods in Immunoloav and Immunochemistry, Vol. 1, Academic Press, New York; each of which are incorporated herein by reference. A typical method involves hyperimmunization of an animal with an antigen. The blood of the animal is then collected shortly after the repeated immunizations and serum or gamma globulin is isolated.
In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. See, Stites, et al. (eds.) Basa and Clinical Immunoloav (4th Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH Press; Goding (1986) Monoclonal Antibodies: Princioles and Practice (2d ed.) Academic Press, New York; and particularly Kohler and Milstein (1975) Nature 256:495-497, each of these references is incorporated herein by reference. Briefly, an immunogen is injected into an animal to induce an immune response. The animal is then sacrificed and cells taken from its spleen, which are fused with myeloma cells to produce a hybridoma. The population of hybridomas is then screened to isolate an individual clone which secretes an antibody which binds to the immunogen.
Other suitable techniques involve in vitro exposure of lymphocytes to the antigenic polypeptides or alternatively to 00 0 selection of libraries of antibodies in phage or similar vectors.
See, Huse, et al. (1989) "Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda," Science 246:1275-1281; and Ward, et al. (1989) Nature 341:544-
O
S 5 546, each of which is hereby incorporated herein by reference.
Chimeric or humanized antibodies may be produced, see Cabilly, C- U.S. Patent No. 4,816,567; or made in transgenic mice, see Mendez, et al. (1997) Nature Genetics 15:146-156. These Sreferences are incorporated herein by reference.
C 10 Polypeptides and antibodies will often be labeled. A wide 00 Svariety of labels and conjugation techniques are known and are C- reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like.
Patents teaching the use of such labels include, U.S.
Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.
The antibodies of this invention can also be used for affinity chromatography in isolating the OX2RH proteins or peptides. Columns can be prepared where the antibodies are linked to a solid support, particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified protein will be released. Alternatively, the protein may be used to purify antibody. Appropriate cross absorptions or depletions may be applied.
The antibodies may also be used to screen expression libraries for particular expression products. Usually the antibodies used in such a procedure will be labeled with a moiety allowing easy detection of presence of antigen by antibody binding.
Antibodies raised against an OX2RH will also be used to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related 00 0 to expression of the protein or cells which express the protein.
They also will be useful as agonists or antagonists of the Sligand, which may be competitive inhibitors or substitutes for naturally occurring ligands.
A receptor homolog protein that specifically binds to or that is specifically immunoreactive with an antibody generated against a defined immunogen, such as an immunogen consisting of the amino acid sequence of SEQ ID NO: 2, is typically determined in an immunoassay. The immunoassay typically uses a polyclonal 0 10 antiserum which was raised, to a protein of SEQ ID NO: 2.
SThis antiserum is selected to have low crossreactivity against C other Ig superfamily receptor members, NKG2D, preferably from the same species, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay.
In order to produce antisera for use in an immunoassay, the protein, of SEQ ID NO: 2, is isolated as described herein.
For example, recombinant protein may be produced in a mammalian cell line. An appropriate host, an inbred strain of mice such as Balb/c, is immunized with the selected protein, typically using a standard adjuvant, such as Freund's adjuvant, and a standard mouse immunization protocol (see Harlow and Lane, supra). Alternatively, a synthetic peptide derived from the sequences disclosed herein and conjugated to a carrier protein can be used an immunogen. Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, a solid phase immunoassay with the immunogen immobilized on a solid support. Polyclonal antisera with a titer of 104 or greater are selected and tested for their cross reactivity against other Ig superfamily receptor members using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573. Preferably at least two receptor family members are used in this determination. These receptor family members can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.
Immunoassays in the competitive binding format can be used 00 o for the crossreactivity determinations. For example, the protein of SEQ ID NO: 2 can be immobilized to a solid support. Proteins Sadded to the assay compete with the binding of the antisera to IN the immobilized antigen. The ability of the above proteins to S 5 compete with the binding of the antisera to the immobilized protein is compared to the proteins. The percent crossreactivity M for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity 8 with each of the proteins listed above are selected and pooled.
OO 10 The cross-reacting antibodies are then removed from the pooled Santisera by immunoabsorption with the above-listed proteins.
The immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein the OX2RH1 like protein of SEQ ID NO: In order to make this comparison, the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined.
If the amount of the second protein required is less than twice the amount of the protein of the selected protein or proteins that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
It is understood that these OX2 receptor homolog proteins are members of a family of homologous proteins that comprise at least 6 so far identified genes. For a particular gene product, such as the OX2RH1, the term refers not only to the amino acid sequences disclosed herein, but also to other proteins that are allelic, non-allelic, or species variants. It is also understood that the terms include nonnatural mutations introduced by deliberate mutation using conventional recombinant technology such as single site mutation, or by excising short sections of DNA encoding the respective proteins, or by substituting new amino acids, or adding new amino acids. Such minor alterations typically will substantially maintain the immunoidentity of the original molecule and/or its biological activity. Thus, these alterations include proteins that are specifically immunoreactive 00 with a designated naturally occurring OX2RH protein. The c-i biological properties of the altered proteins can be determined by expressing the protein in an appropriate cell line and measuring the appropriate effect, upon transfected lymphocytes. Particular protein modifications considered minor would include conservative substitution of amino acids with c-i similar chemical properties, as described above for the receptor homolog family as a whole. By aligning a protein optimally with the protein of the receptor homologs and by using the cI 10 conventional immunoassays described herein to determine 00 iimunoidentity, one can determine the protein compositions of the (Ni invention.
VII. Kits and quantitation Both naturally occurring and recombinant forms of the receptor like molecules of this invention are particularly useful in kits and assay methods. For example, these methods would also be applied to screening for binding activity, ligands for these proteins. Several methods of automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds per year. See, a BIOMEK automated workstation, Beckman Instruments, Palo Alto, California, and Fodor, et al. (1991) Scen- 251:767-773, which is incorporated herein by reference. The latter describes means for testing binding by a plurality of defined polymers synthesized on a solid substrate. The development of suitable assays to screen for a ligand or agonist/antagonist homologous proteins can be greatly facilitated by the availability of large amounts of purified, soluble receptors in an active state such as is provided by this invention.
Purified OX2RH can be coated directly onto plates for use in the aforementioned ligand screening techniques. However, non-neutrali zing antibodies to these proteins can be used as capture antibodies to immobilize the respective receptor homolog on the solid phase, useful, in diagnostic uses.
This invention also contemplates use of OX2RH, fragments 00 0 thereof, peptides, and their fusion products in a variety of Cq diagnostic kits and methods for detecting the presence of the Sprotein or its ligand. Alternatively, or additionally, antibodies against the molecules may be incorporated into the
NO
kits and methods and may be used in quantitating the OX2RH or cells expressing them. Typically the kit will have a compartment CI containing either an OX2RH peptide or gene segment or a reagent which recognizes one or the other. Typically, recognition reagents, in the case of peptide, would be a receptor homolog or S10 antibody, or in the case of a gene segment, would usually be a 00 Shybridization probe.
CI A preferred kit for determining the concentration of OX2RH in a sample would typically comprise a labeled compound, e.g., ligand or antibody, having known binding affinity for OX2RH, a source of OX2RH (naturally occurring or recombinant) as a positive control, and a means for separating the bound from free labeled compound, for example a solid phase for immobilizing the OX2RH in the test sample. Compartments containing reagents, and instructions, will normally be provided. Appropriate nucleic acid or protein containing kits are also provided.
Antibodies, including antigen binding fragments, specific for mammalian OX2RH or a peptide fragment, or receptor homolog fragments are useful in diagnostic applications to detect the presence of elevated levels of homolog and/or its fragments.
Diagnostic assays may be homogeneous (without a separation step between free reagent and antibody-antigen complex) or heterogeneous (with a separation step). Various commercial assays exist, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT), substrate-labeled fluorescent immunoassay (SLFIA) and the like. For example, unlabeled antibodies can be employed by using a second antibody which is labeled and which recognizes the antibody to a receptor homolog or to a particular fragment thereof. These assays have also been extensively discussed in the literature. See, e.g., Harlow and Lane (1988) Antibodies: A Laboratory Manual, CSH., and 00 Coligan (ed. 1991 and periodic supplements) Current Protocols In Immunoloav Greene/Wiley, New York.
Anti-idiotypic antibodies may have similar use to serve as agonists or antagonists of the receptor homologs. These should
NO
be useful as therapeutic reagents under appropriate circumstances.
(c1 Frequently, the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay. For the subject invention, depending upon the nature of the assay, the
C
10 protocol, and the label, either labeled or unlabeled antibody, or labeled ligand is provided. This is usually in conjunction with C other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like. Preferably, the kit will also contain instructions for proper use and disposal of the contents after use. Typically the kit has compartments for each useful reagent, and will contain instructions for proper use and disposal of reagents.
Desirably, the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium having appropriate concentrations for performing the assay.
The aforementioned constituents of the diagnostic assays may be used without modification or may be modified in a variety of ways. For example, labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal. In many of these assays, a test compound, receptor homolog, or antibodies thereto can be labeled either directly or indirectly. Possibilities for direct labeling include label groups: radiolabels such as 1251, enzymes (U.S.
Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization. Both of the patents are incorporated herein by reference. Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
There are also numerous methods of separating the bound from 00 0 the free ligand, or alternatively the bound from the free test compound. The receptor homolog can be immobilized on various matrixes followed by washing. Suitable matrices include plastic such as an ELISA plate, filters, and beads. Methods of S 5 immobilizing the receptor homolog to a matrix include, without limitation, direct adhesion to plastic, use of a capture antibody, chemical coupling, and biotin-avidin. The last step in this approach involves the precipitation of antibody/antigen r complex by any of several methods including those utilizing, Cl 10 an organic solvent such as polyethylene glycol or a salt 00 Ssuch as ammonium sulfate. Other suitable separation techniques -q include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30(9):1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678, each of which is incorporated herein by reference.
The methods for linking protein or fragments to various labels have been extensively reported in the literature and do not require detailed discussion here. Many of the techniques involve the use of activated carboxyl groups either through the use- of carbodiimide or active esters to form peptide bonds, the formation of thioethers by reaction of a mercapto group with an activated halogen such as chloroacetyl, or an activated olefin such as maleimide, for linkage, or the like. Fusion proteins will also find use in these applications.
Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a receptor homolog. These sequences can be used as probes for detecting levels of the respective receptor homolog in patients suspected of having an immunological disorder. The preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. Normally an oligonucleotide probe should have at least about 14 nucleotides, usually at least about 18 nucleotides, and the polynucleotide probes may be up to several 00 kilobases. Various labels may be employed, most commonly radionuclides, particularly 32 p. However, other techniques may also be employed, such as using biotin modified nucleotides for introduction into a polynucleotide. The biotin then serves as the site for binding to avidin or antibodies, which may be labeled with a wide variety of labels, such as radionuclides, fluorescers, enzymes, or the like. Alternatively, antibodies may M be employed which can recognize specific duplexes, including
DNA
duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein 00 duplexes. The antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected. The use of probes to the novel anti-sense RNA may be carried out in conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT), and hybrid arrested translation (HART) This also includes amplification techniques such as polymerase chain reaction (PCR).
Diagnostic kits which also test for the qualitative or quantitative presence of other markers are also contemplated.
Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, Viallet, et al. (1989) Proaress in Growth Factor Res. 1:89-97.
VIII. Therapeutic Utility This invention provides reagents with significant therapeutic value. See, Levitzki (1996) Curr. ODin. Cell Biol. 8:239-244. The receptor homologs (naturally occurring or recombinant), fragments thereof, mutein receptors, and antibodies, along with compounds identified as having binding affinity to the receptor homologs or antibodies, should be useful in the treatment of conditions wherein modulation of function of myeloid lineage cells particularly is desirable. Such abnormality will typically be manifested by immunological Sdisorders, but also by conditions in which myeloid cell Cq activities impact physiological processes, CNS maturation Sor development, etc. Additionally, this invention should provide therapeutic value in various diseases or disorders associated
IO
with abnormal expression or abnormal triggering of response to the ligand.
Cl In cases where leukocytes, including macrophage/myeloid lineage cells, expressing the OX2R are involved in pathologies and contribute to the disease process, it may be desirable to S 10 inhibit the function of these cells. This may be achieved by 00 0 appropriate stimulation of an OX2R, such that the cell-inhibitory CI activities of receptor signalling are mobilized. This may be achieved using, a ligand OX2 agonist or an antibody to the OX2R that has agonistic activities for the receptor. Suitable conditions would be where the animal exhibits signs or symptoms of an inflammatory, leukoproliferative, neurodegenerative, or post-traumatic condition. Preferred embodiments include where the sign or symptom is in neural tissue; lymphoid tissue; myeloid tissue; pancreas; gastrointestinal tissue; thyroid tissue; muscle tissue; or skin or collagenous tissue. Certain embodiments include where the animal is experiencing signs or symptoms of autoimmunity; an inflammatory condition; tissue specific autoimmunity; degenerative autoimmunity; rheumatoid arthritis; atherosclerosis; multiple sclerosis; vasculitides; delayed hypersensitivities; skin grafting; a transplant; spinal injury; stroke; neurodegeneration; or ischemia. The administering agent may be in combination with: an anti-inflammatory cytokine agonist or antagonist; an analgesic; an anti-inflammatory agent; or a steroid.
By contrast, in cases where leukocytes, including macrophage/myeloid lineage cells, expressing the OX2R are involved in processes of immunization and vaccination, repair mechanisms, limiting pathologies, or controlling infection, particularly of bacterial infections, it may be desirable to enhance the function of these cells. This may be achieved therapeutically by appropriate stimulation of an OX2R, such that 0 76 0 O the cell-activation activities of receptor signalling are mobilized, or by blocking OX2-OX2R interactions completely should n this enable cell-activation to proceed. The latter occurs in O ligand OX2 gene knockout mice where the lack of ligand OX2 leads to myeloid cell activation. This may be achieved using, a ligand OX2 antagonist (such as an antibody against ligand OX2), an antibody to the OX2R that prevents OX2-OX2R interactions, c antisense nucleic acids which may prevent OX2R expression, an Ig- C OX2R fusion protein that, by competitive binding, blocks 00 the capacity of cell-bound OX2 to interact with cell-bound OX2R, or a small molecule antagonist. That this modality has applications in vivo in promotion of myeloid cell functions has been demonstrated by experiment, where i.v. injection to mice of an adenovirus construct producing a human IgG-mouse OX2RH1 fusion protein known to bind mouse OX2 resulted in accelerated onset of the autoimmune disease experimental autoimmune encephalomyelitis (EAE) in these mice, as compared to mice receiving an adenovirus construct producing only the backbone human IgG-fusion protein. The degree of disease acceleration was comparable to that seen in mice in which a ligand for OX2R, namely OX2, had been inactivated by gene targeting.
Alternatively, if the various OX2R molecules described herein have activating vs. inhibitory function, specific activation of the OX2R that induces cellular activation may be appropriate. This may be achieved, by the use of specific antibody with agonistic activities of a given OX2R. In various embodiments, the method is applied where the animal experiences signs or symptoms of wound healing or clot formation in which enhanced macrophage activation may be desirable, or where an animal experiences a bacterial infection where enhanced phagocytic activity by granulocytes and/or macrophages is desirable. The administering will often be in combination with: an angiogenic factor; a growth factor, including FGF or PDGF; an antibiotic; or a clotting factor.
00 Recombinant receptors, muteins, agonist or antagonist antibodies N thereto, or antibodies can be purified and then administered to a Spatient. These reagents can be combined for therapeutic use with ksO additional active ingredients, in conventional pharmaceutically acceptable carriers or diluents, along with physiologically innocuous stabilizers and excipients. These combinations can be sterile, filtered, and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations. This invention also 00 10 contemplates use of antibodies or binding fragments thereof which are not complement binding.
c Ligand screening using receptor or fragments thereof can be performed to identify molecules having binding affinity to the receptors. Subsequent biological assays can then be utilized to determine if a putative ligand can provide competitive binding, which can block intrinsic stimulating activity. Receptor fragments can be used as a blocker or antagonist in that it blocks the activity of ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of ligand, inducing signaling. This invention further contemplates the therapeutic use of antibodies to receptors as antagonists.
The quantities of reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, reagent physiological life, pharmacological life, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, in Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; each of 00 which is hereby incorporated herein by reference. Methods for administration are discussed therein and below, for oral, intravenous, intraperitoneal, or intramuscular administration, transdermal diffusion, and others. Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, in the Merk Idex Merck Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 100 itM concentrations, typically less than about 1 mM concentrations, usually less than about 100 pM, 00 10 preferably less than about 1 pM, and most preferably less than about 10 nM, with an appropriate carrier. Slow release formulations, or slow release apparatus will often be utilized for continuous administration.
Receptor homologs, fragments thereof, and antibodies or its fragments, antagonists, and agonists, may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumfin or serum albumin prior to their administration. Therapeutic formulations may be administered in many conventional dosage formulations. W'hile it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation. Formulations comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier must be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient. Formulations include those suitable for oral, rectal, nasal, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. The formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, Gilman, et al.
