CA2388032A1 - Polynucleotides encoding human and murine adhesion proteins (bigr) - Google Patents

Abstract

The present invention relates to isolated and purified polynucleotides encoding for a human adhesion protein or a murine adhesion protein.

Description

POLYNUCLEOTIDES ENCODING HUMAN AND MURINE
ADHESION PROTEINS (131g~
Technical Field of the Invention The present invention relates to molecular biology. More specifically, the present invention relates to polynucleotides which encode a human or a murine adhesion protein, polypeptides encoded by said polynucleotides and recombinant vectors expressing said polypeptides.
Background of the Invention Cell adhesion is of prime importance for the formation and functional maintenance of multicellular organisms. Adhesion proteins can be classified as cell surface molecules that mediate intercellular bonds and/or participate in cell-substratum interactions. Their intracellular domains provide a functional link to the cytoskeleton and this appears to be important for efficient cell-cell adhesion to take place. They are expressed in characteristic spatiotemperal sequences. Different superfamilies have been described including immunoglobulin (hereinafter, "Ig"), cadherin, integrin, selectin (Aplin AE, Howe A, Alahari SK, Juliano RL, (1998) Pharmacol. Rev. 50:197-263). Adhesion proteins belonging to the immunoglobulin superfamily may operate in both a homotypic and/or heterotypic manner.
The common building block is the Ig domain and the prototype is neural cell adhesion molecule (hereinafter, "NCAM") which possesses five Ig domains. This family participates in diverse biological functions including leukocyte-endothelial cell interactions, neural crest cell migration, neurite guidance and tumor invasion.
It is well demonstrated that during inflammation members of the Ig superfamily interact with and participate in leukocyte adhesion, invasion and migration through the vessel wall (Gonzalez-Amaro R, Diaz-Gonzalez F, Sanchez-Madrid F, (1998) Drugs 56:977-88).
Selectins are involved in the initial interactions (tethering/rolling) of leukocytes with activated endothelium, whereas integrins and Ig superfamily CAMS mediate the firm adhesion of these cells and their subsequent extravasation.
SUBSTITUTE SHEET (RULE 26) Tight junctions (hereinafter, "TJ") and adherens junctions (hereinafter, "AJ") arc specialized structures that occur between opposing endothelial and epithelial cells. They form a semipermeable intercellular diffusion barrier that is both dynamic and regulated. Obviously these structures must be disrupted, or reorganized, in order to facilitate leukocyte passage from the circulation. The platelet endothelial cell adhesion molecule, PECAM-1, a member of the Ig superfamily of adhesion proteins, localizes to the lateral membranes between endothelial cells (Zocchi MR, Ferrero E, Leone BE, Rovere P, Bianchi E, Toninelli E, Pardi R, ( 1996) Eur. J. Immunol. 26:759-67). However, it is not associated with the TJ and AJ
structures (Ayalon O, Sabanai H, Lampugnani MG, Dejana E, Geiger B, ( 1994) J.
Cell. Bio.
126(1):247-58). The crucial role of PECAM-1 in paracellular migration of leukocytes to extravascular sites has been established (Muller WA, Weigl SA, Deng X, Phillips DM, (1993) J. Exp. Med. 178:449-60). Another cell-cell adhesion molecule of the Ig superfamily, nectin (hereinafter, "PRR"), is recruited to the AJ through interaction with a PDZ-domain in the novel protein afadin (Takahashi K, Nakanishi H, Miyahara M, Mandai K, Satoh K, Satoh A, Nishioka H, Aoki J, Nomoto A, Mizoguchi A, Takai Y, ( 1999) J. Cell. Biol.
145:539-49).
Tight junctions are crucial structures for maintenance of the blood-brain (hereinafter, "BBB") and blood-retinal (hereinafter, "BRB") barriers. In some instances it may be desirable to selectively disrupt TJs. For example, disruption of the BBB may provide a method for transvascular delivery of therapeutic agents to the brain (Muldoon LL, Pagel MA, Kroll RA, Roman-Goldstein S, Jones RS, Neuwelt EA, (1999) Am. J. Neuroradiol.
20:217-22). On the other hand, breakdown of the BBB is part of the pathology of multiple sclerosis and stroke. In this instance it would be desirable to prevent reorganization of the TJs.
In 1998, a novel mouse functional adhesion molecule (hereinafter, "JAM") was cloned and identified as an additional transmembrane protein component of the tight junction (Martin-Padura I, Lostaglio S, Sehneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E, ( 1998) J. Cell.
Biol. 142( 1 ):117-27). JAM possesses two Ig domains; a single transmembrane and a short intracellular domain. Thus it belongs to the Ig superfamily of adhesion molecules and evidence suggests that it also influences the paracellular transmigration of immune cells.
Whether its SUBSTITUTE SHEET (RULE 26) extracellular domain engages in heterotypic interactions remains to be elucidated.
The nucleotide sequence of mouse JAM was utilized to identify novel putative adhesion proteins belonging to the Ig superfamily.
Summary of the Invention The present invention relates to an isolated and purified human adhesion polynucleotide which encodes a human adhesion polypeptide or fragment thereof.
Moreover, the present invention further relates to an isolated and purified polynucleotide having the nucleotide sequence of SEQ ID NO: I . The human adhesion polypeptide has the amino acid sequence shown in SEQ ID N0:2.
The present invention relates to an isolated and purified murine adhesion polynucleotide which encodes a murine adhesion polypeptide or fragment thereof.
Moreover, the present invention further relates to an isolated and purified polynucleotide having the nucleotide sequence of SEQ ID N0:3. The murine adhesion polypeptide has the amino acid sequence shown in SEQ ID N0:4.
The present invention also relates to a recombinant vector. This vector contains a polynucleotide having the nucleotide sequence of SEQ ID NO:1 or SEQ ID N0:3, which encodes for either a human or murine adhesion protein. The polynucleotide is operatively linked to a promoter that controls expression of the nucleotide sequence and a termination segment.
The present invention also relates to a host cell containing the recombinant vector.
The host cell can be a bacterial cell, an animal cell or a plant cell. The present invention also relates to transgenic mammals carrying a null mutation of the polynucleotide or animals overexpressing the polynucleotide described herein.
The present invention also relates to a transgenic mammal comprising a null mutation of the nucleotide sequence of SEQ ID NO: I or SEQ ID N0:3. Additionally, the present SUBSTITUTE SHEET (RULE 26) invention also relates to a transgenic mammal overexpressing the nucleotide sequence of SEQ ID NO:1 or SEQ ID N0:3.
Finally, the present invention relates to an antibody that binds to one or both of the hereinbefore described polypeptides.
Brief Description of the Figures Fig. 1A shows the alignment of a homologous Expressed Sequence Tag (hereinafter referred to as "EST") obtained from the databases accessed through the home page of the National Center for Biotechnology Information at www.ncbi.nlm.nih.gov. with the open reading frame of a murine functional adhesion protein (hereinafter referred to as "mouse JAM"). Identity is shown on the amino acid level. Fig 1B shows the alignment of overlapping ESTs that encode various sections of a human brain immunoglobulin superfamily receptor (hereinafter referred to as "human BIgR") including the stop codon.
Fig. 1 C summarizes the Rapid Amplification of cDNA Ends (hereinafter referred to as "RACE") procedure employed to obtain the full open reading frame of human BIgR. The longest clones identified from each reaction are aligned.
Fig. 2 shows the alignment of the sequences from RACE reactions 1 and 2 with GenBank Accession AF062733. RACE reaction 2 isolated a clone with a 34 amino acid deletion compared to AF062733.
Fig. 3 shows the full cDNA and amino acid sequence of the human BIgR. The predicted signal sequence and transmembrane domain are underlined. N-linked glycosylation sites are highlighted as are cysteine residues which form disulfide bonds within the immunoglobulin-like folds of the extracellular domain ~, position and sequence (below) of the 34 amino acid insert.

SUBSTITUTE SHEET (RULE 26) Fig. 4 shows the alignment of the intracellular domains of human BIgR with glycophorin C and drosophila Ncurcxin. Residues that arc construed between at least two of~
the sequences are highlighted.
Fig. 5 shows the complete cDNA and protein sequence of murine adhesion protein (hereinafter referred to as "mouse BIgR"). Construed cysteine residues are highlighted.
Fig. 6 shows the alignment of mouse (upper) and human (lower) BIgR amino acid sequences.
Fig. 7 shows transcripts that were identified on a multiple tissue Northern blot probed under high stringency with a [a-3zP]dCTP BIgR probe. Arrows highlight human BIgR transcripts.
Fig. 8 shows that transcripts were identified on a normalized human brain multiple tissue Northern blot probed under high stringency with a [a-32P]dCTP BIgR (A) or (3-actin (B). Arrows highlight human BIgR transcripts.
Fig. 9 is a Western blot of Cos cells, control and expressing BIgR, probed with a rabbit anti-BIgR antibody.
Fig. 10 defines the subcellular localization of BIgR when expressed in CHO
cells.
Immunofluoresence was performed with the rabbit anti-BIgR antibody and viewed using a NoranTM Confocal laser-scanning microscope (Noran Instruments, Middleton, WI).
Fig. 11 shows screening for BIgR counter-receptors on various leukocyte cell lines.
Calcein loaded cells were added to BIgR-Fc captured in 96 well plates. Binding was performed in TBS (n=6) or TBS+1Mn (n=6); Wells were washed, retained cells lysed and fluorescence quantitated with a fluorimeter at excitation 485/emission 530 nm.
Data from a representative experiment. Average ~SEM.
SUBSTITUTE SHEET (RULE 26) Detailed Description of the Invention The present invention relates to isolated and purif ed polynucleotide sequences which encode for a human adhesion protein (referred to herein as "human BigR") and a murine adhesion protein (referred to herein as "mouse BIgR"). In another embodiment, the present invention relates to polypeptidcs for human BIgR and mouse BIgR. In yet another embodiment, the present invention relates to recombinant vectors which, upon expression, produce human BIgR or mouse BIgR. The present invention also relates to host cells transformed with these recombinant vectors.
II. Sequence Listing The present application also contains a sequence listing that contains 10 sequences.
The sequence listing contains nucleotide sequences and amino acid sequences.
For the nucleotide sequences, the base pairs are represented by the following base codes:
m 1 Meaning A A; adenine C C; cytosine G G; guanine T T; thymine U U; uracil M A or C

R Aorta W A or T/U

S C or G

S,wmbol Meaning Y C or T/U

K G or T/U

V A or C or G; not T/U

H A or C or T/U; not G

D A or G or T/U; not C

B C or G or T/U; not A

N (A or C or G or T/U) The amino acids shown in the application are in the L-form and are represented by the following amino acid-three letter abbreviations:

SUBST1ITUTE SHEET (RULE 26) AbbreviationAmino acid name Ala L-Alanine Arg L-Arginine Asn L-Asparagine Asp L-Aspartic Acid Asx L-Aspartic Acid or Asparagine Cys L-Cysteine Glu L-Glutamic Acid Gln L-Glutamine Glx L-Glutamine or Glutamic Acid Gly L-Glycine His L-Histidine Ile L-Isoleucine Leu L-Leucine Lys L-Lysine Met L-Methionine Phe L-Phenylalanine Pro L-Proline Ser L-Serine Thr L-Threonine Trp L-Tryptophan Tyr L-Tyrosine Val L-Valine Xaa L-Unknown or other III. Polvnucleotides In one aspect, the present invention provides an isolated and purified polynucleotide which encodes human BIgR. In another aspect, the present invention provides an isolated and purified polynucleotide which encodes mouse BIgR. These polynucleotides can be DNA
molecules, such as gene sequences, cDNAs or synthetic DNAs. The DNA molecules can be double-stranded or single-stranded, and if single stranded may be the coding strand. In addition, the polynucleotides can be RNA molecules such as mRNAs.
The present invention also provides non-coding strands (antisense) which are complementary to the coding sequences as well as RNA sequences identical to or complementary to those coding sequences. One of ordinary skill in the art will readily appreciate that corresponding RNA sequences contain uracil (U) in place of thymidine (T).
In one embodiment, the polynucleotide of the present invention is an isolated and purified cDNA molecule that contains the coding sequence of human BIgR. An exemplary SUBSTITUTE SHEET (RULE 26) cDNA molecule is shown as SEQ ID NO:1. In a second embodiment, the polynucleotide of the present invention is an isolated and purified cDNA molecule that contains the coding sequence of mouse BIgR. An exemplary cDNA molecule is shown as SEQ ID N0:3.
As is well known in the art, because of the degeneracy of the genetic code, there are numerous other DNA and RNA molecules that can code for the same polypcptidcs as those encoded by SEQ ID NO: 1 or SEQ ID N0:3 or portions or fragments thereof. The present invention also contemplates homologous polynucleotides having at least 70%
homology to the sequence shown in SEQ ID NO:1 or SEQ ID N0:3, preferably at least 80%
homology, and most preferably at least 90% homology. The term "homology", as used herein, refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). A partially complementary sequence is one that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid; it is referred to using the functional term "substantially homologous." The inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or Northern blot, solution hybridization and the like) under conditions of low stringency. A substantially homologous sequence or probe will compete for and inhibit the binding (i.e., the hybridization) of a completely homologous sequence or probe to the target sequence under conditions of low stringency. This is not to say that conditions of low stringency are such that non-specific binding is permitted;
low stringency conditions require that the binding of two sequences to one another be a specific (i.e., selective) interaction. The absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% identity); in the absence of non-specific binding, the probe will not hybridize to the second non-complementary target sequence. Moreover, the present invention also contemplates naturally occurring allelic variations and mutations of the cDNA
sequence set forth above so long as those variations and mutations code, on expression, for the human or murine adhesion protein of the present invention. The present invention also encompasses splice variations of the human and murine BIgR polynucleotides.