(eds. 1990) Goodman and Gilmnan' The Pharmacoloaical Bases f Therapeutics, 8th Ed., Pergamon Press; and Reminaton's Pharmaceutical Science-s, 17th ed. (1990), Mack Publishing Co., Easton, Penn.; Avis, et al. (eds. 1993) Pharmaceutical Dosaae 00 O Forms: Parenteral Medications Dekker, NY; Lieberman, et al. (eds.
eCq 1990) Pharmaceutical Dosaae Forms: Tablets Dekker, NY; and Lieberman, et al. (eds. 1990) Pharmaceutical Dosage Forms: Disperse Systems Dekker, NY. The therapy of this invention may be combined with or used in association with other therapeutic agents, particularly agonists or antagonists of other receptor C- family members.
g IX. Screening C- 10 Drug screening using OX2RH or fragments thereof can be 00 Sperformed to identify compounds having binding affinity to the Cq j. receptor homolog, including isolation of associated components.
Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity and is therefore a blocker or antagonist in that it blocks the activity of the ligand. Likewise, a compound having intrinsic stimulating activity can activate the receptor and is thus an agonist in that it simulates the activity of a ligand, OX2. This invention further contemplates the therapeutic use of antibodies to the receptor as agonists or antagonists.
Similarly, complexes comprising multiple proteins may be used to screen for ligands or reagents capable of recognizing the complex. Some receptors comprise at least two subunits, which may be the same, or distinct. Alternatively, the transmembrane receptor may bind to a complex comprising a ligand associated with another soluble protein serving, as a second receptor subunit.
One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing the OX2RH in combination with another receptor subunit. Cells may be isolated which express a receptor in isolation from other functional receptors.
Such cells, either in viable or fixed form, can be used for standard antibody/antigen or ligand/receptor binding assays. See also, Parce, et al. (1989) Science 246:243-247; and Owicki, et al. (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which
I
00 O describe sensitive methods to detect cellular responses.
Competitive assays are particularly useful, where the cells C' (source of putative ligand) are contacted and incubated with a labeled receptor or antibody having known binding affinity to the ligand, such as 125I-antibody, and a test sample whose binding C-M affinity to the binding composition is being measured. The bound and free labeled binding compositions are then separated to Sassess the degree of ligand binding. The amount of test compound CI bound is inversely proportional to the amount of labeled receptor binding to the known source. Many techniques can be used to c, separate bound from free ligand to assess the degree of ligand binding. This separation step could typically involve a procedure such as adhesion to filters followed by washing, adhesion to plastic followed by washing, or centrifugation of the cell membranes. Viable cells could also be used to screen for the effects of drugs on OX2 mediated functions, second messenger levels, cell proliferation; inositol phosphate pool changes; and others. Some detection methods allow for elimination of a separation step, a proximity sensitive detection system. Calcium sensitive dyes will be useful for detecting Ca++ levels, with a fluorimeter or a fluorescence cell sorting apparatus.
X. Ligands The descriptions of the OX2RHs herein provides means to identify ligands, as described above. Such ligand should bind specifically to the respective receptor with reasonably high affinity. Various constructs are made available which allow either labeling of the receptor to detect its ligand. For example, directly labeling receptor, fusing onto it markers for secondary labeling, FLAG or other epitope tags, etc., will allow detection of receptor. This can be histological, as an affinity method for biochemical purification, or labeling or selection in an expression cloning approach. A two-hybrid selection system may also be applied making appropriate constructs with the available receptor sequences. See, e.g.,
I
81 00 O Fields and Song (1989) Nature 340:245-246.
The broad scope of this invention is best understood with reference to the following examples, which are not intended to ND limit the inventions to the specific embodiments.
00 00 C-I EXAMPLES I. General Methods 5 Some of the standard methods are described or referenced, in Maniatis, et al. (1982) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al. (1989) Molecular Cloning: A Laboratory Manual, (2d vols. 1-3, CSH Press, NY; or Ausubel, et al. (1987 and 00 10 Supplements) Current Protocols in Molecular Bioloay, SGreene/Wiley, New York. Methods for protein purification include C such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, Ausubel, et al. (1987 and periodic supplements); Coligan, et al. (ed. 1996) and periodic supplements, Current Protocols In Protein Science Greene/Wiley, New York; Deutscher (1990) "Guide to Protein Purification" in Methods in Enzvmoloav, vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, Pharmacia, Piscataway, or Bio- Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments, to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
See, Hochuli (1990) "Purification of Recombinant Proteins with Metal Chelate Absorbent" in Setlow Genetic Enaineerina. Principle and Methods 12:87-98, Plenum Press, N.Y.; and Crowe, et al. (1992) OTAexoress: The High Level Exoression Protein Purification System QUIAGEN, Inc., Chatsworth, CA.
Computer sequence analysis is performed, using available software programs, including those from the GCG (U.
Wisconsin) and GenBank sources. Public sequence databases were also used, from GenBank and others.
Many techniques applicable to IL-10 receptors may be applied to the OX2RHs, as described, in USSN 08/110,683 receptor), which is incorporated herein by reference.
00 SII. Monoclonal Antibody which blocks rat OX2/OX2RH interaction CI on macrophages SA bead assay was set up using recombinant OX2-CD4 protein and rat peritoneal macrophages. See Preston, et al. (1997) EJr.
J. Immunol. 27:1911-1918. Macrophages bound to fluorescent beads coated with recombinant OX2-CD4 proteins. Six wk old BALB/c mice C1 were immunized 6 times with either 0.1-0.25 mg crude membrane fraction (Williams and Barclay (1986) in Handbook of Experimental STmmunology vol. 1, 22.1-22.24, Blackwell Scientific Publications) 00 10 or resident rat peritoneal exudate cells (5 million). Mice were O screened for high titers of antibodies recognizing macrophages by CI testing various dilutions of the sera for labeling of macrophages by indirect immunofluorescence and flow cytometry. Mice producing good immune responses to the rat macrophages were finally boosted by injection of peritoneal exudate cells. Four days later, spleens were removed and fused to NS-1 myeloma cells to produce hybridomas. The final injection before screening was intrasplenic. Hybridoma supernatants were screened for the ability to label rat macrophages and for the ability to block the rat OX2 interaction with macrophages. One antibody, designated OX102, was obtained and cloned. This antibody gave clear blocking. This hybridoma was grown in bulk and the antibody was purified by standard procedures.
III. Purification of the antigen for the OX102 mAb Purified OX102 mAb was covalently coupled to CNBr activated sepharose-4B (Pharmacia) as recommended by the manufacturer.
Membrane proteins were solubilised using Tween 40 and sodium deoxycholate and incubated with the Sepharose beads coupled to OX102 mAb, for 70 hours. Williams and Barclay (1986) in Handbook of Experimental Immunoloav vol. 1, 22.1-22.24, Blackwell Scientific Publications. The OX102 mAb-coupled Sepharose beads were pelleted by centrifugation and washed in 0.1% sodium dodecyl sulphate (SDS) and finally eluted in 0.5% SDS at 550 C for 15 min The eluted fraction was analysed by SDS polyacrylamide gel electrophoresis
(SDS-PAGE).
00 c IV. N-terminal sequence of the antigen for the OX102 mAb Z Amino terminal sequencing was performed using automated ND Edman degradation in an Applied Biosystems Procise 494A protein sequencer (Perkin-Elmer Ltd., UK). The N-terminal sequence was confirmed, as shown in Table 1. Blank cycles are assumed to be asparagine due to the presence of asparagine modified by N-linked Sglycosylation. The purified polypeptide was identified as novel Sby screening known protein databases with the N-terminal 20 amino 00 10 acids of the antigen for the OX102 mAb. This protein is the rat SOX2RH1.
V. Isolation of cDNA clones coding for the antigen of the OX102 mAb Total RNA was extracted from rat peritoneal exudate cells using RNAzol B (Biogenesis) and then the poly-A fraction purified using oligo dT beads (Oligotex, QIAGEN) as recommended by the manufacturer. Approximately 50 ng of polyA+ purified mRNA was treated with 200 U of Superscript II reverse transcriptase
(GIBCO
BRL) in the presence of 1 pM of selected sense and antisense oligonucleotides, 1 mM dNTPs, and 2 mM DTT, 50 mM Tris-HCl pH 8.3, 75 mM KC1, 3 mM MgC12, and incubated at 420 C for 1 h.
This cDNA was then used as a template in a PCR reaction, 40 pl of provided 10x Advantage Taq thermophilic PCR buffer (provided by Clontech); 8 ul of 10 mM dNTPs; 8 1l of Advantage Taq (Clontech); 2 pl of cDNA prepared as described above; 318 pl of distilled water; 16 p1 of an antisense, degenerate oligonucleotide corresponding to the N-terminal peptide at 10 pM; and 8 pl 10 pM sense oligonucleotide. Both oligonucleotide primers were synthesized (Genosys) with a 5' terminal phosphate to facilitate cloning.
The PCR mix was aliquoted into 8 x 50 p1 samples and subjected to PCR conditions in a Robocycler PCR machine (Stratagene) which allows the operator to vary the annealing temperature in separate samples simultaneously. Example parameters are: 930 C 30 sec; followed by 35 cycles of: 930 C 00 0 sec; 42-56° C 1 min; 720 C 30 sec; and a final cycle of 720 C for (C 8 min.
STen pl of the PCR products were analyzed by agarose gel S electrophoresis by standard procedures. PCR products of lengths ranging between 100 and 300 base pairs in the 3 samples which had an annealing temperature of about 420, 440, and 460 C were excised from the gel and the nucleic acid purified using QIAquick (QIAGEN). These purified products were ligated at about 160 C Sfor 48 h using standard procedures into PCRScript vector 0O 10 (Stratagene) which had been SmaI digested and phosphatase treated.
C-I Transformants were screened initially by colony PCR and colonies containing appropriately-sized inserts were grown up in LB broth in the presence of 50 pg/ml ampicillin and plasmids purified by a QIAGEN robot. Inserts were sequenced using the BIGDYE fluorescent dideoxy-terminator technology and ABI-PRISM Model 377 (Perkin-Elmer Ltd., UK). Inserts containing nucleotide sequence that coded for the N-terminal sequence of antigen for the OX102 mAb were used to design oligonucleotides for 3' RACE reactions.
The full cDNA sequence of the antigen for OX102 was obtained by 3'RACE PCR (using the same protocol as above) but modified by using appropriate oligonucleotides at a final concentration of 0.2 uM each. PCR conditions, were: 930 C 30 sec; followed by 30 cycles of: 930 C 30 sec; 51°-650 C 1 min; 72° C 3.5 min; and a final cycle of 72° C for 12 min.
A band of approximately 2.3 Kb was excised from the 65° C PCR reaction, gel purified, digested with NotI and XhoI, and ligated into NotI/XhoI digested vector (PCRScript, Stratagene) using standard procedures. Inserts were sequenced as above.
The cDNA sequence of the OX102 protein, also referred to herein as rat OX2RH1, is shown in Table 1. Full length isolates of the other homolog embodiments are similarly cloned and sequences confirmed. Standard methods are readily applicable.
00 VI. Obtaining other OX2RH cDNAs The knowledge of the rat OX2RH1 nucleotide and predicted amino acid sequences allows one to obtain homologous functional IND equivalents from other species, including mouse or human OX2RHI, on the basis of sequence similarity and because the rat OX2RHl provides a tool for the isolation of such equivalents.
Thus, to identify human OX2RH1, one can search existing Mdatabases of nucleotide and amino acid sequences for polypeptides NI of unknown identity databases storing sequences obtained 00 1 10 from the Human Genome Project) for sequences with homology to the rat OX2RHI nucleic acid and amino acid sequences provided herein.
The databases, which are stored and updated at many sites, including the European Bioinformatics Centre (http://www.ebi.ac.uk/) and National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/), can be accessed by widely-used programs such as FASTA or BLAST. These databases include sequences for expressed sequence tags (ESTs) which are short regions of nucleotide sequence sequenced from random cDNA clones. This allows partial cDNA clones for mouse or human OX2RH to be isolated by comparison with the rat OX2RH sequence information provided herein. Full length clones can then be isolated by screening macrophage cDNA or genomic libraries or by primer extension techniques such as those described herein for obtaining the full-length rat OX2RHl clones.
Mouse and human sequences related to the rat OX2RHI were identified from genomic sequence database using, the BLAST server (Altschul, et al. (1994) Nature Genet. 6:119-129).
Standard analysis programs may be used to evaluate structure, PHD (Rost and Sander (1994) Proteins 19:55-72) and DSC (King and Sternberg (1996) Protein Sci. 5:2298-2310). Standard comparison software includes, Altschul, et al. (1990) J ol..o. L 215:403-10; Waterman (1995) Introduction to Computational Bioloav: Maps. Secuences, and Genomes Chapman Hall; Lander and Waterman (eds. 1995) Calculatina the Secrets of Life: Applications of the Mathematical Sciences in Molecular Bioloav National Academy Press; and Speed and Waterman (eds.
00 0 1996) Genetic MaDpina and DNA Secuencing (IMA Volumes in C Mathematics and Its Applications, Vol 81) Springer Verlag.
Nucleic acid sequence for rat and human OX2RH1 will have between 50 and 98% homology, as can be observed in Table
NO
For the other homologs, the similarity may be less, especially with the homolog 3.
CI As an alternative to database screening, the rat OX2RH1 nucleic acid sequence as provided herein can be used to screen macrophage cDNA or genomic libraries to identify human sequences 00 10 sufficiently homologous to hybridize under conditions of Sappropriate stringency. This approach was utilized to isolate CI the human OX2 gene using rat OX2 nucleic acid as a probe.
McCaughan, et al. (1987) Immunoaenetics 25:329-335). PCR based methods using templates of cDNA, genomic DNA, cDNA clones, or genomic clones as templates are also widely used, the general approach being exemplified by the isolation of mouse OX2 from cDNA. See Preston, et al. (1997) Eur. J. Immunol. 27:1911-1918.
PCR primers derived from the provided OX2RH sequences are used to probe a human, or other species or tissue, cDNA library.
Sequences may be derived, from Tables 1-4, preferably those adjacent the ends of sequences. Full length cDNAs for primate, rodent, or other species OX2RHs are cloned, by DNA hybridization screening of XgtlO phage. PCR reactions are conducted using T. aquaticus Taqplus DNA polymerase (Stratagene) under appropriate conditions.
Further, the rat OX2RH1 sequence can be used to isolate the corresponding mouse OX2RH1 sequence, and to identify regions conserved between them. Particularly with the discovery of a group of homologs, regions of similarity may be identified.
Conserved-region sequences provide useful reagents for identifying a given gene in a range of species. For instance domain 1 of mouse and rat OX2 are 90% identical at the amino acid level, compared to 77% identity between the same human and rat domains. Preston, et al. (1997) Eur. J. Immunol. 27:1911-1918.
Yet another method is to use antibody reagents to expression clone or identify crossreacting proteins expressed in cDNA D libraries from appropriate cell types, macrophages, or from other species.
0 VII. Chromosomal localization The genes will be mapped. For example, chromosome spreads are prepared. In situ hybridization is performed on chromosome preparations obtained from phytohemagglutinin-stimulated Slymphocytes, from human, cultured for 72 h. bromodeoxyuridine is added for the final seven hours of culture 00 10 (60 pg/ml of medium), to ensure a posthybridization chromosomal Sbanding of good quality.
A PCR fragment, amplified with the help of primers, is cloned into an appropriate vector. The vector is labeled by nick-translation with 3 H. The radiolabeled probe is hybridized to metaphase spreads at final concentration of 200 ng/ml of hybridization solution as described in Mattei, et al. (1985) RuL.
Genet. 69:327-331.
After coating with nuclear track emulsion (KODAK NTB2), slides are exposed. To avoid any slipping of silver grains during the banding procedure, chromosome spreads are first stained with buffered Giemsa solution and metaphase photographed.
R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG) method and metaphases rephotographed before analysis.
Similar appropriate methods are used for other species.
VIII. Localization of various OX2RH mRNA While the expected expression patterns of the OX2RH1 are primarily on macrophages, granulocytes, and mast cells, the homologs H2, H3, and/or H4 may not be so closely related functionally. Thus, the distribution of those will be of particular interest. Distribution may be evaluated at the nucleic acid level, by hybridization or PCR methods, or at the protein level, by histology or immunochemical methods.
Human multiple tissue (Cat 2) and cancer cell line blots (Cat #7757-1), containing approximately 2 ig of poly(A) RNA per lane, are purchased from Clontech (Palo Alto, CA). Probes are S radiolabeled with [a- 32 p] dATP, using the Amersham C, Rediprime random primer labeling kit (RPN1633). Prehybridization and hybridizations are performed, at 650 C in 0.5 M Na2HPO4, 7% SDS, 0.5 M EDTA (pH High stringency washes are 5 conducted, at 650 C with two initial washes in 2 x SSC, 0.1% SDS for 40 min followed by a subsequent wash in 0.1 x SSC, 0.1% SDS for 20 min. Membranes are then exposed at -700 C to X- Ray film (Kodak) in the presence of intensifying screens. More c detailed studies by cDNA library Southerns are performed with C 10 selected appropriate mammalian OX2RH clones to examine their 00 S expression in hemopoietic or other cell subsets.
Alternatively, two appropriate primers are selected, e.g., from Tables 1-4. RT-PCR is used on an appropriate mRNA sample selected for the presence of message to produce a cDNA, a sample which expresses the gene.
Full length clones may be isolated by hybridization of cDNA libraries from appropriate tissues pre-selected by PCR signal.
Northern blots can be performed.
Message for genes encoding OX2RHs will be assayed by appropriate technology, PCR, immunoassay, hybridization, or otherwise. Tissue and organ cDNA preparations are available, from Clontech, Mountain View, CA. Identification of sources of natural expression are useful, as described.
For mouse distribution, Southern Analysis can be performed: DNA (5 jig) from a primary amplified cDNA library was digested with appropriate restriction enzymes to release the inserts, run on a 1% agarose gel and transferred to a nylon membrane (Schleicher and Schuell, Keene, NH).
Samples for mouse mRNA isolation may include: resting mouse fibroblastic L cell line (C200); Braf:ER (Braf fusion to estrogen receptor) transfected cells, control (C201); T cells, TH1 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IFN-y and anti IL-4; T200); T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and anti-IFN-y; T201); T cells, highly TH1 polarized (see Openshaw, et al. (1995) J. ExD. Med. 182:1357-1367; activated 00 with anti-CD3 for 2, 6, 16 h pooled; T202); T cells, highly TH2 polarized (see Openshaw, et al. (1995) J. ExD. Med. 182:1357- 1367; activated with anti-CD 3 for 2, 6, 16 h pooled; T203); CD44- O CD25+ pre T cells, sorted from thymus (T204); TH1 T cell clone D1.1, resting for 3 weeks after last stimulation with antigen (T205); TH1 T cell clone Dl.1, 10 g/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for 3 weeks after last stimulation with antigen (T207); TH2 T cell clone CDC35, 10 4g/ml 0 ConA stimulated 15 h (T208); Mell4+ naive T cells from spleen, 0 10 resting (T209); Mell4+ T cells, polarized to Thl with IFN-y/IL- S 12/anti-IL-4 for 6, 12, 24 h pooled (T210); Mell4+ T cells, polarized to Th2 with IL-4/anti-IFN-y for 6, 13, 24 h pooled (T211); unstimulated mature B cell leukemia cell line A20 (B200); unstimulated B cell line CH12 (B201); unstimulated large B cells from spleen (B202); B cells from total spleen, LPS activated (B203); metrizamide enriched dendritic cells from spleen, resting (D200); dendritic cells from bone marrow, resting (D201); monocyte cell line RAW 264.7 activated with LPS 4 h (M200); bonemarrow macrophages derived with GM and M-CSF (M201); macrophage cell line J774, resting (M202); macrophage cell line J774 LPS anti-IL-1 0 at 0.5, 1, 3, 6, 12 h pooled (M203); macrophage cell line J774 LPS IL-10 at 0.5, 1, 3, 5, 12 h pooled(M204); aerosol challenged mouse lung tissue, Th2 primers, aerosol
OVA
challenge 7, 14, 23 h pooled (see Garlisi, et al. (1995) Clinical Immunoloav and Immunonatholoav 75:75-83; X206); Nippostrongulusinfected lung tissue (see Coffman, et al. (1989) Sience 245:308- 310; X200); total adult lung, normal (0200); total lung, rag-l (see Schwarz, et al. (1993) Tmmunodeficiency 4:249-252; 0205); K.O. spleen (see Kuhn, et al. (1991) £all 75:263-274; X201); total adult spleen, normal (0201); total spleen, rag-i (0207); IL-10 K.O. Peyer's patches (0202); total Peyer's patches, normal (0210); IL-10 K.O. mesenteric lymph nodes (X203); total mesenteric lymph nodes, normal (0211); IL-10 K.O. colon (X203); total colon, normal (0212); NOD mouse pancreas (see Makino, et al. (1980) Jikken Dobutsu 29:1-13; X205); total thymus, rag-i (0208); total kidney, rag-i (0209); total heart, rag-i (0202); 00 D total brain, rag-i (0203); total testes, rag-i (0204); total C liver, rag-i (0206); rat normal joint tissue (0300); and rat Sarthritic joint tissue (X300).