SUBSTITUTE SHEET (RULE Z6) The polynucleotidc of the present invention can be used in marker-aided selection using techniques which arc well-known in the art. Marker-aided selection does not require the complete sequence of the gene. Instead, partial sequences can be used as hybridization probes or as the basis for oligonucleotide primers to amplify by PCR or other methods to identify nucleotides specific for human or murine adhesion proteins in other mammals.
IV. Poly~e tn ides The present invention also provides for polypeptides which encode for human BIgR
and mouse BIgR. The amino acid sequence for human BIgR protein is provided in SEQ ID
N0:2 and contains 398 amino acid residues. The amino acid sequence for the mouse BIgR
adhesion protein is provided in SEQ ID N0:4 and contains 396 amino acid residues.
The present invention also contemplates amino acid residue sequences that are substantially duplicative of the sequences set forth herein such that those sequences demonstrate like biological activity to the disclosed sequences. Such contemplated sequences include those sequences characterized by a minimal change in amino acid residue sequence or type (e.g., conservatively substituted sequences) which insubstantial change does not alter the basic nature and biological activity of the polypeptides.
It is well known in the art that modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide. For example, certain amino acids can be substituted for other amino acids in a given polypeptide without any appreciable loss of function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substitutents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like.
As detailed in United States Patent No. 4,554,101, incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues:
Arg (+3.0);
Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gln (+0.2); Gly (0); Pro (-0.5);
Thr (-0.4); Ala (-0.5); His (-0.5); Cys (-1.0); Met (-1.3); Val (-1.5); Leu (-1.8); IIe (-1.8); Tyr (-2.3); Phe (-2.5); and Trp (-3.4). It is understood that an amino acid residue can be SUBSTITUTE SHEET (RULE 26) substituted for another having a similar hydrophilicity value (c.g., within a value of plus or minus 2.0) and still obtain a biologically equivalent polypeptide.
In a similar manner, substitutions can be made on the basis of similarity in hydropathic index. Each amino acid residue has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those hydropathic index values arc:
Ile (+4.5); Val (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4);
Thr (-0.7); Ser (-0.8); Trp (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gln (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In making a substitution based on the hydropathic index, a value of within plus or minus 2.0 is preferred.
V. Recombinant Vectors The present invention also relates to recombinant vectors which contain the polynucleotide of the present invention, host cells which are genetically engineered with recombinant vectors of the present invention and the production of the polypeptide of the present invention by recombinant techniques.
The polynucleotide of the present invention can be employed for producing polypeptides using recombinant techniques which are well known in the art. For example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. One of the most popular vectors for obtaining genetic elements is from the well known cloning vector pBR322 (available from the American Type Culture Collection, Manassas, Virginia as ATCC Accession Number 37017). The pBR322 "backbone" sections can be combined with an appropriate promoter and the structural sequence to be expressed. However, any other vector may be used as long as it is replicable and viable in the host.
SUBSTITUTE SHEET (RULE 26) The polynucleotide sequence of the present invention may be inserted into one of the hereinbcforc mentioned recombinant vectors, in a forward or reverse orientation. A variety of procedures, which are well known in the art may be used to achieve this. In general, the polynuclcotidc is inserted into an appropriate restriction endonucleasc site(s).
When inserted into an appropriate expression vector, the polynucleotidc of the present invention is operatively linked to an appropriate expression control sequence(s), such as a promoter, to direct mRNA synthesis. As used herein, the term "operatively linked" includes reference to a functional linkage between a promoter and a second sequence, wherein the promoter sequence initiates and mediates transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleotide sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in the same reading frame. The heterologous structural sequence can encode a fusion protein including either an N-terminal or C-terminal identification peptide imparting desired characteristics, such as stablization or simplified purification of expressed recombinant product.
Promoter regions can be selected from any desired gene using chloramphenicol transferase (CAT) vectors or other vectors with selectable markers. Such promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), a-factor, acid phosphatase, or heat shock proteins. Examples of bacterial promoters which can be used include, but are not limited to, IacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Examples of other promoters that can be used include the polyhedrin promoter of baculovirus.
Typically, recombinant expression vectors contain an origin of replication to ensure maintenance of the vector. They preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Examples of selectable marker genes which can be used include, but are not limited to, dihydrofolate reductase, SUBSTITUTE SHEET (RULE 26) neomycin or blasticidin resistance for eukaryotic cell culture, tetracycline or ampicillin resistance for E. colt. and the TRP 1 gene for S. cerevi.riac. The expression vector may also contain a ribosome binding site for translation initiation and a transcription termination segment. The vector may also include appropriate sequences for amplifying expression.
Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described in Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., ( 1989), which is herein incorporated by reference. Large numbers of suitable vectors and promoters are commercially available and can be used in the present invention. Examples of vectors which can be used include, but are not limited to:
Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDlO, phagescript, psiX174, pBluescript SK, pSKS, pNHBA, pkrH 16a, pNH 18A, pNH46A (Stratagene), ptrc99a, PKK223-3, pKK233-3, pDR540, pRITS (Pharmacia), pGEM (Promega). Eukaryotic: pWLNEO, pSV2CAT, p)G44, pXTI, pSG (Stratagene), pSVK3, pBPV, pMSG, pSVL (Pharmacia), pcDNA3, pcDNA6 (InVitrogen).
In another embodiment, the present invention relates to host cells containing the hereinbefore described recombinant vectors. The vector (such as a cloning or expression vector ) containing the hereinbefore described polynucleotide, may be employed to transform, transduce or transfect an appropriate host to permit the host to express the protein.
Appropriate hosts which can be used in the present invention, include, but are not limited to prokaryotic cells such as E. colt, Streptomyces, Bacillus subtilis, Salmonella typhimurium, as well as various species within the general Pseudomonas, Streptomyces, and Staphylococcus.
Lower eukaryotic cells such as yeast and insect cells such as Drosophila S2 and Spodoptera Sf9 and Sf21. Introduction of the recombinant construct into the host cell can be effected by calcium phosphate, DEAE-Dextran or liposome mediated transfection, or electroporation (see, Davis, L., Dibner, M., Battey, L. Basic Methods in Molecular Biology, ( 1986), herein incorporated by reference).
Various higher eukaryotic cells such as mammalian cell culture systems can also be employed to express recombinant protein. Examples of mammalian expression systems SUBSTITUTE SHEET (RULE 26) include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, C.'ell, 23:175 ( I 981 ), and other cell lines capable of expressing a compatible vector, for example, the C 127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors will contain an origin of replication, a suitable promoter and enhancer, and any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribcd sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying genes encoding for the human functional adhesion protein of the present invention. The culture conditions, such as temperature, pH and the like, are those previously used with the host cell selected for expression and can be determined experimentally, using techniques which are well known in the art.
Transcription of the polynucleotide encoding the polypeptides of the present invention by higher eukaryotes can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA which are about from 10 to about 300 base pairs in length, which act on a promoter to increase its transcription.
Examples of suitable enhancers which can be used in the present invention include the SV40 enhancer on the late side of the replication origin base pairs 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter is induced by appropriate means (such as temperature shift or chemical induction) and cells are cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification. Microbial cells employed in expression of proteins can be disrupted by any convenient method, including freeze-thaw SUBSTITUTE SHEET (RULE 26) cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods arc well-known to those skilled in the art.
The polypcptides of the present invention can be recovered and purified from recombinant cell cultures, the cell mass or otherwise according to methods of protein chemistry which are known in the art. For example, ammonium sulfate or ethanol precipitation, acid extraction, and various forms of chromatography e.g. anion / cation exchange, phosphocellulose, hydrophobic interaction, affinity chromatography including immunoaffinity, lectin and hydroxylapatite chromatography. Other methods may include dialysis, ultrafiltration, gelfiltration, SDS-PAGE and isoelectric focusing.
Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (hereinafter, "HPLC") on normal or reverse systems or the like, can be employed for final purification steps.
BIgR may be used to inactivate the endogenous gene by homologous recombination and thereby create a BIgR deficient cell, tissue or animal. Such cells, tissue or animals may then be used to define specific in vivo processes normally dependent upon BIgR.
The cDNA sequence can be used to prepare stable cell lines expressing either wt BIgR or BIgR mutated at pertinent positions to determine which part of the molecule is responsible for function. Stable or transient cell lines can be created with BIgR possessing a tag at either the 5' or 3' end, e.g. VS or HA epitope, to enable monitoring of BIgR
function/modification/cellular interactions.
The extracellular sequence of BIgR can be used to make recombinant protein fused to the Fc region of human or mouse IgG. This protein can be used:
a) To screen for a BIgR ligand. If BIgR is an adhesion protein, interactions with, but not limited to, leucocytes/neutrophils will be analyzed. Briefly, BIgR-Fc fusion can be captured on ELISA plates. Cultured cells, control or stimulated with inflammatory cytokines, can be labeled with calcien dye, incubated with the immobilized BIgR-Fc, SUBSTITUTE SHEET (RULE 26) washed and fluorescence monitored. Alternatively, BIgR can be coupled to a solid support and then used to prepare a column for purification of solubilized proteins derived from various cells/tissues. Peptide sequencing could then be used to identify the ligand. Another approach would be to bind the BIgR-Fc to cell lysates and perform cross-linking with DSS.
b) Upon identification of a BIgR ligand, the BIgR-Fc can be used to screen for a small molecule inhibitor of BIgR heterotypic or homotypic interactions.
c) As a tool to neutralize BIgR function, either heterotypic or homotypic interactions.
d) If BIgR displays homotypic/heterotypic interactions, the fusion can be used to bind to either BIgR or its ligand to initiate a cellular response.
Recombinant protein derived from the extracellular domain can be used to analyze homotypic interactions. Such protein would not possess a Fc Tag. Single immunoglobulin-like domains can be made to determine which one is responsible for homotypic interactions.
The interactions of the separate domains with each other or with a recombinant form possessing all three Ig-like domains may be assessed by various means.
Examples are cross-linking with DSS, analytical ultracentrifugation or sizing columns.
The BIgR sequence can be used to identify antisense oligonucleotides for inhibition of BIgR function in cell systems. Further, degenerate oligonucleotides may be designed to aid in the identification of additional members of this family by the polymerase chain reaction. Alternatively, low stringency hybridization of cDNA libraries may be performed with BIgR sequence to identify closely related sequences.
The intracellular domain of BIgR can be used to "fish" for novel interacting partners in the yeast two-hybrid system.
SUBSTITUTE SHEET (RULE 25) Additionally, recombinant purified BIgR, and cell lines expressing recombinant BIgR, can be used to screen for small molecule inhibitors of BIgR.
Apart from low expression in the placenta, BIgR is located exclusively in the brain.
BIgR may be located in glial, neuronal or endothelial cells. If expressed in brain endothelial cells then it may play a role in the blood brain barrier. If BIgR is an adhesion protein, it is believed that it might play roles in, but not be limited to, neuronal plasticity, growth cone guidance and neurite outgrowth. It might possibly be involved in stroke and multiple sclerosis.
VI. Antibodies The polypeptides of the present invention, fragments thereof, or cells expressing said polypeptides can be used as an immunogen to produce antibodies. These antibodies can be, for example, polyclonal or monoclonal antibodies. The present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of a Fab expression library.
Antibodies generated against the polypeptides of the present invention can be obtained by administering the polypeptides to an animal, preferably a nonhuman. Even a sequence encoding only a fragment of a polypeptide of the present invention can be used to generate antibodies binding to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used. Examples include the hybridoma technique (described by Kohler and Milstein, 1975, Nature, 256:495-497, herein incorporated by reference), the trioma technique, the human B-cell hybridoma technique (described by Kozbor et al., 1983, Immunology Today 4:72, herein incorporated by reference), and the EBV-hybridoma technique to produce human monoclonal antibodies (described by Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R.
Liss, Inc., pp-77-96, herein incorporated by reference).