^0 Samples for human mRNA isolation may include: peripheral blood mononuclear cells (monocytes, T cells, NK cells, granulocytes, B cells), resting (T100); peripheral blood mononuclear cells, activated with anti-CD3 for.2, 6, 12 h pooled (T101); T cell, THO clone Mot 72, resting (T102); T cell, THO 0 clone Mot 72, activated with anti-CD28 and anti-CD3 for 3, 6, 12 00 10 h pooled (T103); T cell, THO clone Mot 72, anergic treated with S specific peptide for 2, 7, 12 h pooled (T104); T cell, TH1 clone HY06, resting (T107); T cell, TH1 clone HY06, activated with anti-CD28 and anti-CD3 for 3, 6, 12 h pooled (T108); T cell, TH1 clone HY06, anergic treated with specific peptide for 2, 6, 12 h pooled (T109); T cell, TH2 clone HY935, resting (T110); T cell, TH2 clone HY935, activated with anti-CD28 and anti-CD3 for 2, 7, 12 h pooled (T1ll); T cells CD4+CD45RO- T cells polarized 27 days in anti-CD28, IL-4, and anti IFN-y, TH2 polarized, activated with anti-CD3 and anti-CD28 4 h (T116); T cell tumor lines Jurkat and Hut78,'resting (T117); T cell clones, pooled AD130.2, Tc783.12, Tc783.13, Tc783.58, Tc782.69, resting (T118); T cell random y6 T cell clones, resting (T119); Splenocytes, resting (B100); Splenocytes, activated with anti-CD40 and IL-4 (B101); B cell EBV lines pooled WT49, RSB, JY, CVIR, 721.221, RM3, HSY, resting (B102); B cell line JY, activated with PMA and ionomycin for 1, 6 h pooled (B103); NK 20 clones pooled, resting (K100); NK clones pooled, activated with PMA and ionomycin for 6 h (K101); NKL clone, derived from peripheral blood of LGL leukemia patient, IL-2 treated (K106); NK cytotoxic clone 640-A30-1, resting (K107); hematopoietic precursor line TF1, activated with PMA and ionomycin for 1, 6 h pooled (C100); U937 premonocytic line, resting (M100); U937 premonocytic line, activated with PMA and ionomycin for 1, 6 h pooled (M101); elutriated monocytes, activated with LPS, IFNy, anti-IL-10 for 1, 2, 6, 12, 24 h pooled (M102); elutriated monocytes, activated with LPS, IFNy, IL-10 for 1, 2, 6, 12, 24 h pooled (M103); elutriated monocytes, activated 00 Swith LPS, IFNy, anti-IL-10 for 4, 16 h pooled (M106); elutriated monocytes, activated with LPS, IFN,. IL-10 for 4, 16 h pooled c- (M107); elutriated monocytes, activated LPS for 1 h (M108); elutriated monocytes, activated LPS for 6 h (M109); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, resting (D101); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, activated with PMA and ionomycin for 1 hr (D102); DC 70% CDla+, from CD34+ GM-CSF, TNFa 12 days, Sactivated with PMA and ionomycin for 6 hr (D103); DC 95% CDla+, from CD34+ GM-CSF, TNFa 12 days FACS sorted, activated with PMA 00 10 and ionomycin for 1, 6 h pooled (D104); DC 95% CD14+, ex CD34+ GM-CSF, TNFa 12 days FACS sorted, activated with PMA and ionomycin 1, 6 hr pooled (D105); DC CDla+ CD86+, from CD34+ GM- CSF, TNFa 12 days FACS sorted, activated with PMA and ionomycin for 1, 6 h pooled (D106); DC from monocytes GM-CSF, IL-4 5 days, resting (D107); DC from monocytes GM-CSF, 11-4 5 days, resting (D108); DC from monocytes GM-CSF, IL-4 5 days, activated LPS 4, 16 h pooled (D109); DC from monocytes GM-CSF, IL-4 5 days, activated TNFa, monocyte supe for 4, 16 h pooled (D110); leiomyoma L11 benign tumor (X101); normal myometrium M5 (0115); malignant leiomyosarcoma GS1 (X103); lung fibroblast sarcoma line activated with PMA and ionomycin for 1, 6 h pooled (C101); kidney epithelial carcinoma cell line CHA, activated with PMA and ionomycin for 1, 6 h pooled (C102); kidney fetal 28 wk male (0100); lung fetal 28 wk male (0101); liver fetal 28 wk male (0102); heart fetal 28 wk male (0103); brain fetal 28 wk male (0104); gallbladder fetal 28 wk male (0106); small intestine fetal 28 wk male (0107); adipose tissue fetal 28 wk male (0108); ovary fetal 25 wk female (0109); uterus fetal 25 wk female (0110); testes fetal 28 wk male (0111); spleen fetal 28 wk male (0112); adult placenta 28 wk (0113); and tonsil inflamed, from 12 year old (X100).
Similar samples may isolated in other species for evaluation. Histology may also be performed.
00 S IX. Production of mammalian OX2RH proteins An appropriate, GST, fusion construct is engineered for expression, in E. coli. For example, a mouse OX2RH O pGex plasmid is constructed and transformed into E. coli.
Freshly transformed cells are grown, in LB medium containing 50 pg/ml ampicillin and induced with IPTG (Sigma, St.
Louis, MO). After overnight induction, the bacteria are c harvested and the pellets containing the OX2RH protein are 0 isolated. The pellets are homogenized, in TE buffer (50 mM 00 10 Tris-base pH 8.0, 10 mM EDTA and 2 mM pefabloc) in 2 liters.
S This material is passed through a microfluidizer (Microfluidics, Newton, MA) three times. The fluidized supernatant is spun down on a Sorvall GS-3 rotor for 1 h at 13,000 rpm. The resulting supernatant containing the OX2RH protein is filtered and passed over a glutathione-SEPHAROSE column equilibrated in 50 mM Trisbase pH 8.0. The fractions containing the OX2RH-GST fusion protein are pooled and cleaved, with thrombin (Enzyme Research Laboratories, Inc., South Bend, IN). The cleaved pool is then passed over a Q-SEPHAROSE column equilibrated in 50 mM Tris-base. Fractions containing OX2RH are pooled and diluted in cold distilled H20, to lower the conductivity, and passed back over a fresh Q-Sepharose column, alone or in succession with an immunoaffinity antibody column. Fractions containing the OX2RH protein are pooled, aliquoted, and stored in the 7 0 C freezer.
Various fusion constructs are made with OX2RH. Thus, e.g., fusion of the extracellular portions of the OX2RH2, H3, or H4 may be fused to intracellular portions of the DAP12 or to IgG domains or other labeling or functional domains. A portion of the appropriate gene is fused to an epitope tag, a FLAG tag, or to a two hybrid system construct. See, Fields and Song (1989) Nature 340:245-246.
The epitope tag may be used in an expression cloning procedure with detection with anti-FLAG antibodies to detect a binding partner, ligand for the respective receptor homolog. The two hybrid system may also be used to isolate proteins which specifically bind to an OX2RH.
00 Comparison of the CD spectrum with similar Ig superfamily receptor protein may suggest that the protein is correctly folded. See Hazuda, et al. (1969) 1. Biol. Cher. 264:1689-1693; and Campbell, et al. (1979) Nature 282:341-342.
The reactivity of the OX2/OX2R in terms of binding properties, kinetics and functional effects, can be investigated. The interactions of the OX2R cytoplasmic domain can be determined using well established immunoprecipitation 00CI methods or genetic methods such as the yeast two-hybrid system.
Transfection of the OX2RH into cells normally not expressing the ci proteins may be useful in physiological and signaling studies.
X. Preparation of antibodies specific for OX2RHs Appropriate species or strains, inbred Balb/c mice, are immunized intraperitoneally with recombinant forms of the protein, purified OX2RH or stable transfected NIH-3T3 cells. Animals are boosted at appropriate time points with protein, with or without additional adjuvant, to further stimulate antibody production. Serum is collected, or hybridomas produced with harvested spleens.
Alternatively, the animals, Balb/c mice, are immunized with cells transformed with the gene or fragments thereof, either endogenous or exogenous cells, or with isolated membranes enriched for expression of the antigen. Serum is collected at the appropriate time, typically after numerous further administrations. Various gene therapy techniques may be useful, in producing protein in situ, for generating an immune response. Serum or antibody preparations may be cross-absorbed or immunoselected to prepare substantially purified antibodies of defined specificity and high affinity. Thus, antibodies could be prepared which recognize various species counterparts, or antibodies which recognize specific species or groups of subsets, rodent, embodiments.
Monoclonal antibodies may be made. For example, splenocytes are fused with an appropriate fusion partner and hybridomas are selected in growth medium by standard procedures. Hybridoma S supernatants are screened for the presence of antibodies which bind to the rat OX2RH1, by ELISA or other assay.
Antibodies which specifically recognize specific OX2RH embodiments may also be selected or prepared.
In another method, synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, Coligan (ed. 1991) Curren Protocols in Immunolov Wiley/Greene; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press. In OO 10 appropriate situations, the binding reagent is either labeled as S described above, fluorescence or otherwise, or immobilized to a substrate for panning methods. Nucleic acids may also be introduced into cells in an animal to produce the antigen, which serves to elicit an immune response. See, Wang, et al.
(1993) Proc. Nat'l. Acad. Sci. 90:4156-4160; Barry, et al. (1994) BioTechniaues 16:616-619; and Xiang, et al. (1995) Immunit 2: 129-135.
XI. Ligand binding and partner specificity Means for testing of the binding selectivity and affinity are readily available. Surface plasmon resonance (see manufacturer's protocol; BIAcore manual, Pharmacia Biosensor) or other methods may be used to determine the ligand for the OX2RHs.
The rat and mouse HI bind to their species counterpart ligand OX2; the human HI will be similarly tested. The H2 will be similarly tested against similar potential ligands, though the similarity of the extracellular domains of the H2 to the known receptor (rat and mouse HI) suggest the same or closely related ligand.
A receptor can be used as a specific binding reagent to identify its binding partner, by taking advantage of its specificity of binding, much like an antibody would be used. The binding receptor may be an OX2RH, or may involve, a complex of the OX2RH with another subunit. A binding reagent is either labeled as described above, fluorescence or otherwise, or immobilized to a substrate for panning methods.
00 C- The binding composition is used to screen an expression library made from a cell line which expresses a binding partneri ligand, preferably membrane associated. Standard staining techniques are used to detect or sort surface expressed ligand, or surface expressing transformed cells are screened by panning.
CIq Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al. (1991) M 10:2821-2832.
00 Alternatively, receptor reagents are used to affinity purify or sort out cells expressing a putative ligand. See, e.g., Sanbrook, et al. or Ausubel, et al.
Another strategy is to screen for a membrane bound ligand by panning. The receptor cDNA is constructed as described above.
Immobilization may be achieved by use of appropriate antibodies which recognize, a FLAG sequence on an OX2RH fusion construct, or by use of antibodies raised against the first antibodies. Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of receptor expressing clones.
Phage expression libraries can be screened by mammalian OX2RH, labeled forms. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate phage clones.
Upon confirmation of OX2-OX2RH binding, or identification of alternative ligands for the other homologs, signaling pathways will be tested. See, Preston, et al. (1997)
L
I.MUDflQL 27:1911-1918. Implications of the DAP12 involvement are also clear. See, Bakker, et al. (2000) Human IrmunloaV 61:18-27; Lanier, et al. (1998) Irmunity 8:693-701; Smith, et al.
(1998) J. Irmunol. 161:7-10; Gosselin, et al. (1999) J. Lukoc.
R 66:165-171; Tomasello, et al.(1 998 J. Biol. Chem.
273:34115-34119; and McVicar, et al. (1998) J. Biol. Chem.
273:32934-32942. Similarly, or alternatively, DAPl0 may be involved. See, Wu J, et al. (1999) Si 285:730-732; and Bauer, et al. (1999) Science 285:727-729.
00 In particular, the DAP12 coreceptor partner is in the same CN I family as the T cell receptor subunit and the FcERy, which possess ITIM motifs, and signal through the pathway involving the S syk/zap 7 0 protein tyrosine kinases. The DAP10 has the YxxM motif, which signals through or analogously to the PI3 kinase pathway.
(c Certain isoforms of the MHC class I receptors on NK cells Slack ITIM sequences in their cytoplasmic domains and it has been 8 proposed that these isoforms activate, rather than inhibit,
NK
00 10 cells. These activating receptors have very short intracellular S regions lacking any signaling motifs and they all share a C positively charged residue within their transmembrane domain, which suggested the association with an adapter molecule that is capable of signaling. DAP12, a type I disulfide-linked homodimer containing an ITAM non-covalently assembles with the human KIR2DS receptors. DAP12 has a negatively charged aspartic acid residue in its transmembrane region and corresponds to a reported 12-13 kD phosphoprotein that was found to co-immunoprecipitate with KIR2DS.
Upon receptor engagement, DAP12 becomes phosphorylated and recruits the Syk kinase, thus inducing a signaling cascade similar to T cell receptor. Besides being associated with KIR2DS, a receptor for HLA-C, DAP12 is also expressed at the cell surface of NK cells associated with the activating mouse Ly49D and Ly49H receptors recognizing H-2 and with the human CD94/NKG2C heterodimer receptor complex recognizing
HLA-E.
Recent efforts to identify potential membrane signaling proteins by searching the EST databases have led to the identification of DAP10, a novel 10-kD surface adapter primarily expressed in hematopoietic cells. Although DAP10 has only limited homology with DAP12, its transmembrane domain contains a negatively charged residue that is conserved in the transmembrane regions of DAP12 and all of the CD3 subunits of the TCR. In addition, the conserved cysteine residues within the extracellular domain of DAP12 and the CD3 chains are also present in DAP10. Interestingly, the human DAP10 and DAP12 genes lie 00 S adjacent on chromosome 19ql3.1, in opposite transcriptional orientation and separated by only approximately 130 base pairs, presumably as a result of gene duplication. One unique feature ^0 of DAP10 is its short, but conserved, cytoplasmic tail which, contains a YxxM signaling motif, a potential src-homology 2 (SH2) domain-binding site for the p85 regulatory subunit of the S phosphatidylinositol 3-kinase (PI 3-kinase). The physical and functional association of various OX2RH with DAP12 or DAP10 can Sbe determined.
00 S XII. Genetic analysis, animal studies c The sequences make available information and reagents useful for determination of the chromosomal mapping, disease marker correlation, and isolation and determination of the genetic structure of the respective genes. Intron/exon structure will be determined, and transgenic and deletion animals will be prepared.
See, Goodnow (1992) "Transgenic Animals" in Roitt (ed.) Encyclopedia of Immunoloav, Academic Press, San Diego, pp. 1502- 1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989) cence 244:1288; Robertson (ed. 1987) Teratocarcinomas and Embryonic Stem Cells: A Practical Anoroach, IRL Press, Oxford; Rosenberg (1992) J.
Clinical OncolocV 10:180-199; and Cournoyer and Caskey (1993) Ann. Rev. Immunol. 11:297-329.
To determine function of OX2-OX2R interaction in vitro and in vivo, an adenovirus construct was prepared that produced in soluble form, the extracellular region of OX2RHl fused to human IgG. A control construct was prepared that produced in soluble form, only the human IgG backbone. In the first instance, supernatants containing these fusion proteins were produced by cellular infection in vitro to test whether the OX2R fusion protein had biological function on the basis of binding to normally expressed mouse OX2. In the first series of studies, tissue sections of mouse spleen from normal as well as OX2-gene knockout (KO) mice, were prepared and OX2R, or control fusion proteins were added, and these reagents detected by addition of 00 D an antibody binding to the human Fc portion of the fusion protein, and subsequent immunoperoxidase staining procedures to n reveal binding. Weak binding only of the OX2R fusion protein, and only in normal but not OX2 KO mice, was detected on follicular dendritic cells and endothelial cells. Both cell types are know to express very high levels of the ligand OX2.
Thus, the reagent bound to a physiological form of OX2 and no binding was observed in spleen where the ligand OX2 was absent Sdue to gene targeting.
00 10 B cells are known to express the ligand OX2, but at lower 0 level than on follicular dendritic cells and endothelial cells.
C Immunohistochemistry is not a particularly sensitive technique.
Thus in a second study, the same fusion proteins were applied to isolated splenic leukocytes from normal and OX2 KO mouse and binding to B cells determined by flow cytometric analysis using mAb specific to the B220 molecule on B cells, and the more sensitive detection of the fusion proteins by secondary antibodies to human IgG coupled to phycoerythrin. In this case, all B cells in normal mice were labeled by the OX2R fusion protein but not by the control fusion protein. The interaction of the OX2R fusion protein with B cells was blocked by addition of a mAb called OX90 that is known to bind to the part of the mouse OX2 molecule that interacts with the OX2R. In addition, no binding above the background level seen with the control fusion protein was observed when OX2R fusion protein was added to B cells from OX2 KO mice.
The conclusions of these studies were: that the OX2R fusion protein was biologically active and bound to OX2 on hematopoietic and non-hematopoietic cells; and that the major ligand bound by OX2R is indeed the identified ligand OX2, on the basis of anti-OX2 (OX90) binding inhibition and lack of detectable binding to B cells from OX2 KO mice. These data cannot exclude the possibility that there are other ligands bound by the OX2R in addition to the known ligand OX2, as even flow cytometry had not detected cell-surface molecules expressed at very low level.
00 100 Transgenic mice can be generated by standard methods. Such animals are useful to determine the effects of deletion of the gene, in specific tissues, or completely throughout the organism.
Such may provide interesting insight into development of the animal or particular tissues in various stages. Moreover, the c- effect on various responses to biological stress can be evaluated. See, Hogan, et al. (1995) Manipulatin the 1 iMouse Embryo: A Laboratory Manual (2d ed.) Cold Spring Harbor C' Laboratory Press. Likewise, deletion mice, knock out mice 00 C 10 may be generated.
0 These animals will be subject to animal models to study the function of the genes in vivo. See, Gorczynski, et al.
(1999) J. Immunol 163:1654-1660; Mankoo, et al. (1999) Nature 400:69-73; Gorczynski, et al. (1999) Transplant. Proc. 31:577- 578; and Gorczynski L, et al. (1999) J. Immunol. 162:774-781. Of particular interest will be the roles of macrophages or other myeloid cell populations, in the blood, lymphoid tissues, or solid organs, including the microglia in the nervous system.
Tests of susceptibility to infection, autoimmune inflammation, and neural degeneration are indicated. Both antagonist and agonists will be useful reagents in the in vitro or in vivo models described or made available.
XIII. Screening for Substances Likely to Have Therapeutic Value The biological effect of the OX2R/OX2 interaction can be investigated by using antibody reactive with OX2R (an experimental substitute for OX2) to crosslink OX2R molecules in the macrophage cell surface membrane, and looking for changes in, nitric oxide production or phosphorylation of signaling proteins.
The effects of perturbing the OX2R/OX2 interaction can be tested using macrophages carrying OX2R, by exposing them to a cross-linking binding partner such as a mAb for OX2R OX102) or a recombinant multivalent version of OX2 in the presence and absence of the candidate substance. Comparing activity of the macrophages nitric oxide production or 101 00 C phosphorylation of signalling proteins) in the presence or absence of the candidate compound will indicate whether the candidate substance has a modulatory effect inhibition or O enhancement).
Candidate substances can also be tested in well established models of diseases in which macrophages are involved in the pathology of the disease, such as autoimmunity. For instance, c established models such as experimental allergic O encephalomyelitis can be used, as described and exemplified 00 10 above, with the models exhibiting accelerated onset of the Sautoimmune disease experimental autoimmune encephalomyelitis (EAE) in mice. OX2R mimetics may be advantageous in chronic conditions such as chronic granulomatosis. Combinations of agonists or antagonists of the OX2/OX2R signaling may be combined with existing therapeutics for such conditions. See Physicians' Desk Reference Medical Economics Co, Montvale, NJ.
Analyses such as the above will indicate ways in which perturbation of the OX2R/OX2 interaction may be therapeutically beneficial. For example, the invention provides the means to make recombinant versions of OX2RH which can be used in conjunction with the available OX2 proteins to screen for possible pharmacological reagents which block the interaction.
The availability of interacting proteins provides the means for high throughput small molecule screening programs, as are used in the pharmacology industry. See, meetings on High Throughput Screening, International Business Communications, Southborough, MA 01772-1749. Interactions through the cytoplasmic region of OX2RH are likely therapeutic targets and knowledge of the sequence and its interactions provide a means to develop pharmacological reagents through similar screening methods.
XIV. Structure activity relationship Information on the criticality of particular residues is determined using standard procedures and analysis. Standard mutagenesis analysis is performed, by generating many 102 00 O at the different variants at determined positions, at the positions identified above, and evaluating biological activities of the variants. This may be performed to the extent of O determining positions which modify activity, or to focus on specific positions to determine the residues which can be substituted to either retain, block, or modulate biological activity.
SAlternatively, analysis of natural variants can indicate c what positions tolerate natural mutations. This may result from 00 populational analysis of variation among individuals, or across Sstrains or species. Samples from selected individuals are analyzed, by PCR analysis and sequencing. This allows evaluation of population polymorphisms.
All citations herein are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited by the terms of the appended claims, along with the full scope of equivalents to which such clai-ns are entitled; and the invention is not to be limited by the specific embodiments that have been presented herein by way of example.