SUBSTITUTE SHEET (RULE 26) For preparation of polyclonal antibodies any technique known in the art which provides such antibodies can be used.
Techniques for the production of single chain antibodies, such as those described in U.S. Patent 4,946,778, herein incorporated by reference, can be adapted to produce single chain antibodies to immunogenic polypeptides of the present invention.
In yet another embodiment, the present invention relates to a method for identifying human or murine cells expressing endogenous human or murine adhesion proteins.
The method involves identifying cells which express proteins having the same function as the human or murine adhesion proteins described herein and then characterizing the requisite cells producing said proteins using techniques known in the art. Antibodies, prepared pursuant to the techniques described herein, can be used to screen for BIgR
and to identify cells (diseased or normal) in a subject (human or animal) which express endogenous BIgR.
The antibodies of the present invention can be used to:
a) Probe subcellular localization/expression of endogenous BIgR in cells (diseased or normal) in a subject (human or animal).
b) Immunoprecipitate endogenous BIgR protein from cells or recombinant BIgR
from transfected cells, to determine whether it is modified by glycosylation, phosphorylation, etc. Co-precipitation of proteins that associate with BIgR
can also be identified in this manner.
c) As a tool to neutralize BIgR function, either heterotypic or homotypic interactions.
The anti-BIgR antibody may be administered in vivo in various animal models in order to perturb BIgR function. Alternatively, proof of concept studies may be conducted in vitro.
d) Map functional epitopes of the BIgR molecule.
By way of example, and not of limitation, examples of the present invention shall SU8ST1TUTE SHEET (RULE 26) now be given.
EXAMPLE I: CLONING OF HUMAN BIgR cDNA AND MAPPING OF THE BIgR GENE.
The polynucleotide sequences shown in SEQ ID NO:1 and SEQ ID N0:3 were cloned using a combination of electronic and conventional cloning techniques.
The electronic techniques used involved utilizing the Expressed Sequence Tag (EST) databases accessed through the home page of the National Center for Biotechnology Information (NCBI) at www.ncbi.nlm.nih.gov. As a template for electronic cloning the cDNA sequence of a novel mouse Junctional Adhesion Protein (JAM) published by Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E, J. Cell. Biol. ( 1998) 142( 1 ): 117-27, herein incorporated by reference, was used. The mouse JAM cDNA sequence is also available on GenBank (Accession No. U89915). The advanced Basic Local Alignment Search Tool (BLAST
2.0) was used to identify ESTs displaying homology with mouse JAM.
Electronic Cloning The complete mouse JAM peptide sequence (Acc. No. U89915) was searched for homology with human EST sequences using the tblastn program which compares a protein query sequence against a nucleotide sequence database dynamically translated in all reading frames. The complete mouse JAM protein sequence is 300 amino acids in length.
The initiation codon begins at base pair (hereinafter "bp") 71 and the stop codon at 971 (see Fig.
1 A).
Some 116 amino acids encoded within EST 888252 showed 38% similarity and 28%
identity over a 129 amino acid stretch of mouse JAM (see Fig 1A). A subsequent search of the SWISS-PROT protein sequence database revealed that this EST displayed similarities to other known adhesion proteins. Based on this, and the conservation of cysteine residues important in the formation of the immunoglobulin-like fold, EST 888252 was analyzed further. Throughout the assembly of the virtual sequence, the translation was continually monitored in all reading frames in order to identify the putative codons for initiation and SUBST1TL1TE SHEET (RULE 25) termination of the virtual protein. Where available, examination of overlapping ESTs was conducted to verify sequence/errors.
The 5' 150 by of 888252 was blasted through the nr human dbEST (a non-redundant sub-category of the EST database containing only human ESTs) using the blastn program.
EST T08949 showed 100% identity over a 109 by overlap and gave the most additional 5'-sequence (see Fig. 1 B). A blastn analysis of T08949 through the nr human EST
database was unsuccessful in isolating additional bases at the S' end. Thus, a putative initiation codon could not be identified for this protein by this method.
The final 120 by of 888252 was blasted through the nr human dbEST using the blastn program. H 14720 showed 97% identity over 229 base pairs and gave 147 additional bases at the 3' end (see Fig. 1 B).
The 3' 106 by of H 14720 was blasted through the nr human dbEST using the blastn program. The EST 815338 showed 95% identity over 170 base pairs and lengthened the novel sequence by 367 bases (see Fig. 1B). Within this sequence a putative stop codon was identified. Thus, further searching of the database was terminated.
Conventional Cloning In order to obtain further 5' sequence for this cDNA, a RACE technique was performed. Human fetal brain mRNA was reverse transcribed with AMV reverse transcriptase (Clontech Palo Alto, CA.) at 42°C. Amplification reactions were performed using the Marathon cDNA amplification kit (Clontech) and either of two antisense primers (see below). The following program utilized: 1 cycle at 94°C, 30sec; 5 cycles at 94°C, Ssec and 72°C, 4min; 5 cycles at 94°C, Ssec and 70°C, 4min; 25 cycles at 94°C, Ssec and 68°C, 4min. Products were ligated into the pCR-Blunt II-TOPO vector (Invitrogen) and sequenced using an ABI sequencer (Seqwright, TX).
The primer used for the first RACE reaction was directed at a site 176 by into the EST T08949 (5'- CCCAGAAGACTGACAGTTTAGGGTGGCTG-3') (SEQ ID NO:S). A

SUBSTITUTE SHEET (RULE 26) product from this reaction allowed an additiona1~272 by of BIgR to be isolated (see Fig. 1C).
The primer used for the second RACE reaction was directed at a site 86 by into the EST
T08949 (S'-GCGCAGTGACGAGGGACTTGGCAGTTC-3') (SEQ ID N0:6). The longest product sequenced gave an cxtcnsion of 232 by from the S' end of T08949 by (see Fig. 1 C).
Most interestingly, an alignment of the sequences from each RACE reaction showed that at some 204 nucleotides upstream of the 5' end of T08949, the sequences diverged (see Fig. 3). Neither product possessed a putative consensus initiation ATG with upstream stop codon.
Coincident with this result, further searching of the databases showed that a recent release of GenBank (March 4'~' 1999) contained a cDNA sequence (Accession No.
AF062733) the alignment of which showed that the product of RACE reaction 1 fell 41 amino acids short of the full-open reading frame clone (see Fig. 2). Further, a comparison of the sequence obtained from RACE reaction 2 demonstrated that a splice variant of AF062733 with a 34 amino acid deletion (see Fig. 2) had been isolated.
Construction of a Full-Length BIg.R
In order to construct full-length human BIgR, a sense oligonucleotide directed against Accession No. AF062733 in the 5'-untranslated region was designed, some 45 by upstream of the ATG. This oligonucleotide 5'-TTCAGGCTCGCCAGCGCCCAG-3' (SEQ ID N0:7) was coupled with an antisense primer 5'- TAGATGAAATATTCCTTCTTGTCGTC-3' (SEQ ID N0:8) that incorporated the TAG stop codon. Human fetal brain mRNA was reverse transcribed and amplified using the following program: 1 cycle, 95°C for 7 min; 35 cycles, 95°C for 20 s, 60°C for 20 s, 72°C for 20 s; 1 cycle, 72°C for 5 min. Products were ligated into an E. coli vector and sequenced using an ABI sequencer (Seqwright, TX).
Sequence Features The polymerase chain reaction allowed the identification of two different full-length isoforms of BIgR. Consistent with the RACE reaction, clones were isolated with an SU8ST1TUTE SHEET (RULE 26) additional 34 amino acids just following the signal sequence, and clones in which these residues were spliced out. In Figure 3, the complete coding region of the shorter splice variant that possesses 398 amino acids is displayed. Below this, the sequence corresponding to the 34 amino acid (SEQ ID NO:11 ) insert is shown. Sequencing proved these residues to be identical to those of GenBank accession AF062733. BIgR features a putative signal sequence (underlined), three immunoglobulin-like folds, a single transmcmbrane domain (underlined) and a short intracellular domain (see Fig. 3). Thus, the protein belongs to the immunoglobulin superfamily. The cysteine residues predicted to form disulfide bonds within each immunoglobulin-like domain are highlighted. The 1 S' and 2"d cysteine are located in the first, the 3'd and 4'" in the second, and the 5'" and 6'" in the third immunoglobulin-like fold respectively. Highlighted are two consensus N-linked glycosylation sites (NxS/T) at amino acid 25 and 353 (although the first falls within the predicted signal sequence).
Differences between BIgR poly,~eptide and GenBank Accession No. AF062733 The BIgR polypeptide shown in SEQ ID N0:2 is different from AF062733 at two places. First, the polypeptide of the present invention is a smaller isoform because it has 34 amino acids which are deleted (see Figs. 2, 3). Second, a single glutamic acid deletion occurs at position 104 in SEQ ID N0:2, regardless of whether the 34 amino acids are retained or spliced out. Thus SEQ ID N0:2 reads, from amino acid 100, ALAD EGEY while reads ALADEEGEY in this same region.
Proteins Homologous to BIgR Extracellular and Intracellular Domains BIgR is not a member of the family of functional adhesion molecules.
Nevertheless, a blast analysis demonstrates that its various immunoglobulin-like domains show similarity to other novel putative adhesion molecules.
Most interestingly, the intracellular domain of BIgR showed 66% similarity and 53%
identity to glycophorin C, a protein required for anchoring protein 4.1 in red blood cells.
Further, this homology is also displayed with the Drosophila transmembrane protein Neurexin IV, which is required for localization of the drosophila 4.1 protein, coracle, to SUBSTITUTE SHEET (RULE 26) WO 01/29083 PCT/USUO/28b42 septate junctions (see Fig. 4).
Chromosomal Mat~aing Segments of chromosomal sequence, identical to BIgR cDNA, were retrieved from the public non-redundant database. Results required minor manual modification due to dual designation of isolated bases at the end of some exon boundaries. The correct designation was based on 5' and 3' splice-site consensus sequences. Using the public database, 10 exons for BIgR (see Table 1 below) were identified. The exon/intron structure was determined from Acc No. AL035403 which was derived from human chromosome 1q21.2-22. All intron/exon boundaries conform to the GT/AG rule. Most pertinent, the structure of the BIgR
gene shows that exclusion of exon 2 during splicing would result in the 34 amino acid deletion described here.
Table 1.
Intron/Exon Boundaries of human BIgR
3'splice Exon No. Exon 5'splice Intron (bp) (bp) nnnnnn/(N) 1 >_224 AGGACG/gtgagt 17,917 ccccag/GCTACT 2 103 GTCAAG/gtgaga 2,063 ctgcag/ACAGCC 3 141 AGAGAG/gtagta 511 ccccag/CCCTTC 4 153 TGCTAG/gtgaga 691 atggag/GAATTC 5 138 TCCACG/gtgagt 308 ctctag/GAGAAC 6 171 TTTTAT/gtatgt 2,323 ccacag/ACACAC 7 91 TCCAGT/gtaaga 435 cgacag/CCCCCA 8 170 TTAATG/gtaagc 2,679 ttccag/ACCCCA 9 126 ACAAAG/gtcaga 926 acacag/GAACCT 10 >1,208 TP~NNNN/(n) EXAMPLE II: CLONING OF MOUSE BIgR
A search of the databases (see Example 1 ) did not result in identification of any mouse ESTs encoding BIgR. Using the primers described in Example 1 to clone full length human BIgR, a product from mouse brain mRNA (Clontech) was amplified.
Sequencing of three independent PCR reactions showed that the isoform with a 34 amino acid deletion in the extracellular domain (see Fig. 5) was isolated. Fig. 6 shows that at the amino acid level the mouse and human sequences display 95% identity and 96% similarity. A
notable SUBSTITUTE SHEET (RULE 26) difference is the deletion of a serine and leucine, in the signal sequence.
Further, mouse BIgR
does not possess the additional glutamic acid residue in the ALAD_EGEY
sequence in the extracellular domain.
EXAMPLE III: EXPRESSION PATTERN OF BIgR
Tissue expression of BIgR was examined on a normalized human Multiple Tissue Northern blot (Clontech) with an [a-'ZP]dCTP labeled probe derived from the extracellular domain. The results show that BIgR is expressed as two transcripts of approximately 2.6 and 3.8 kb (see Fig. 7). The blot was probed at high stringency and thus these two species likely represent alternatively spliced products. Fig.7 shows that human BIgR is abundantly expressed in the brain with extremely low levels in the placenta. This has been independently confirmed in this latter tissue using PCR (see Table 2, below). To further localize BIgR, the expression in various regions of the brain (see Fig. 8) was examined. Clearly, the cerebellum expresses this gene to a higher level than other areas although expression is noted throughout. In the cerebellum an additional minor transcript of approximately 6.5 kb is apparent. The mRNA is barely detectable in the medulla and spinal cord. In all cases the 3.8 kb transcript is expressed to a higher level than the 2.6 kb species.
The EST database confirms that BIgR is a brain specific transcript that is expressed in both infant and adult brain (see Table 2, below). All EST sequence data was derived from brain cDNA libraries.
Table 2 Expression Characteristics of Human BIgR
mRNA Source EST, GenBank Acc. No. RT-PCR