权利要求:
Claims (11)
[1] 1. A composition of matter selected from: O al) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 2; a2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to Ssegments of SEQ ID NO: 2; a3) a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 2; a4) a fusion polypeptide comprising rat OX2RH1 sequence; bl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 4; b2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 4; b3) a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 4; b4) a fusion polypeptide comprising human OX2RH1 sequence; cl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 6; c2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 6; c3) a natural sequence rodent OX2RH1 polypeptide comprising mature SEQ ID NO: 6; c4) a fusion polypeptide comprising mouse OX2RH1 sequence; 104 00 Sdl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping Ssegments of at least four amino acids identical to 0 segments of SEQ ID NO: 8; d2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to M segments of SEQ ID NO: 8; d3) a natural sequence rodent OX2RH1 polypeptide comprising 00 10 mature SEQ ID NO: 8; d4) a fusion polypeptide comprising human OX2RH2 sequence; el) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: e2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: e3) a natural sequence rodent OX2RH2 polypeptide comprising mature SEQ ID NO: e4) a fusion polypeptide comprising mouse OX2RH2 sequence; fl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 12; f2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 12; f3) a natural sequence rodent OX2RH3 comprising mature SEQ ID NO: 12; f4) a fusion polypeptide comprising mouse OX2RH3 sequence; gl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping 105 00 D segments of at least four amino acids identical to segments of SEQ ID NO: Sg2) a substantially pure or recombinant polypeptide IND comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: g3) a natural sequence primate OX2RH1.2 polypeptide c comprising mature SEQ ID NO: g4) a fusion polypeptide comprising primate OX2RH1.2 0 0 10 sequence; Shl) a substantially pure or recombinant polypeptide comprising at least three distinct nonoverlapping segments of at least four amino acids identical to segments of SEQ ID NO: 23; h2) a substantially pure or recombinant polypeptide comprising at least two distinct nonoverlapping segments of at least five amino acids identical to segments of SEQ ID NO: 23; h3) a natural sequence rodent OX2RH4 polypeptide comprising mature SEQ ID NO: 23; or h4) a fusion polypeptide comprising mouse OX2RH4 sequence.
[2] 2. A binding compound comprising an antigen binding site from an antibody, which specifically binds to a natural OX2RH polypeptide of Claim 1, wherein: a) said binding compound is in a container; b) said OX2RH polypeptide is from a rodent or primate; c) said binding compound is an Fv, Fab, or Fab2 fragment; d) said binding compound is conjugated to another chemical moiety; or e) said antibody: i) is raised against a peptide sequence of a mature polypeptide of Tables 1-3; ii) is raised against a mature OX2RH; iii) is raised to a purified mammalian OX2RH; iv) is immunoselected; 00 cZ O c 00 10 O o 0D 0q v) is a polyclonal antibody; vi) binds to a denatured OX2RH; vii) exhibits a Kd to antigen of at least 30 M; viii) is attached to a solid substrate, including a bead or plastic membrane; ix) is in a sterile composition; or x) is detectably labeled, including a fluorescent label.
[3] 3. An isolated or recombinant nucleic acid encoding said OX2RH polypeptide of Claim 1, wherein said: a) OX2RH is from a mammal; or b) said nucleic acid: i) encodes an antigenic peptide sequence of Tables 1-
[4] 4. 3; ii) encodes a plurality of antigenic peptide sequences of Tables 1-3; iii) exhibits identity over at least thirteen nucleotides to a natural cDNA encoding said segment; iv) is an expression vector; v) further comprises an origin of replication; vi) is from a natural source; vii) comprises a detectable label; viii) comprises synthetic nucleotide sequence; ix) is less than 6 kb, preferably less than 3 kb; x) is from a primate or rodent; xi) comprises a natural full length coding sequence; xii) is a hybridization probe for a gene encoding said OX2RH; xiii) further encodes DAP12 or DAP10; or xiv) is a PCR primer, PCR product, or mutagenesis primer.
[5] A nucleic acid which: 107 00 a) hybridizes under wash conditions of 30 minutes at 400 C C and less than 2M salt to the coding portion of SEQ ID SNO: 1, 3, 5, 7, 9, 11, 19, or 22; or O b) exhibits identity over a stretch of at least about nucleotides to a primate or rodent OX2RH cDNA. A method of modulating physiology or development of a cell or tissue culture cells comprising contacting said cell with San agonist or antagonist of a mammalian OX2RH. 00
[6] 6. The method of Claim 5, wherein said: a) modulating physiology is: i) enhancing meloid function; or ii) enhancing immunity; b) agonist or antagonist attentuates OX2 mediated signaling to said cell; or c) said antagonist is: i) an antibody to said OX2RH; ii) a soluble OX2RH construct; iii) a soluble OX2RH-Ig fusion; or iv) an OX2R antisense nucleic acid.
[7] 7. The method of Claim 6, wherein: a) said modulating of physiology is enhancement of meloid cell function in vitro, and said antagonist is an OX2 mutein; or b) said modulating of physiology is enhancement of immunity in an animal being systemically treated with said antagonist.
[8] 8. A method for identification of a non-OX2 ligand for an OX2R, said method comprising screening a library of genes from an OX2 knock out mouse for binding to an OX2R-Ig fusion protein, and identifying genes which bind to said fusion protein. 00 0 SEQUENCE SUBMISSION SEQ ID NO: 1 is rodent OXR nucleotide sequence. SEQ ID NO: 1 is rodent OX2R nucleotide sequence. Z SEQ ID NO: 2 is rodent OX2R homolog 1 nucleoypep tide sequence. SEQ ID NO: 3 is primate OX2R homolog 1 nucleotide sequence. S SEQ ID NO: 5 is rodent OX2R homolog 1 nucleotide sequence. SEQ ID NO: 6 is rodent OX2R homolog 1 polypeptide sequence. SEQ ID NO: 7 is primate OX2R homolog 2 nucleic acid sequence. SEQ ID NO: 8 is primate OX2R homolog 2 polypeptide sequence. SEQ ID NO: 9 is rodent OX2R homolog 2 nucleic acid sequence. SEQ ID NO: 10 is rodent OX2R homolog 2 polypeptide sequence. SSEQ ID NO: 11 is rodent OX2R homolog 3 nucleic acid sequence. SSEQ ID NO: 12 is rodent OX2R homolog 3 polypeptide sequence. C SEQ ID NO: 13 is rodent OX2R polypeptide encoding sequence. 00 SEQ ID NO: 14 is primate OX2R homolog 1 polypeptide encoding sequence. SSEQ ID NO: 15 is rodent OX2R hiomolog 1 polypeptide encoding sequence. SEQ ID NO: 16 is primate OX2R homolog 2 polypeptide encoding sequence. SEQ ID NO 17 is rodent OX2R homolog 2 polypeptide encoding sequence. SEQ ID NO: 18 is rodent OX2R homolog 3 polypeptide encoding sequence. SEQ ID NO: 19 is primate OX2R homolog 1.2 nucleotide sequence. SEQ ID NO: 20 is primate OX2R homolog 1.2 polypeptide sequence. SEQ ID NO: 21 is primate OX2R homolog 1.2 polypeptide encoding sequence. SEQ ID NO: 22 is rodent OX2R homolog 4 nucleic acid sequence. SEQ ID NO: 23 is rodent OX2R homolog 4 polypeptide sequence. SEQ ID NO: 24 is rodent OX2R homolog 4 polypeptide encoding sequence. <110> Medical Research Council Schering Corporation <120> OX2 Receptor Homologs <130> DX01052K1 PCT <140> PCT/US 00/12998 <141> 2000-05-11 <150> GB 9911123.9 <151> 1999-05-13 <150> GB 9925989.7 <151> 1999-11-03 <160> 24 <170> PatentIn Ver. <210> 1 <211> 1574 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:rodent; surmised rattus rattus <220> <221> CDS <222> (9i)..(1071) <220> <221> mat peptide <222> (162)..(1071) <400> 1 agcggaggga toctggtcat. ggtoaccgct gotcccctac otgtgaagag aaagagcaoo gagtgagccg otgaaaacca gaaaaccgaa atg ctc tgc ttt tgg aga act tot Met Leu Cys Phe Trp Arg Thr Ser cac His tgt Cys 1 gaa Glu tgc Cys ata Ile aca Thr gc Al a ctc Leu aat Asn aco Thr gg a Gly 145 4ta Jal cct Pro gtt Val tgo Cys acc Thr agg Arg tcc Ser cag Gin ttc Phe cac His 130 aaa Lys gca Ala gat Asp aao Asn Oct Pro ctc Leu gag Giu aca Thr cat His caa Gin 115 ttt Phe Cct Pro gt a Val1 aag Lys act Thr tct. Ser aga Arg ac c Thr cct Pro gaa Glu 100 aac Asn ca Pro got Al a ctc LeuI aat Asn 5 aca Thr att. Ilie gga. Gly cat His gac Asp ggg Gly atc Ile ggg *Gly gog *Ala :tg eu :aa 31n gtg J7a1 tca Ser cag Gin gaa Glu 70 cto Leu cgt Arg tat Tyr gaa Glu o ag Gln 150 ato Ilie -10 aca Thr ttt Phe ctg Leu Oct Pro 55 ago Ser got Al a tao Tyr gao aat Asn 135 atc Ile tgg TrpC atg MetC gta Val aoa Thr 40 too Ser aa 0 Asn oct Pro tca Ser oto pLeu 120 aga Arg tot Ser 9gg 'ly cag 31n cag Gln 25 aaa Lys tgc Cys tgo Cys gao Asp tgt Cys 105 caa Gin act Thr tgg Trp gt 0 Val1 aac Asn 10 atg Met gta Val1 ata Ile tog Ser Ott Leu 90 gat Asp gtg *Val goa *Ala acc Thi tto gog Phe Ala -5 aat tca Asn Ser ggt aaa Giy Lys ata tta Ile Leu ata too Ile Ser gao aga Asp Arg 75 cag ato Gin Ile ata gca Ile Ala ctg gtg Leu Val gtt tgt Vai Cys 140 oca gat Pro Asp 155 ;ct kl a toa Ser aag Lys ata Ile tao Tyr ago S er agt. Ser gta Val coo Pro 125 gag Gil. gg9 G1') 3ag toa agt Glu act Thr got Ala aoa Thr aaa Lys ato Ile goa Al a cot Pro 110 cot Pro gog IAla gat Asp Ser atg otg Leu tgg Trp goa Al a aco Thr gtg Val gac Asp gaa Git att Ile tgt cy Ser -i1 aca Thr oto Leu aca Thr gao Asp tgg Trp goo Ala ggg Gly gta Val goa Aia gto 3Val 160 114 162 210 258 306 354 402 450 498 546 594 642 690 got aag aat gaa tca cac ago aat ggo aco gtg act gtc cgg ago aca Ala Lys Asn Glu Ser His Ser Asn Gly Thr Val Thr Val Arg ser Thr 00 00 tgc cac tgg Cys His Trp cac ttg aca His Leu Thr 2.95 ggt gac caa Gly Asp Gin gag Giu 180 act Thr agc cac gtg Ser His Val gtg ttc tgt Val. Phe Cys 175 gtt gtc tct Vai Val Ser 190 ggt aga ggg Gly Arg Giy cca tct att Pro Ser Ile ggt aac cag Gly Asn Gin tct Ser 200 tac Tyr tct ata gaa Ser Ile Giu tta tta gga Leu Leu Giy 210 att att Ile Ile t ca Ser 215 gga Gly att caa tac Ile Gin Tyr atc Ile 220 ttg Leu ttg atc atc Leu Ile Ile tgc att tgt Cys Ile Cys ctt Leu 235 gct Aila aaa atc agt Lys Ile Ser 738 786 834 882 930 978 1026 1.071. aga aaa tgt Arg Lys Cys a aa Lys 245 cag Gin cca aaa tcg Pro Lys Ser gga Giy 250 tac Tyr act cca gat Thr Pro Asp att gag Ile Giu 255 gag gat gaa Giu Asp Giu ctc tat gat Leu Tyr Asp 275 aaa gtc aat Lys Val Asn 290 atg Met 260 act Thr ccg tat gct Pro Tyr Ala agc Ser 265 gag Glu aca gag aag Thr Giu Lys agc aat cca Ser Asn Pro 270 tca caa ggc Ser Gin Gly gtg acc acg Vai Thr Thr acg Thr 280 ctt Leu gca cac cca Ala His Pro geg Ala 285 atg gga atc Met Gly Ile ggc aca gac tgt Gly Thr Asp Cys act ttg tca gcc Thr Leu Ser Aia 300 tagaaccaag ctccaatgga tttgccttta taggaagaac gtgcaataca tactaaagca tatggtatgt ttatcaagac tgagtattgg gaaaagaagt gggaaattag agttcttcta tgatttaatt atgatgaaaa tttcatgtga gtgtgccttt atctctgtca agatgttttt caagagacat ctcttctgaa tcattggaag attacaaaga c tgagt t tcc gttcttccaa atatgtttgt aaaactcact ata cataattact gttcttagaa tttggaatct aagcacattg tcaagaaata aaaagaaaat ttacaaatgt ggcgttccag gcttttcttt agcacaaatg ttgctgctac ttatggtaaa actgcagaag ccctgtgtat gtatatatgc atttatgaaa ctttaaactt ttctaatgga ctgttaattc a tat caa at t gaacaatcat acgacatgat acacatctga gctaataaag
11232.