Fetal brain +

Infant brain T08949, H14720, H08308, 243981, 836062, Adult brain 888252, T32071, H38530, 888963, H40798 MS lesions N45514 Placenta +

SUBSTITUTE SHEET (RULE 26) EXAMPLE IV: EXPRESSION OF EXTRACELLULAR DOMAIN IN INSECT
CELLS AND POLYCLONAL ANTIBODY GENERATION.
Oligonucleotides were designed to amplify the extracellular domain of human BIgR
from the full-length clone. Sense 5'- CCG A AT GCGATGGGGGCCCCA -3'(SEQ ID
N0:9) and antisense 5'-GATGGTACCGTGGTAGGTGCTGGAGGA-3' (SEQ ID NO: 10) oligonucleotides with EcoRV and KpnI restriction sites (underlined) respectively, were used to enable subcloning into a SacI/KpnI restricted pFastBac 1 (Life Technologies, GIBCO BRL, Grand Island, NY ) vector containing the constant region of mouse IgG-2a (Fc;
Cunningham SA, Tran TM, Arrate MP, Brock TA, (1999) J. Biol. Chem. 274:18421-7). This vector drives protein expression from the polyhedrin promotor.
Secreted recombinant BIgR-Fc was purified from the media of infected Sf21 cells using Hi-Trap protein A columns (Amersham Pharmacia Biotech., Piscataway, NJ).
The Fc-fusion was removed following thrombin digestion. A female New Zealand White rabbit ( 12-weeks old, Myrtle's Rabbitry, Thompson Station, TN) was immunized with SOO~g of BIgR
emulsified with an equal volume of Freund's Adjuvant. Six weeks thereafter, the animal was boosted with another SOO~g BIgR emulsified with Incomplete Freund's Adjuvant.
Serum was collected 14 days following the boost.
To test the specificity of the antibody, the BIgR cDNA was subcloned into the pcDNA6 vector and lOpg transfected into Cos cells using FuGENE 6 (Roche).
Three days following transfection the cells were lyzed in Tris buffered saline (pH 7.5) /
1% Triton X-100 with the inclusion of Protease Inhibitor Cocktail Set III (Calbiochem, La Jolla, CA). Figure 9 shows that the BIgR polyclonal serum detects a protein species of 46kDa in transfected Cos cells. The antibody does not cross-react with mock-transfected Cos cells.
EXAMPLE V: EXPRESSION OF THE FULL LENGTH CLONE IN MAMMALIAN
CELLS AND SUBCELLULAR LOCALIZATION.
The full-length clone of BIgR, in the pcDNA6 vector, was modified at its C-terminus by PCR mutagenesis to incorporate a VS-epitope Tag for additional detection purposes.