11292. 1.252. 213112 2.371. 2.431. 2.492. 1.551. 2.574 <210> 2 <2112> 327 '<212> PRT <z213> Unknown <400> 2 M ,et Leu Cys Phe Trp Arg Thr Ser His Val Ala Val Leu Leu Ile Trp Gly Gin Gin Lys Cys Cys Asp Cys 105 Gin Thr Trp Giy Ser 185 Leu Ile Ile Ser Ser 265 Val1 Asn Met Val Ile Ser Leu Asp Val1 Aila Thr Thr 170 Val Ser Gin Cys Gly 250 Tyr Phe Asn Giy Ile Ile Asp Gin Ile Leu Val Pro 155 Vali Val1 Ile Tyr Leu 235 Al a Thr Ala Ser Lys Leu Ser Arg Ile Ala Val1 Cys 140 Asp Thr Phe Glu Ile 220 Leu Thr Glu Al a Ser Lys Ile Tyr Ser Ser Val Pro 125 Giu Gly Vai Cys Leu 205 Ile Lys Pro Lys Giu Thr Al a 30 Thr Lys Ile Al a Pro 110 Pro Ala Asp Arg Vai 190 Gly Pro Ile Asp Ser 270 Ser Met 15 Leu Trp Al a Thr Val1 95 Asp Giu Ile Cys Ser 175 Val1 Arg Ser Ser I le 255 Asn Ser Cys Thr Glu Leu Cys Thr Ile Asp Thr 65 Trp Ala 80 Ala Leu Gly Asn Val Thr Ala Gly 145 Val Ala 160 Thr Cys Ser His Gly Gly Ile Ile 225 Gly Cys 240 Glu Giu Pro Leu Pro Val Cys Thr 50 Arg Ser Gin Phe His 130 Lys Lys His Leu Asp 210 Ile Arg Asp Tyr Asp Asn Pro 35 Leu Giu Thr His Gin 115 Phe Pro Asn Trp Thr 195 Gin Leu Lys Giu Asp 275 Lys Thr Ser Arg Thr Pro Giu 100 Asn Pro Aia Giu Giu 180 Thr Leu Ile Cys Met 260 Thr Asn Thr Ile Gly His Asp Gly Ile Gly Ala Ser 165 Gin Gly Leu Ile Lys 245 Gin Val1 Gin Val Ser Gin Glu Leu Arg Tyr Giu Gin 150 His Ser Asn Giy Ile 230 Leu Pro Thr Thr Phe Leu Pro Ser Ala Tyr Asp Asn 135 Ile Ser His Gin Ser 215 Gly Pro Tyr Thr Met Val Thr Ser Asn Pro Ser Leu 120 Arg Ser Asn Val Ser 200 Tyr Cys Lys Ala Thr 280 Giu Ala His Pro Ala Ser Gin Gly Lys Val Asn Gly Thr Asp Cys Leu 285 290 295 Thr Leu Ser Ala Met Gly Ile 300 <210> 3 <211> 1604 <212> DNA <213> Unknown 00 <220> <223> <220> Description of homo sapiens Unknown organism:primate; surmised <221>. CDS <222> (217)..(1101) <220> <221> mat_peptide <222> (295)..(1101) <400> 3 cagagaaaag cttctgttcg tccaagttac taaccaggct aaaccacata gacgtgaagg aaggggctag aaggaaggga gtgccccact gttgatgggg taagaggatc ctgtactgag 120 aagttgacca gagagggtct caccatgcgc acagttcctt ctgtaccagt gtggaggaaa 180 agtactgagt gaagggcaga aaaagagaaa acagaa atg ctc tgc cct tgg aga Met Leu Cys Pro Trp Arg 234 282 act gct Thr Ala aac cta ggg Asn Leu Gly cta Leu ctg ttg att ttg act atc ttc tta gtg gcc Leu Leu Ile Leu Thr Ile Phe Leu Val Ala gaa gcg gag Giu Ala Giu agc aag gag Ser Lys Glu ggt gct gct caa cca aac aac tca tta atg ctg caa act Gly Ala Ala Gin Pro Asn Asn Ser Leu Met Leu Gin Thr -1 1 5 aat cat gct tta Asn His Ala Leu tca agc agt tta Ser Ser Ser Leu tgt Cys atg gat gaa Met Asp Glu aaa cag Lys Gin att aca. cag aac Ile Thr Gin Asri tcg aaa gta ctc Ser Lys Val Leu gaa gtt aac act Giu Val Asn Thr 426 474 tca Ser tgg cct gta aag Trp Pro Val Lys gct aca aat gct Ala Thr Asn Ala ctt tgt tgc cct Leu Cys Cys Pro cct Pro atc gca tta aga aat ttg atc ata ata Ile Ala Leu Arg Asn Leu Ile Ile Ile aca Thr 70 tgg gaa at~a atc Tr Giu Ile Ile ctg aga Leu Arg 522 570 ggc cag cct tcc tgc aca aaa gcc tac aag aaa gaa aca aat gag acc Gly Gin Pro Ser Cys Thr Lys Ala Tyr Lys Lys Giu Thr Asn Glu Thr 6 00 80 85 aag gaa acc aac tgt act gat gag aga ata acc tgg gtc tcc aga cct 618 Lys Glu Thr Asn Cys Thr Asp Giu Arg Ile Thr Trp Val Ser Arg Pro 100 105 gat cag aat tcg gac ctt cag att cgt acc gtg gcc atc act cat gac 666 IND Asp Gin Asn Ser Asp Leu Gin Ile Arg Thr Val Ala Ile Thr His Asp 110 115 120 ggg tat tac aga tgc ata atg gta aca cct gat ggg aat ttc cat cgt 714 Gly Tyr Tyr Arg Cys Ile Met Val Thr Pro Asp Gly Asn Phe His Arg 125 130 135 140 gga tat cac ctc caa gtg tta gtt aca cct gaa gtg acc ctg ttt caa 762 Gly Tyr His Leu Gin Val Leu Vai Thr Pro Giu Val Thr Leu Phe Gin 00 145 150 155 aac agg aat aga act gca gta tgc aag gca gtt gca ggg aag cca gct 810 Asn Arg Asn Arg Thr Ala Val Cys Lys Ala Val Ala Gly Lys Pro Ala 160 165 170 gcg cat atc tcc tgg atc cca gag ggc gat tgt gcc act aag caa gaa 858 Ala His Ile Ser Trp lie Pro Glu Gly Asp Cys Ala Thr Lys Gin Glu 175 180 185 tac tgg agc aat ggc aca gtg act gtt aag agt aca tgc cac tgg gag 906 Tyr Trp Ser Asn Gly Thr Val Thr Val Lys Ser Thr Cys His Trp Glu 190 195 200 gtc cac aat gtg tct acc gtg acc tgc cac gtc tcc cat ttg act ggc 954 Val His Asn Val Ser Thr Val Thr Cys His Val Ser His Leu Thr Gly 205 210 215 220 aac aag agt ctg tac ata gag cta ctt cct gtt cca ggt gcc aaa aaa 1002 Asn Lys Ser Leu Tyr Ile Giu Leu Leu Pro Val Pro Gly Ala Lys Lys 225 230 235 atc agc aaa att ata tat tcc ata tat cat cct tac tat tat tat tta 1050 Ile Ser Lys Ile Ile Tyr Ser Ile Tyr His Pro Tyr Tyr Tyr Tyr Leu 240 245 250 gac cat cgt ggg att cat ttg gtt gtt gaa agt caa tgg ctg cag aaa 1098 Asp His Arg Gly Ile His Leu Val Val Giu Ser Gin Trp Leu Gin Lys 255 260 265 ata taaattgaat aaaacagaat ctactccagt tgttgaggag gatgaaatgc 1151 Ile agccctatgc cagctacaca gagaagaaca atcctctcta tgatactaca aacaaggtga 1211 aggcatctga ggcattacaa agtgaagttg acacagacct ccatacttta taagttgttg 1271 gactctagta ccaagaaaca acaacaaacg agatacatta taattactgt ctgattttct 1331 tacagttcta gaatgaagac ttatattgaa attaggtttt ccaaggttct tagaagacat 1391 tttaatggat tctcattcat acccttgtat aattggaatt tttgattctt agctgctacc 1451 7 00 agctagttct ctgaagaact gatgttatta caaagaaaat acatgcccat gaccaaatat 1511 tcaaattgtg caggacagta aataatgaaa accaaatttc ctcaagaaat aactgaagaa 1571 ggagcaagtg tgaacagttt cttgtgtatc ctt 1604 <210> 4 <211> 295 <212> PRT <213> Unknown <400> 4 Met Leu Cys Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu -20 00 Thr Ile Phe Leu Val Ala Glu Ala Giu Gly Ala Ala Gin Pro Asn Asn -5 -1 1 Ser Leu Met Leu Gin Thr Ser Lys Glu Asn His Ala Leu Ala Ser Ser 15 Ser Leu Cys Met Asp Glu Lys Gin Ile Thr Gin Asn Tyr Ser Lys Val 30 Leu Ala Giu Val Asn Thr Ser Trp Pro Val Lys Met Ala Thr Asn Ala 45 Val Leu Cys Cys Pro Pro Ile Ala Leu Arg Asn Leu Ile Ile Ile Thr 60 65 Trp Glu Ile Ile Leu Arg Gly Gin Pro Ser Cys Thr Lys Ala Tyr Lys 80 Lys Giu Thr Asn Glu Thr Lys Giu Thr Asn Cys Thr Asp Giu Arg Ile 95 100 Thr Trp Val Ser Arg Pro Asp Gin Asn Ser Asp Leu Gin Ile Arg Thr 105 110 115 Val Ala Ilie Thr His Asp Giy Tyr Tyr Arg Cys Ile Met Vai Thr Pro 120 125 130 Asp Giy Asn Phe His Arg Gly Tyr His Leu Gin Val Leu Val Thr Pro 135 140 145 150 Giu Val Thr Leu Phe Gin Asn Arg Asn Arg Thr Ala Val Cys Lys Ala 155 160 165 Val Ala Gly Lys Pro Ala Aia His Ile Ser Trp Ile Pro Giu Giy Asp 170 175 180 Cys Ala Thr Lys Gin Glu Tyr Trp Ser Asn Gly Thr Val Thr Val Lys 185 190 195 Ser Thr Cys His Trp Glu Vai His Asn Val Ser Thr Val Thr Cys His 200 205 210 Val Ser His Leu Thr Gly Aso Lys Ser Leu Tyr Ile Glu Leu Leu Pro Val Pro Gly Ala Lys Lys Ile Ser Lys Ile Ile Tyr Ser Ile Tyr His 235 240 245 Pro Tyr Tyr Tyr Tyr Leu. Asp His Arg Gly Ile His Leu Val Val Giu 250 255 260 Ser Gin Trp Leu. Gin Lys Ile 265 <210> <211> <212> <213> <220> <223> 1490 DNA Unknown Description of Unknown Organism:rodent; surmised mus musculus <220> <22i> CDS <222> (10)..(987) <220> <221> matypeptide <222> (85)..(987) <400> aaaaccgaa atg ttt tgc ttt tgg aga act tct gcc cta gca gtg ctc tta 51 Met Phe Cys Phe Trp Arg Thr Ser Ala Leu Ala Vai Leu Leu. ata tgg Ile Trp ggg gtc ttt gtg Gly Val Phe Val gct Ala -5 ggg tca agt tgt act gat aag aat Gly Ser Ser Cys Thr Asp Lys Asn -1 1 aca aca cag aac aac agt tca tct cct Thr Thr Gin Asn Asn Ser Ser Ser Pro aca caa gtg aac Thr Gin Val Asn act aca Thr Thr gtg tct gta Vai Ser Val ata ggt aca aag Ile Gly Thr Lys gct Ala ctg ctc tgc tgc Leu. Leu Cys Cys ttt tct att Phe Ser Ile ctc aga ggc Leu Arg Gly cca ctg aca aaa gca gta tta Pro Leu Thr Lys Ala Val Leu aca tgg ata ata Thr Trp Ile Ile aag Lys ctg cca Leu Pro tcc tgc aca ata Ser Cys Thr Ile gca Ala tac aaa gta gat Tyr Lys Val Asp aag acc aat gaa Lys Thr Asn Giu agc tgc ttg ggc Ser Cys Leu Gly agg Arg aac atc acc tgg Asn Ile Thr Trp tcc aca cct gac Ser Thr Pro Asp agt cct gaa ctt cag atc agt gca gtg acc ctc cag cat gag ggg act Ser tac Tyr Leu gag Giu Gin aca Thr Ser aca Thr Al a cct Pro Leu aat Asn Glu Gly Thr 100 aaa aac tat Lys Asn Tyr 115 105 gac Asp aac Asn atc Ile 150 agc Ser aat Asn tct Ser tat Tyr tgc Cya 230 aaE Ly~ gcl Al. gt Va. ctc c Leu G 1 aga t Arg S 135 tct t Ser 'I aat Asn gtg1 Val ctg Leu att Ile 215 att Ile tta 3Leu tagc a Ser g gag 1. Giu aa in 20 .ct er gg rp ~gc 'ly tct Ser tcc Ser 200 cca Pro tgt CyE ga~ Gli ta gc Al gtg c Val L gca Ala tct Ser act Thr gat Asp 185 ata Ile tac Tyr ctt 3Leu a gct ui Ala t aca r Thr 265 a ttt a Phe :tg ~eu t C l ca ro gtg Val1 1.70 gig Val1 gaa Glu atc Ile tt s Let act Th 251 ga~ Gli cc Pr gtg C Val P tgt Cys C I gat S Asp 155 act Thr tcc Ser ctg Leu ata Ile aaa 1Lys 235 tca c- Ser 0 g aag u Lys a gta o Val cc ci gaa gta acc tac ttt cca ~ro ~ag lu 40 'iy jt c Iai tgc Cys agt Ser cca Pro 220 atc Ile gct Al ag Se: tc 1 Se Pro 125 gca Al a gac Asp agg Arg att Ile aga Arg 205 tca Sex agt Sex. att IIl~ caal r* As a ca. r Gl: Glu V atg g Met P tgt S Cys agc Ser gtc Val 190 ggt Gly att Ile ggC Gly :gag a Giu t cca ni Pro 270 a ggc n Gly 'al rca ~la ~tc Tal ica rhr 175 tct Ser ggt Gi atc IlE tic Ph ga Gl 25 ct Le ga Gl Thr T ggc a Gly L 1. act a Thr T 160 tgc C Cys I cat His aac Asn att Ile *aga Arg 240 g gat u Asp 5 c tat u Tyr a gic u Val yr ag ~ys ~cg ~hr ~ac is ttg Leu caa Gin ttg LeL 225 aaE Ly~ ga~ Cii ga As' aa As Phe P 130 cct g Pro A agt g Ser G tgg
[9] 9 Trp C act Thr tca Ser 210 atc Ile Stgc Cys a atg :i Met t act p Thr t ggc n Cly ro ct l1a aa lu rag 1u ~gt 3i1Y 1.95 tta Lei. atc Il aa Ly cal Gi gt Va 27 ac Tli gag a Glu L gca c Ala G tca c Ser I- cag Gin 180 aac Asn cga 1Arg ata a Ile a ttg s Leu g cct n Pro 260 g act 1 Thr a gac *r Asp aa lys ag in :ac Iis qaac %sn caa Gin cca Pro gga Gly cca Pro 245 tat Tyr aag Lys tgc Cys 483 531 579 627 675 723 771 819 867 915 963 280 285 290 ctt act ttg tcg gcc att gga atc tagaaccaag aaaaaagaag tcaagagaca Leu Thr Leu Ser Ala Ile Cly Ile 295 300 tcataattac tgcittgctt tctttaaaai tcgacaatgg aaggactact tggaaattag ctcttccaaa gciattaaaa agcacaaatg ttctaatgaa aitgcattta aattctatca 1017 1077 1137 00 ttggaagttt ggaatctctg ctgctacctg ttaattttag gaagaactga tttaattatt. 1197 acaaagaaag cacatggtta tggtgaaata tcaagttgtg caataaagta tgatgaaaac 1257 tgagtttcct caagaaataa ctgcaggagg aacaatcatc actaaagaat ttcatgtgag 1317 IND ttcttacaaa aaaattccta tgtatacatg actatggtat gtgtgtccaa ttacatgttt 1377 atttacaaat gtgtatatat gcacacattt. gcttttcagg acatctcctt gtaaaaaaca 1437 cactggagtt ttggatttat aaaagcttat aaagtgagca ttggagatat ttt 1490 <210> 6 <211> 326 00 <212> PRT 00 <213> Unknown (N2~ <400> 6 Met Phe Cys Phe Trp Arg Thr Ser Ala Leu Ala Val Leu Leu Ile Trp -20 -15 Gly Val Phe Vai Ala Gly Ser Ser Cys Thr Asp Lys Asn Gin Thr Thr -1 1 Gin Asn Asn Ser Ser Ser Pro Leu Thr Gin Val Asn Thr Thr Val Ser 15 Val Gin Ile Gly Thr Lys Ala Leu Leu Cys Cys Phe Ser Ile Pro Leu 30 Thr Lys Ala Val Leu Ilie Thr Trp Ile Ile Lys Leu Arg Gly Leu Pro 45 50 Ser Cys Thr Ile Ala Tyr Lys Val Asp Thr Lys Thr Asn Giu Thr Ser 65 Cys Leu Gly Arg Asn Ile Thr Trp Ala Ser Thr Pro Asp His Ser Pro 80 Giu Leu Gin lie Ser Ala Val Thr Leu Gin His Glu Gly Thr Tyr Thr 95 100 Cys Glu Thr Val Thr Pro Giu Gly Asn Phe Giu Lys Asn Tyr Asp Leu 105 110 115 Gin Val Leu Val Pro Pro Giu Val Thr Tyr Phe Pro Giu Lys Asn Arg 120 125 130 135 Ser Ala Val Cys Glu Ala Met Ala Gly Lys Pro Ala Ala Gin Ile Ser 140 145 150 Trp Ser Pro Asp Gly Asp Cys Val Thr Thr Ser Giu Ser His Ser Asn 155 160 165 Gly Thr Val Thr Val Arg Ser Thr Cys His Trp Glu Gin Asn Asn Val 170 175 180 Ser Asp Val Ser Cys Ile Vai Ser His Leu Thr Gly Asn Gin Ser Leu 00 185 190 195 Ser Ile Giu Leu Ser Arg Giy Gly Asn Gin Ser Leu Arg Pro Tyr Ile K1200 205 210 215 Pro Tyr Ile Ile Pro Ser Ile Ile Ile Leu Ile Ile Ile Gly Cys Ile 220 225 230 Cys Leu Leu Lys Ile Ser Giy Phe Arg Lys Cys Lys Leu Pro Lys Leu 235 240 245 Giu Ala Thr Ser Ala Ile Giu Giu Asp Giu Met Gin Pro Tyr Ala Ser 250 255 260 Tyr Thr Giu Lys Ser Asn Pro Leu Tyr Asp Thr Val Thr Lys Val Giu rl265 270 275 00 Ala Phe Pro Val Ser Gin Gly Giu Vai Asn Giy Thr Asp Cys Leu Thr 280 285 290 295 Leu Ser Ala Ile Giy Ile 300 <210> 7 <211> 1010 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:primate; surmised homo sapiens <220> <221> CDS <222> <400> 7 atg ggt gga aag cag atg aca cag aac tat tca aca att ttt gca gaa 48 Met Gly Gly Lys Gin Met Thr Gin Asn Tyr Ser Thr Ile Phe Ala Giu 1 5 10 ggt aac atit tica cag cct gta ctg atg gat ata aat gct gtg ctt tgt 96 Gly Asn Ile Ser Gin Pro Val Leu Met Asp Ile Asn Ala Val Leu Cys 25 tgc cct cct att gca tta. aga aat ttg atc ata ata aca tgg gaa ata 144 Cys Pro Pro Ile Ala Leu Arg Asn Leu Ile Ile Ile Thr Trp Giu Ile 40 atic ctg aga ggc cag cct tcc tgc aca aaa gcc tac aag aaa gaa aca 192 Ile Leu Arg Gly Gin Pro Ser Cys Thr Lys Ala Tyr Lys Lys Giu Thr 55 aat gag acc aag gaa acc aac tgt act gtt gag aga ata acc tgg gtc 240 Asn Giu Thr Lys Giu Thr Asn Cys Thr Val Giu Arg Ile Thr Trp Val 70 75 tct aga cct gat cag aat tcg gac ctt cag att cgt ccg gtg gac acc 288 Ser Arg Pro act cat gac Thr His Asp Asp Gin Asn Ser Asp Leu 12 Gin Ile Arg Pro Vai Asp Thr 90 9gg Gly 1.00 tat tac aga ggc Tyr Tyr Arg Gly ata Ile 105 gtg gta aca cct Val Val Thr Pro gat ggg aat. Asp Gly Asn 110 ttc cat cgt gga tat cac ctc caa gtg tta gtt aca ccc gaa gtg aac Phe His Arg Gly Tyr His Leu Gin Val. Leu Val. Thr Pro Giu Va). Asn 115 120 125 cta ttt Leu Phe 130 caa agc agg aat Gin Ser Arg Asn ata Ile 135 act gca gta tgc Thr Aia Vai Cys aag gca Lys Ala 140 gtt aca ggg Vai Thr Giy aag Lys 145 cca gct gcc cag Pro Ala Ala Gin at c Ile 150 tcc tgg atc cca Ser Trp Ile Pro gga tct att ctt Giy Ser Ile Leu 432 480 528 act aag caa gaa Thr Lys Gin Giu tac Tyr 165 tgg ggc aat ggc Trp Gly Asn Gly aca Thr 170 gtg acg gtt aag Vai Thr Val Lys agt aca Ser Thr 175 tgc ccc tgg Cys Pro Trp ttg act ggc Leu Thr Gly 195 gag Giu 180 ggc cac aag tct Giy His Lys Ser act Thr 185 gtg acc tgc cat Val Thr Cys His gtc tcc cat Vai Ser His 190 ggt ctc aga Gly Leu Arg 576 624 aac aag agt ctg Asn Lys Ser Leu gta aag ttg aat Va. Lys Leu Asn acc tca Thr Ser 210 gga tct cca gcg Giy Ser Pro Ala tcc tta ctg atc Ser Leu Leu Ile ctt tat gtg aaa Leu Tyr Val Lys 672 720 ctc Leu 225 tct ctt ttt gtg Ser Leu Phe Val att ctg gtc acc Ile Leu Vai Thr aca gga Thr Gly 235 ttt gtt ttc Phe Val Phe ttc Phe 240 cag agg ata aat cat gtc aga aaa gtt Val. Arg Lys Va). Gin Arg Ile Asn His 245 ctt taaagaagaa ggaagggtct Leu 250 tcttttgctt ctcctccttg tctctggact gcaacattgg tgagatgagt gatggtccag 830 cagtgaactt gggccatgga tgatgttaag gatagaagcc actcagtagg atagaagaaa 890 agaaagatgg aagaaggatc ctgggcttga tgaccatgaa gtttccctat aaaccctcaa 950 ccacctattc attgacttct tttgtgttag agtgaataaa attttgttca tgccagtgtt 1010 <210> 8 <211> 250 <212> PRT <213> Unknown <400> 8 Met Giy Gly Lys Gin Met Thr Gin Asn Tyr Ser Thr Ile Phe Ala Giu Gly Asn Ile Ser Gin Pro Val Leu M~ Cys Pro Ile Leu Asn Glu Ser Arg Thr His Phe His Leu Phe 130 Lys Pro 145 Thr Lys Cys Pro Leu Thr Thr Ser 210 Leu Ser 225 Pro Arg Thr Pro Asp Arg 115 Gin Aila Gin Trp Gly 195 Giy Leu Ile Giy Lys Asp Giy 100 Gly Ser Ala Glu Glu 180 Asn Ser Phe Al a Gin Giu Gin Tyr Tyr Arg Gin Tyr 165 Giy Lys Pro Val1 Leu Arg Pro Ser 55 Thr Asn 70 Asn Ser Tyr Arg His Leu Asn Ile 135 Ile Ser 150 Trp Gly His Lys Ser Leu Ala Leu 215 Val Ile 230 Asn 40 Cys Cys Asp Gly Gin 120 Thr Trp Asn Ser Ser 200 S er Leu L L V A I G T V L V 13 let Asp 25 ~eu Ile 'hr Lys 'hr Val *eu Gin 90 le Vai 05 al Leu la Val le Pro ly Thr 170 hr Vai 85 al Lys eu Leu al Thr Ile Ile Al a Giu 75 Ile Val Val Cys Glu 155 Val Thr Leu Ile Thr 235 Asn Ile Tyr Arg Arg Thr Thr Lys 140 Gly Thr Cys Asn Ile 220 Gly Ala Thr Lys Ile Pro Pro Pro 125 Ala Ser Val His Ser 205 Leu Phe Val Trp Lys Thr Val Asp 110 Glu Val Ile Lys Val 190 Gly Tyr Val Leu Giu Giu Trp Asp Giy Val1 Thr Leu Ser 175 Ser Leu Val1 Phe Cys Ile Thr Val1 Thr Asn Asn Gly Al a 160 Thr His Arg Lys Phe 240 Gln Arg Ile Asn His Vai Arg Lys Val Leu 245 23;0 <210> 9 <211> 1085 <212> DNA <213> Unknown <220> <223> Description of Unknown Organism:rodent; surmised mus inusculus <220> <221> CDS 00 222> c~K1 <400> 9 aga ggc cag cct tcc tgc ata atg gcc tac aaa gta gaa aca aag gag 48 Arg Giy Gin Pro Ser Cys Ilie Met Ala Tyr Lys Val Glu Thr Lys Giu 1 510 acc aat gaa acc tgc ttg ggc agg aac atc acc tgg gcc tcc aca cct 96 Thr Asn Giu Thr Cys Leu Gly Arg Asn Ile Thr Trp Ala Ser Thr Pro 25 gac cac att cct gac ctt cag atc agt gcg gtg gcc: ctc cag cat gag 144 Asp His Ilie Pro Asp Leu Gin Ile Ser Ala Vai Ala Leu Gin His Giu 40 00 ggg aat tac tta tgt gag ata aca aca cct gaa ggg aat ttc cat aaa 192 Gly Asn Tyr Leu Cys Giu Ile Thr Thr Pro Giu Gly Asn Phe His Lys 55 gtc tat gac ctc caa gtg ctg gtg ccc cct gaa gta acc tac ttt ctc 240 Vai Tyr Asp Leu Gin Vai Leu Val Pro Pro Giu Val Thr Tyr Phe Leu 70 75 ggg gaa aat aga act gca gtt tgt gag gca atg gca ggc aag cct gct 288 Giy Giu Asn Arg Thr Ala Vai cys Giu Ala Met Ala Gly Lys Pro Ala 90 gca cag atc tct. tgg act cca gat ggg gac tgt gtc: act aag agt gag 336 Ala Gin Ilie Ser Trp Thr Pro Asp Gly Asp Cys Val Thr Lys Ser Glu 100 105 110 tca cac agc aat ggc act gtg act gtc agg agc act tgc cac tgg gag 384 Ser His Ser Asn Giy Thr Val Thr Val Arg Ser Thr cys His Trp, Glu 115 120 125 cag aac aat gtg tct gct gtg tcc tgc att gtc tct cat tcg act ggt 432 Gin Asn Asn Vai Ser Ala Val Ser Cys Ilie Val Ser His Ser Thr Gly 130 135 140 aat cag tct ctg tcc ata gaa ctg agt aga ggt acc acc agc: acc: acc 480 Asn Gin Ser Leu Ser Ile Giu Leu Ser Arg Gly Thr Thr Ser Thr Thr 145 150 155 160 cct tcc ttg ctg acc att ctc tac gtg aaa atg gtc ctt ttg ggg att 528 Pro Ser Leu Leu Thr Ile Leu Tyr Vai Lys Met Vai Leu Leu Giy Ile 165 170 175 att ctt ctt aaa gtg gga ttt gct ttc ttc cag aag aga aat gtt acc 576 Ile Leu Leu Lys Val Gly Phe Ala Phe Phe Gin Lys Arg Asn Vai Thr 180 185 190 aga aca tgaatatcca gatttctgga agctcattag tctgatgaca cataccagaa 632 Arg Thr aacagcattt gtaatcaact ttctcattgg aatccagctt acccgtccct gctgtcttca 692 tgtttgttag acactcacct ccaaattctt aactgagaag ggctcctgtc taaaggaaat 752 atggggacaa attgtggagc atagaccaaa agaaaggcca tccagagact gccccaccta 812 aggacccatc ccatatacag acaccaaaCC cagacactac tgaagatgct gcgaagcgtt 872 tgctgacagg agcctgttat agctgtctcc tgagaggctc agccagagcc tgacaaatac 932 ataggtagat gcttgcagcc aacaactgga ctgagcaaaa aatctccatt ggaggagtta 992 gagaaaggac tgaagagggt gaaagggttt gcagccccat aggaagaaca acaatatcaa 1052 ccaaccagat ctcccagagc tcccagggac taa 1085 <210> <211> 194 <212> PRT <213> Unknown <400> Arg Gly Gin Pro Ser Cys Ile Met Ala Tyr Lys Val Glu Thr Lys Giu 1 5 Thr Asp Gly Val Gly Al a Ser Gin Asn 145 Pro Asn His Asn Tyr Giu Gin His Asn 130 Gin Ser Giu Ile Tyr Asp Asn Ile Ser 115 Asn Ser Leu Thr Pro Leu Leu Arg Ser 100 Asn Val Leu Leu Cys Asp Cys Gin Thr Trp Giy Ser Ser Thr 165 Leu Leu Giu Val 70 Ala Thr Thr Ala Ile 150 Ile Gly Gin Ile 55 Leu Val Pro Val Val 135 Giu Leu %.rg Ile 40 Thr Val Cys Asp Thr 120 Ser Leu Tyr Asn 25 Ser Thr Pro Giu Gly 105 Val Cys Ser Val
[10] 10 Ile Thr Trp Ala Ser Thr Pro Ala Val Ala Leu Gin His Giu Pro Pro Al a 90 Asp Arg Ile Arg Lys 170 Giu Giu 75 Met Cys Ser Val Gly 155 Met Gly Val1 Ala Val Thr Ser 140 Thr Val Asn 'Ihr Gly Thr Cys 125 His Thr Leu Phe Tyr Lys Lys 110 His Ser Ser Leu His Phe Pro Ser Trp Thr Thr Gly 175 Lys Leu Ala Glu Giu Gly Thr 160 Ile Ile Arg Leu Leu Lys 180 Thr Val Gly Phe Ala Phe 185 Phe Gin Lys Arg Asn Val Thr 190 <210> 11 00 <211> 1354 <212> DNA <213> Unknown <220> <223> Description of Unknown organism:rodent; surmised INO mus musculus <220> <221.> CDS <222> (42)..(875) <220> <221> mat peptide <222> (117)..(875) 00 <400> 11 ggcacgagtt acgatttgtg cttaacctga ctccactcca g atg cat gct ttg ggg 56 Met His Ala Leu Gly agg Arg act ctg gct ttg atg tta ctc atc ttc Thr Leu Ala Leu Met Leu Leu Ile Phe act att, ttg gtg Thr Ile Leu Val gag tca agt Giu Ser Ser tca ttt cct Ser Phe Pro tgt tca Cys Ser -1 1 gtg aaa gga Val Lys Gly gag gag atc cca Giu Giu Ile Pro ccg gat gat Pro Asp Asp gtg ggc gtc Val Gly Val ttt tca gat gat Phe Ser Asp Asp aat Asn 20 atc ttc cct gat Ile Phe Pro Asp gga Gly acc atg Thr Met gag att gag att Glu Ile Giu Ilie act cca gtg tct Thr Pro Val Ser gta Val cag ata ggt atc Gin Ile Gly Ile gct cag ctt ttc Ala Gin Leu Phe cat cct agt cca His Pro Ser Pro aaa gaa gca aca Lys Giu Ala Thr aga ata tgg gaa Arg Ilie Trp Giu act ccc aga gac Thr Pro Arg Asp cct tcc tgc aga Pro Ser Cys Arg cta ccc Leu Pro tac aga gca Tyr Arg Ala gga acc act Gly Thr Thr ttg cag cag atc Leu Gin Gin Ilie aaa aaa atc tgt Lys Lys Ilie Cys act gag aga Thr Glu Arg ctt ccc atc Leu Pro Ile agg gtc cct gca Arg Val Pro Ala cac cag agt tct His Gin Ser Ser gac Asp 105 aaa tca Lys Ser 110 atg gcc ctc aag Met Ala ieu Lys gat ggg cat tac Asp Giy His Tyr tca Ser 120 tgt cgg ata gaa Cys Arg Ile Giu aca aca gat ggg att ttc caa gag aga cat Thr Thr Asp Giy Ilie Phe Gin Giu Arg His agc atc caa gtg cca ggg Ser Ile Gin Val Pro Gly 125 gaa Giu 135 gca aat aga act Asn Arg Thr gta Val 145 act Thr tgt gag gca Cys Giu Ala agc aag Oct got Ala Ser Lys Pro Ala Met cag atc ttg Gin Ilie Leu cac aat gac His Asn Asp 175 aat ggc cac Asn Giv His tgg Trp, 160 acc Thr cca gat gag Pro Asp Giu gtc act aag Val Thr Lys agt aaa tca Ser Lys Ser 170 gag aaa aac Giu Lys Asn gat aac tgg Asp Asn Trp atg att gtC met Ile Val agg Arg 180 ttt Phe aag tgc cac Lys Cys His agt gtg ttc Ser Val Phe 190 att ctc Ile Leu tgc Cys 195 aat Asn atc too cat Ile Ser His t tg Leu 200 ago Ser 584 632 680 728 776 824 872 too atg gaa Ser. Met Giu 205 ttg Leu cag Gin 210 tat Tyr oga ggt aoa Arg Giy Thr ac Thr 215 gta Val ato otg oct Ile Leu Pro too Ser 220 otg ago att Leu Ser Ile cto Leu 225 ttc Phe gtg aaa otg Vai Lys Leu aot gtt oto Thr Val Leu atc gta Ile Val 235 gaa ggc Giu Gly gga ttt got ttt Gly Phe Ala Phe 240 oag aag aga aat tat tto aga gtg coa Gin Lys Arg Asn Tyr Phe Arg Val Pro too tgaggag Ser atottgtota gaoaaatctg tgaggttgtg octoatggtt aagoatttta catggaatgg atatgagott cccagtcco agt ggtotgtggt taagatgaga tttaooaoca totgaaagao cogogoagog atgtggttgt otggaoooag tatotaoaaa tocacagoat aaaattgcaa cagaaagtta tgotgagatt caatcctttc gagccaggct tootaaatto aaoacottta catggtttac caggaactgt oogagaagoa aatggacctg aggtoatata tttoaottco aagaaaotct aagaoagtgo ot tgggacga gccacagaac agtaottota tgt tgatttt agt tttaaaa oooacggaaa aaacoaagca gaaagaagga otaotaggaa 985 taaacoogag 1045 tgotgoaaga 1105 ottttggooo 1165 otgotggtto 1225 goattooaag 1285 ttaaatagt'c 1345 1354 <210> i2 <211> 278 <212> PRT <213> Unknown <400> 12 Met His Ala Leu Gly Arg Thr Leu Ala Leu Met Leu Leu Ile Phe Ile -20 -15 Thr Ile Leu Val Pro Giu Ser Ser Cys Ser -s
[11] -1 1 Val Lys Gly Arg Giu Glu Ile Asp Val Lys Ser Ile Ser Ser 120 Ile Ser Thr His Leu 200 Ser Thr ro ly Glm lu Cys Cys Asp 105 Cys Gin Lys Lys Arg 185 Thr Ile Val Pro F Val C Ile Ala Arg Thr Leu Arg Val Pro Ser 170 Glu Asp Len Leu sp ;ly ;ly Thr Leu 3lu Pro Ile Pro Ala 155 Lys Lys Asi Prc Il Asp S Val Ile I Leu Pro Arg Ile Glu Gly 140 Met Ser Asn i Trp Ser 220 e Val er rhr -ys 45 %rg ryr Gly Lys Thr 125 Glu Gin His Asr Ihf 20! Le Gi' Phe P Met G 30 Ala C Ile Arg I Thr Ser I 110 Thr Asn Ile Asn Gly 190 Leu a Leu y Phe 'ro 15 ;lu In Crp la rhr 95 Met Asp Arg Leu Asp 175 His S ex Sei Al Phe Ile Leu Glu Glu 80 Arg Ala Gly Thr Trp 160 Thr Sei Met Il 3, Ph Ser A Glu I Phe C Ile 65 Len Val Leu Ile Val 145 Thr Met Val Glu e Leu 225 e Phe ~sp :1e :ys 50 Chr 31n Pro Lys Phe 130 Val Pro Ile Phe Glr 21C Ty Gl Asp Ile His Pro Gin Ala His 115 Gin Cys Asp Val CyE 19! As Va Asn I Thr I Pro Arg 2 Ile His 100 Asp Glu Glu Glu Arg 180 Phe i Arg 1 Lys :le ,ro er ksp Ser His Gly Arg Ala Asp 165 Sex IlE Gil Le Phe P Val S Pro S Trp 1 Lys I Gin His His Ile 150 Cys Lys Ser Thr .i Ala 230 n Tyr ro er er 'ro -ys ;er ryr Ser 135 Ala Va1 Cys His Thr 215 Val Phe I Lys Arg As 24! 235 Arg Val Pro 250 Glu Gly Ser <210> 13 c2ll> 981 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t <400> 13 atgytntgyt gcngarwsnw gargtnaaya athwsnytna tgyathathw wsnathacnt ytnc arcayg a thtaygayy acngcngtnt ggngaytgyg tgycaytggg ggnaaycarw athcartaya aarathwsng gargaygara gtnacnacna acnytnwsng tytggmgnac nwsncaygtn sntgyccnga yaaraayCar cnacngtntt ygtncaratg cnaargtnat hytnathacn sntayaargc ngayacnmgn gggcnwsnac nccngayytfl arggnmgnta ywsntgygay tncargtnyt ngtnccnccfl gygargcnat hgcnggnaar tngcnaaraa ygarwsncay arcarwsnca ygtnwsngtn snytnwsnat hgarytnggn thathccnws nathathath gntgymgnaa rtgyaarytl tgcarccnta ygcnwsfltay cngargcnca yccngcnwsn cnatgggnat h gcngtnytny acnatgcara ggnaaraarg tggacnatha ga racnc ayg gcnccngayy athgcngtnc gargtnacnc ccngcngcnc wsnaayggna gtnttytgyg mgnggnggng ytnathatha ccnaarwsng *acngaraarw *carggnaarg tnathtgggg ayaaywsnws cnytnytntg cnytnmgngg arwsnaaytg tncarathws cngayggnaa ayttyccngg ar athws ntg cngtnacngt tngtnwsnca aycarytnyt thggntgyat gngcnacncc snaayccnyt tnaayggnac ngtnttygcn nacnatgacn 120 y .gyccnwsn 180 ncarccnwsn 240 ywsngaymgn 300 ngcngtngcn 360 yttycaraay 420 ngaraaymgn 480 gacnccngay 540 nmgnwsnacn 600 yytnacnacn 660 nggnwsntay 720 htgyytnytn 780 ngayathgar 840 ntaygayacl 900 ngaytyyytn 960 981 <210> 14 <211> 885 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t <400> 14 atgytntgyc cntggrngnac ngcnaayytn ggnytnytny tnathytnac nathttyytn gtngcngarg cngarggngc ngcncarccn aayaaywsny tnatgytnca racnwsnaar 120 garaaycayg cnytngcnws nwsnwsnytn tgyatggayg araarcarat hacncaraay 180 taywsnaarg tnytngcnga rgtnaayacn wsntggcclg tnaaratggc nacnaaygcn 240 gtnytntgyt gyccnccnat hgcnytnmgn aayy~natha thathacntg ggarathath 300 ytrimgrggnc acnaaytgya carathmgna gayggnaayt ttycaraaym. athwsntgga gtnacngtla gtnwsncayy aa ra ara thw mgnggnathc arccflwsftg cngaygarmg cngtrigcfat tycaymgrigg gnaaymgflac thccngargg arwsnacfltg tnacnggnaa sna aratha t ayytngtflgt yacflaargcfl nathacfltgg hacnc ayg ay ntaycayytl ngcflgtfltgy ngaytgygCfl ycaytgggar yaarwsflytl htaywsnath ngarwsncar tayaaraarg gtnwsnmgnc ggntaytaym cargtnytng aargcngtlg acnaarcarg gtncayaayg tayathgary taycaycclt tggytncara ara coa ayga cngaycaraa gntgyathat tnacnc cnga cnggnaarcc artaytggws tnwsia cngt tnytnc cngt aytaytayta arath racnaargar 360 ywsngayytn 420 ggtnacnccn 480 rgtnacnytn 540 ngcngcncay 600 naayggnacl 660 nacntgycay 720 nccnggnycn 780 yytngaycay 840 885 <210> <211> 978 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t <400> atgttytgyt tytggmgnac nwsngclytl gcnggnwsflw sntgyacnga yaaraaycar gcngtlytfly acnacflcara tnathtgggg ayaaywsflws ngtnttygtfl nwsnccnytn 120 acncargtna wsnathccny wsntgyacna aayathacnt ytn car cayg aaytaygayy wsngcngtnt ggngaytgyg tgycaytggg ayacnacngt nwsngtncar athggnacna argcnytnyt ntgytgytty 180 tnacnaargc thgcntayaa gggcnwsflac arggnacflta tncargtflyt gygargcflat tnacnacnws arcaraayaa ngtnytna th rgtngayacfl nccflgaycay yacritgygar ngtnccflccf ggcflggflaar ngarwsncay yg tnwsngay acntggatha aa ra cna ayg wsnccngary acngtlacflc gargtlacflt ccngcngcnc wsnaayggna gtnwsfltgya thaarytnmg a ra cnwsntg tncarathws cngarggflaa ayttyccnga arathwsntg cngtnacngt thgtnwsnca nggnytnccfl 240 yytnggntngn 300 ngcngtlacf 360 yttygaraar 420 raaraaymgrl 480 gwsnccngay 540 nmgnwsnacn 600 yytnacnggn 660 aaycarwsny tnwsnacthga rytnwsnmgn ggnggnaayc arwsnytnmg nccntayath 720 ccntayatha thccnwsnat hathathytn athathathg gntgyathtg yytnytnaar 780 21 athwsnggnt tymgnaartg yaarytnccn aarytngarg cnacflwsngc nathgargar 840 gaygaratgc arccntaygc nwsntayacn garaarwsna ayccnytnta ygayacflgtn 900 acnaargtng argcnttycc ngtnwsncar ggngargtna ayggnacnga ytgyytnacn 960 ytnwsngcna thggnath 978 <210> 16 <211> 750 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t <400> 16 atgggnggna arcaratgac ncaraa carccngtny tnatggayat haaygc ytnathatha thacntggga rathat aaraargara cnaaygarac naarga wsnmgnccng aycaraayws ngayyt taytaymgng gnathgtngt nacncc gtnytngtna cnccngargt naayyt gcngtnacng gnaarccngc ngcnca acnaarcarg artaytgggg naaygg ggncayaarw snacngtnac ntgycz gtnaarytna aywsnggnyt nmgnac ytntaygtna arytnwsnyt nttygt carmgnatha aycaygtnmg naargl ytay wsnacnatht tygcngargg naayathwsn ngtn hytn racn ncar :ngay .ntty rath nacn ~ygtn :nwsn :ngtn tnytn ytntgytgyc mgnggncarc aaytgyacng athmgnccng ggnaayt tyc carwsnmgna wsntggathc gtnacngtna wsncayytna ggnwsnccng cnccnathgc nytnmgnaay 120 cnwsntgyac tngarrngnat tngayacnac aymgnggnt a ayathacngc cngarggnws arwsnacntg cnggnaayaa cnytnwsnyt naargcntay hacntgggtn ncaygayggn ycayytncar ngtntgyaar nathytngcn yccntgggar rwsnytnwsn nytnathath 180 240 300 360 420 480 540 600 660 720 750 athytngtna cnacnggntt ygtnttytty <210> 17 <211> 582 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t <400> 17 mgnggncarc cnwsntgyat hatggcntay aargtngara cnaargarac naaygaracn GO tgyytnggflm wsngcngtflg aayttycaya ggngaraaym tggacnccng gtnmgnwsna caywsnacng c cnwsnytny gtnggnttyg gnaayathac cnytncarca argtfltayga gnaclgcflgt ayggngaytg cntgycaytg gnaaycarws tnacnathyt cnttyttyca 22 ntgggcflwsf acnccngayc ygarggflaay tayytntgyg yytncargtl ytflgtflcfc ntgygargCfl atggclggfla ygtnacnaar wsngarwsrlc ggarcaraay aaygtnwsng nytnwsnath garytnwsnm ntaygtflaar atggtnytny raarmgflaay gtnacflmgfla ayathc cnga arathacnac crigargtflac arccngcngc aywsnaaygg crigtnwsftg gnggnacnac tnggnathat cn yytncarath nccngarggl ntayttyytn nc ara thws n nacngtnacl yathgtnwsn nwsnaclacf hytnytnaar 120 180 240 300 360 420 480 540 582 <210> 18 <211> 834 <212> DNA <213> reverse translation <220> <221> misc feature <222> <223> n may be a, c, g, or t atgcaygcny ccngarwsnw ttywsngayg acnccngtnw aargargcna ccntaymgng mgngtnccflg gayggncayt athcargtnc atgcarathy acnatgathg tnggnmgflac sntgywsflgt ayaayathtt sngtncarat cnytnmgflat cngarytflca cncaycayca aywsfltgymg cnggngaraa tntggaclcc tnmgnwsflaa nytngcnytl naarggnmgl yccngayggl atgytlytfla gargarathc gtngglgtfla hggnathaar gcncarytnt htgggarath rc ara thwsn rwsnwsngay nathgaracn ymgflacfgtfl ngaygargay rtgycaymgfl acnccnmgng aaraaratht ytnccnatha acngayggna gtntgygarg tgygtflacna garaaraaya ytnwsnatgg thttyathac cnc cngayga cnatggarat tytgycaycc aytggc cnws gyacngarmg arwsnatggc thttycarga cnathgcnws arwsnaarws nathytngtn ywsnttyccn 120 hgarathath 180 nwsnccnwsn 240 ntgymgnytn 300 nggnacnacl 360 nytnaarcay 420 rmgncaywsl 480 naarccngcn 540 ncayaaygay 600 ayggncayws ngtnttytgy 660 ttyathwsnc ayytnacnga yaaytggath arcaraaymg tngcngtnac nggnacnacn 720 ngtnytnath 780 wsnathytnc cnwsriytnyt nwsnathytn taygtnaary gtnggnttyg cnttyttyca raarmgnaay tayttymgng tnccngargg nwsn '210> 19 ':211> 1047 '212> DNA (213> Unknown <:220> <:223> Description of Unknown Organism:primate; surmised homo sapiens <:220> <:221> CDS <:222> (1)..(1044) <:220> <:221> mat peptide <:222> (79)..(1044) ':400> 19 atg ctc tgc Met Leu Cys cct tgg aga Pro Trp Arg gct aac cta ggg cta Ala Asn Leu Gly Leu ctg ttg att ttg Leu Leu Ile Leu act atc ttc tta gtg Thr Ile Phe Leu Val gcc Ala -5 gaa gcg gag Glu Ala Glu ggt gct Gly Ala -1 1 gct caa cca Ala Gin Pro aac aac Asn Asn tca tta atg Ser Leu Met agt tta tgt Ser Leu Cys caa act agc aag gag aat cat gct tta Gin Thr Ser Lys Giu Asn His Ala Leu gct tca agc Ala Ser Ser tcg aaa gta Ser Lys Val atg gat gaa aaa Met Asp Giu Lys cag Gin att aca cag aac Ile Thr Gin Asn ctc gca Leu Ala gaa gtt aac act Giu Val Asn Thr tca Ser tgg cct gta aag Trp Pro Val Lys gct. aca aat gct Xla Thr Asn Ala gtg Val ctt tgt tgc cct. Leu Cys Cys Pro cct Pro 60 atc gca tta aga Ile Ala Leu Arg ttg atc: ata ata Leu Ilie Ile Ile aca Thr 240 288 336 tgg gaa ata atc Trp Gu Ile Ile ctg Leu aga ggc cag cct Arg Gly Gin Pro tcc Ser 80 tgc aca aaa gcc Cys Thr Lys Ala tac agg Tyr Arg aaa gaa aca Lys Giu Thr acc tgg gtc Thr Trp Val 105 aat gag acc aag gaa acc Asn Giu Thr Lys Giu Thr 95 aac tgt act gat Asn Cys Thr Asp gag aga ata Giu Arg Ile 100 tcc aga cct gat cag aat tcg gac ctt cag att cgt cca Ser Arg Pro Asp Gin Asn Ser Asp Leu Gin Ile Arg Pro gtg gcc atc act cat gac ggg tat tac aga tgc ata atg gta aca cct Vai Ala Ilie Thr His Asp Gly Tyr Tyr Arg Cys Ile Met Val Thr Pro 120 125 130 ga t Asp 135 ggg aat ttc cat Gly Asn Phe His c9t. gga tat cac Arg Gly Tyr His 140 caa aac agg aat Gin Asn Arg Asn 24 Ctc caa gtg Leu Gin Val 145 tta gtt aca cct Leu Val Thr Pro 150 gaa gtg acc ctg Glu Val Thr Leu ttt Phe 155 act gca gta tgc Thr Ala Val Cys aag gca Lys Ala 165 gtt gca ggg Val Ala Gly tgt gcc act Cys Ala Thr 185 aag Lys 170 cca gct gcg cag Pro Ala Ala Gin atc tcc tgg atc cca gag ggc gat Ile Ser Trp Ile Pro Giu Gly Asp 175 180 aag caa gaa tac Lys Gin Glu Tyr tgg Trp 190 agc aat ggc aca Ser Asn Gly Thr gtg Vai 195 act gtt aag Thr Val Lys agt aca Ser Thr 200 tgc cac tgg gag Cys His Trp Giu cac aat. gtg tct His Asn Val Ser gtg acc tgc cac Val Thr Cys His 672 720 768 816 gtc Val 21S tcc cat ttg act Ser His Leu Thr ggc Gly 220 aac aag agt ctg Asn Lys Ser Leu ata gag cta ctt. Ile Glu Leu Leu gtt cca ggt gcc Val Pro Gly Ala aaa Lys 235 aaa tca gca aaa Lys Ser Ala Lys tat att cca tat Tyr Ile Pro Tyr atc atc Ile Ile 245 ctt act att Leu Thr Ile gtc aat ggc Val Asn Gly 265 att Ile 250 att, ttg acc atc Ile Leu Thr Ile gga ttc att tgg Gly Phe Ile Trp ttg ttg aaa Leu Leu Lys 260 tct act cca Ser Thr Pro tgc aga aaa tat Cys Arg Lys Tyr aaa Lys 270 ttg aat aaa aca Leu Asn Lys Thr gtt gtt Val Vai 280 gag gag gat gaa Glu Glu Asp Giu atg Met 285 cag ccc tat gcc Gin Pro Tyr Ala tac aca gag aag Tyr Thr Giu Lys aac aat cct Asn Asn Pro 295 ctc tat gat Leu Tyr Asp 300 act aca aac aag Thr Thr Asn Lys aag gca tct cag Lys Ala Ser Gin 1008 tta caa agt gaa Leu Gin Ser Glu gac aca gac ctc Asp Thr Asp Leu cat act tta taa His Thr Leu 320 1047 'z210>' <211> 348 <212> PRT <213> Unknown <400> Met Leu Cys Pro Trp Arg Thr Ala Asn Leu Gly Leu Leu Leu Ile Leu -20 Thr Ilie Phe Leu Val Ala Glu Ala Glu-Gly Ala Ala Gln Pro Asn Asn 00-10 -5 -1 Ser Leu Met LeU Gin Thr Ser Lys Giu Asn His Ala Leu Ala Ser Ser 15 Ser Leu Cys Met Asp Giu Lys Gin Ile Thr Gin Asn Tyr Ser Lys Val 30 Leu Ala Giu Val Asn Thr Ser Trp Pro Val Lys Met Aia Thr Asn Ala 45 Val Leu Cys Cys Pro Pro Ile Ala Leu Arg Asn Leu Ile Ile Ile Thr 60 65 Trp Glu Ile Ilie Leu Arg Gly Gin Pro Ser Cys Thr Lys Ala Tyr Arg 00 75 80 Lys Glu Thr Asn Giu Thr Lys Glu Thr Asn Cys Thr Asp Giu Arg Ile C190 95 100 Thr Trp Val Ser Arg Pro Asp Gin Asn Ser Asp Leu Gin Ile Arg Pro 105 110 115 Val Ala Ile Thr His Asp Gly Tyr Tyr Arg Cys Ile Met Val Thr Pro 120 125 130 Asp Gly Asn Phe His Arg Gly Tyr His Leu Gin Val Leu Val Thr Pro 135 140 145 150 Giu Val Thr Leu Phe Gin Asn Arg Asn Arg Thr Ala Val Cys Lys Ala 155 160 165 Val Ala Gly Lys Pro Ala Ala Gin Ile Ser Trp Ile Pro Glu Gly Asp 170 175 180 Cys Ala Thr Lys Gin Giu Tyr Trp Ser Asn Giy Thr Val Thr Vai Lys 185 190 195 Ser Thr Cys His Trp Giu Val His Asn Val Ser Thr Val Thr Cys His 200 205 210 Val Ser His Leu Thr Gly Asn Lys Ser Leu Tyr Ile Glu Leu Leu Pro 215 220 225 230 Val Pro Gly Ala Lys Lys Ser Ala Lys Leu Tyr Ile Pro Tyr Ile Ile 235 240 245 Leu Thr Ile Ile Ile Leu Thr Ile Val Gly Phe Ile Trp Leu Leu Lys 250 255 260 Val Asn Gly Cys Arg Lys Tyr Lys Leu Asn Lys Thr Giu Ser Thr Pro 265 270 275 Val Val Giu Giu Asp Giu Met Gin Pro Tyr Ala Ser Tyr Thr Giu Lys 280 285 290 Asn Asn Pro Leu Tyr Asp Thr Thr Asn Lys Val Lys Ala Ser Gin Ala 295 300 305 310 26 Leu Gin Ser Glu Val Asp Thr Asp Leu His Thr Leu 315 320 <210> <211> <212> <213> <220> <221> <222> <223> <400> 21 1044 DNA reverse translation misc feature (1)..(1044) n may be a, c, g, or t 21 atgytntgyc cntggmgnac ngcnaayytn ggnytnytny tnathytnac nathttyytn gtngcngarg garaaycayg cngarggngc cnytngcnws ngcncarccl nwsnwsnytn taywsnaarg tnytngcnga rgtnaayacn gtnytntgyt ytnmgnggnc acnaaytgya ca rathmgn c gayggnaayt ttycaraaym athwsntgga gtnacngtna g tnwsncayy aaraarwsng gtnggnttya garwsnacnc aayaayccny gyccnccnat arccnwsntg cngaygarmg cngtngcnat tycaymgngg gnaaymgnac thccrigargg arwsnacntg tnacnggnaa cnaarytnta thtggytnyt cngtngtnga tntaygayac hgcnytrimgn yacnaargcl nathacntgg hacncaygay ntaycayytn ngcngtntgy ngaytgygcn ycaytgggar yaarwsnytl yathccntay naargtnaay rgargaygar nacnaayaar aayaaywsfly tgyatggayg wsntggccng aayytnatha taymgnaarg gtnwslmgrc ggntaytayn cargtnytng aargcngtng acnaarcarg gtncayaayg tayathgary athathytna ggntgymgna atgcarccnt gtnaargcnw araarcarat tnaaratggc thathacntg aracnaayga cngaycaraa gntgyathat tnacnccnga cnggnaarcc artaytggws tnwsnacngt tnytnccrigt cnathathat. artayaaryt aygcnwsnta sncargcnyt racncaraay 180 nacnaaygcn 240 ggarathath 300 racnaargar 360 ywsngayytn 420 ggtnacnccn 480 rgtnacnytn 540 ngcngcncar 600 naayggnacn 660 nacntgycay 720 nccnggngcn 780 hytnacnath 840 naayaaracn 900 yacngaraar 960 ncarwsngar 1020 tnatgytnca racnwsnaar 120 gtngayacng ayytncayac nytn <210> 22 <211> 813 <212> DNA <213> Unknown <22 0> <223> Description of Unknown organism:rodent; surmised 1044 <220> <221> <222> <220> <221> <222> mus musculus CDS mat peptide (76)..(810) <400> 22 atg Met cat gct ctg ggg His Ala Leu Gly agg Arg att ccg act ttg act ttg ctg atc ttc atc Ile Pro Thr Leu Thr Leu Leu Ile Phe Ile aat att ttt gtg Asn Ile Phe Val cag aat gac agt Gin Asn Asp Ser ggg tca agt Gly Ser Ser tgt act gat gag Cys Thr Asp Glu -1 1 aca caa gtt aac Thr Gin Val Asn tca tct tct Ser Ser Ser ctg Leu is aat caa aca ata Asn Gin Thr Ile S act aca atg tct Thr Thr Met Ser tct agt cca. ctg Ser Ser Pro Leu gta cag Vai Gin atg gat aaa aag Met Asp Lys Lys gc t Ala ctg ctc tgc tgc Leu Leu Cys Cys ata Ile aat gca. gta. tta. Asn Ala Val Leu aca tgg ata ata Thr Trp Ile Ile cac aga cac ctg His Arg His Leu tcc tyc aca ata Ser Cys Thr Ile gca. Al a tac aac cta gat Tyr Asn Leu Asp aag acc aat gaa Lys Thr Asn Giu acc agc Thr Ser tgc ttg ggc Cys Leu Gly gaa ctt cag Glu Leu Gin agg Arg aac atc acc tgg Asn Ile Thr Trp tcc aca cct gac Ser Thr Pro Asp cac agt cct His Ser Pro act tac aca Thr Tyr Thr atc agt gca gtg Ile Ser Ala Val ctc cag cat gag Leu Gin His Giu ggg Gly i100 tgt gag Cys Giu 105 ata gta aca. cct Ile Val Thr Pro gaa Giu 110 ggg aat tta gaa Gly Asn Leu Glu gtc tat gac ctc Val Tyr Asp Leu 432 480 caa Gin 120 gtg ctg gtg ccc- Vai Leu Val Pro) gag gta acc tac Glu Val Thr Tyr cca ggg aaa aac Pro Gly Lys Asn act gca gtc tgt Thr Ala Val Cys gag gca Glu Ala 140 atg gca ggc aag cct gct Met Ala Gly Lys Pro Ala 145 gca cag atc tct Ala Gin Ile Ser 150 tgg act cca Trp Thr Pro gat Asp 155 ggg gac tgt gtc Gly Asp Cys Vai act aag agt gag tca cac agc aat Thr Lys Ser Giu Ser His Ser Asn 160 165 ggc Gly tct Ser tcc Ser 200 acc Thr gta Val1 act Thr gtt Val1 185 ata Ile att Ile gga dly gtg Val 170 gtg Val1 gaa Giu ctc Leu ttt Phe act Thr tcc Ser ctg Leu tat Tyr gct Al a 235 gtc agg Val Arg tgc Cys agt Ser gtg Val1 220 ttc Phe tta Leu caa Gin 205 aaa Lys ttc Phe agc Ser gtc Vai 190 ggt Gly atg Met cag Gin acg Thr 175 tct Ser aca Thr gcc Aila aag Lys 28 tgc cac Cys His cat tcg His Ser atg acc Met Thr ott ttg Leu Leu 225 aga aat Arg Asn 240 tgg TrD act Thr acc Thr 210 gtg Val1 ttt Phe gag cag Glu GIn 180 ggt aat Gly Asn 195 ccc cgt Pro Arg att att Ile Ile gcc aga Ala Arg aa c Asn cag Gin tc Ser ctt Leu aca Thr 245 aat Asn tct Ser ttg Leu ctt Leu 230 tga gtg Vai ctg Leu ctg Leu 215 aac Asn <210> 23 <211> 270 '<212> PRT <213> Unknown <400> 23 Met His Aia Leu Gly Arg Ile Pro Thr Leu Thr Leu Leu Ile Phe Ile Asn Gin Vai Ile Ser Cys Giu Cys Gin 120 Ile Asn Gin Asn Cys Leu Leu Giu 105 Val Phe Asp Met Ala Thr Gly Gin Ile Leu -20 Val. Ser Gly Ser Ser Ser Asp Lys Lys Val Leu Ile 45 Ile Ala Tyr Arg Asn Ile Ile Ser Ala Vai Thr Prc Vai Pro Prc 12~ 5 Ser Ser Al a 30 Thr Asn Thr Val1 Giu 110 Giu Ser Leu 15 Leu Trp Leu Trp Ala 95 Giy Vali Cys Thr Asp Glu -1 Thr Leu Ile Asp Ala 80 Leu Asn Thr 1 Gin Cys Ile Lys 65 Ser Gin Leu Tyr Val1 Cys Lys 50 Lys Thr His Glu Phe 130 Asn Phe His Thr Pro Glu Lys 115 Pro Asn Thr Ser Arg Asn Asp Giy 100 Val Gly Gin Thr Ser His Giu His Thr Tyr Lys Thr Met Pro Leu Thr Ser Tyr Asp Asn Ile Ser Leu Pro S er Pro Thr Leu Arg 135 Thr Aia Vai Cys Glu Ala Met Ala Giy 140 Pro Ala Ala Gin Ile Ser 150 Tru Thr Pro Gly Thr Val 170 Ser Val Val Asp Gly Asp 155 Thr Val Arg 29 Cys Val Thr Lys 160 Ser Thr Cys His Ser Giu Ser Trp Giu His Ser Asn 165 Asn Asn Val Gin Ser Leu 175 Leu Val Her Ser Cys His Ser Thr 125 Ser Ile 200 Thr Ile Vai Gly 190 Gly Glu Leu Ser Leu Tyr Vai 220 Phe Ala Phe Thr Met Thr Met Ala Leu Leu 225 Asn Thr Pro Arg 210 Val Ile Ile Phe Ala Arg Ser Leu Leu Leu 230 Leu 215 Asn Phe Gin Lys 235 Arg 240 <210> 24 <211> 810 <212> DNA .213> reverse translation 220> <221> misc feature <222>' <223> n may be a, c, g, or t '400> 24 atgcaygcny wsnggnwsnw acncargtna wsnwsnccny wsntgyacna aayathacnt ytncarcayg gtntaygayy acngcngtnt ggngaytgyg tgycaytggg aaycarwsny acnathytnt tnggnmgnat sntgyacnga ayacnacnat tnathaaygc thgcntayaa gggcnwsnac arggnacnta tncargtnyt gygargcnat tnacnaarws arc ara ayaa tnwsnathga aygtnaarat hccnacnytn acnytnytna thttyathaa yathttygtn ygaraaycar gwsngtncar ngtnytnath yytngayaar nccngaycay yacntgygar ngtnc cic cn ggcnggnaar ngarwsncay ygtnwsngtn rytnwsncar ggcnytnytn acnathcara atggayaara acntggatha aara cna ayg wsnccngary athgtnacnc gargtnacnt ccngcngcnc wsnaayggna gtnwsntgyy ggnacnatga aygaywsnws argcnytnyt thaarcaymg ara cnwsntg tncarathws cngarggnaa ayttyccngg arathwsntg cngtnacngt tngtnwsnca cna cn ccnmg nwsnwsnytn 120 ntgytgytty 180 ncayytnccn 240 yytnggnmgn 300 ngcngtngcn yytngaraar 420 naaraaymgn 480 gacnccngay 540 nmgnwsnacn 600 ywsnacnggn 660 nwsnytnytn 720 gtnathathy tnytnaaygt nggnttygcn 780 ttyttycara arrngnaaytt ygcnmgnacn

类似技术:

---

公开号 | 公开日 | 专利标题

---

EP1918377B1|2013-12-04|Mammalian cytokine receptor subunit proteins, related reagents and methods

---

US20120231003A1|2012-09-13|Ox2 Receptor Homologs

---

US20050009145A1|2005-01-13|Mammalian receptor proteins; related reagents and methods

---

US20050106673A1|2005-05-19|Mammalian receptor proteins; related reagents and methods

---

WO1999040195A1|1999-08-12|Mammalian receptor proteins; related reagents and methods

---

EP1181366B1|2009-03-04|Mammalian receptor proteins; related reagents and methods

---

CA2392109A1|2001-05-25|Mammalian receptor proteins; related reagents and methods

---

AU2008203172B2|2012-04-05|OX2 receptor homologs

---

CN101255425A|2008-09-03|OX2 receptor homolog

---

AU2007202362A1|2007-06-14|Mammalian receptor proteins; related reagents and methods

同族专利:

---

公开号 | 公开日

---

AU2008203172B2|2012-04-05|

---

AU2005205753A1|2005-09-29|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

法律状态:
2012-08-02| FGA| Letters patent sealed or granted (standard patent)|

---

2012-10-11| DA2| Applications for amendment section 104|Free format text: THE NATURE OF THE AMENDMENT IS: AMEND THE PATENTEE TO READ MEDICAL RESEARCH COUNCIL AND MERCK SHARP& DOHME CORP. . |

---

2013-12-05| MK14| Patent ceased section 143(a) (annual fees not paid) or expired|

优先权:

---

申请号 | 申请日 | 专利标题

---

GB9911123||1999-05-13||

---

GB9925989||1999-11-03||

---

AU50072/00A|AU5007200A|1999-05-13|2000-05-11|Ox2 receptor homologs|

---

AU2005205753A|AU2005205753A1|1999-05-13|2005-08-31|OX2 receptor homologs|

---

AU2008203172A|AU2008203172B2|1999-05-13|2008-07-16|OX2 receptor homologs|AU2008203172A| AU2008203172B2|1999-05-13|2008-07-16|OX2 receptor homologs|