SUBSTITUTE SHEET (RULE 26) Some l0pg of pcDNA6-BIgR was transfected into CHO cells using FuGENE 6 (Ruche).
Three days following transfection, cells were split and maintained in 10 ug/ml Blasticidin for clone selection.
CHO-K1, control or expressing BIgR, grown on glass slides to confluence, were fixed with 1 % paraformaldehyde and stained with 1:10 times dilution of either preimmunc or anti-BIgR rabbit polyclonal serum. GAR-FITC at 1:100X was used as secondary.
Fluorescence was viewed using a NoranTM Confocal laser-scanning microscope (Noran Instruments, Middleton, WI) equipped with argon laser and appropriate optics and filter module for FITC
detection. Digital images were obtained at x400 optical magnification.
Figure 10 shows that BIgR partitions to sites of cell-cell contact in addition to distributing to the plasma membrane of CHO cells. This pattern of localization provides evidence towards BIgR operating through homotypic interactions. As previously mentioned, the intercellular distribution is a characteristic of the JAM family and thus this feature is maintained.
EXAMPLE VI: ADHESION OF BigR-Fc TO LEUKOCYTE CELL LINES.
In vitro adhesion assays were performed in 96 well plates essentially as described in Todderud, G., J. Leukoc. Biol., 52:85 (1992), herein incorporated by reference. Briefly, 50 ,u1 of goat anti-mouse IgG2a was coated at 5 ~cg/ml in PBS and used to capture 4.8 pmoles of BIgR-Fc or mIgG2a (control). Various leukocyte cell lines, i.e., T
lymphocytes, HSB, TK-1;
B-lymphocytes, RAMOS; monocytic cells, HL60 and the erythroleukemic, K562 lines were labeled with calcein (Molecular Probes Inc., Eugene, OR) at 50 ,ug/ml for 25 minutes at 37 °C with 250,000 cells/well in binding buffer that consisted of Tris buffered saline with and without the addition of 1mM MnCl2. Wells were washed 3X, lysed with 50 mM Tris (pH
7.5), 5 mM EDTA, 1 % NP40, and fluorescence read in a Cytofluor with excitation at 485/20 nm and emission at 530/25 nm. Specific binding was calculated as fluorescence with BIgR-Fc minus fluorescence with mIgG2a.
SUBSTITUTE SHEET (RULE 26) Fig. 1 1 shows that BIgR-Fc is able to capture the RAMOS B-lymphocyte cell line but not the T-lymphocyte, monocyte or crythroleukemic cells tested. The interaction of BIgR
with these cells was dependent upon the addition of manganese in the buffer.
These results support the hypothesis that BIgR is an adhesion protein and may operate by engaging with an integrin counter-receptor.
BIgR displays similarities to proteins of known function in both its intracellular and extracellular domain. However, BIgR aligns most closely throughout its length with IgSF4 (Ace. No. NP 055148) a putative adhesion protein of unknown function and ubiquitous expression localized on chromosome l 1q23.2 (Gomyo et al., Genomics 62:139-146, 1999).
Another member of this family would appear to be Acc. No. AAC32740: a hypothetical gene identified on chromosome 19q13.2. The percent identity for IGSF4 and AAC32740 is 38.5%
and 35% respectively. Thus these three proteins likely form a new family.
The function of BIgR can be extrapolated from its brain specific expression and its homologies to other proteins of known function in conjunction with its capacity to partition to inter-cellular membranes (see Figures 4, 7, 8, 10). BIgR shows 45%
similarity with the poliovirus receptor (PVR) from (3-sheet F in the first Ig fold through to the end of the transmembrane domain. The cellular function of PVR is unknown. However, the binding site for poliovirus appears to be contained within Ig-fold 1, and thus it is not likely that BIgR
shares this binding capacity. Other members of the PVR family are the poliovirus-related receptors (PRR), otherwise known as nectin-1, nectin-2 and nectin-3. Nectin-1 and nectin-2 serve as a-herpesvirus entry mediators. The nectin family are calcium independent homophilie adhesion molecules. They display cis-homo-dimerization in addition to trans-homointeraction and trans-heterointeraction. For nectin-2, Ig-fold 1 is crucial for adhesion of nectin-2 between cells, but not for cis homo-dimerization. Nectin-1 and nectin-2 show 43%
and 41% similarity with BIgR over amino acid overlaps of 237 and 231: this region corresponds mainly to Ig-folds 2 and 3. Nectin-3 shows 43% similarity over a 280 amino acid overlap encompassing all three Ig folds. Most interestingly, the nectin family localize to adherens junctions. Thus, the inventors believe that BIgR is capable of homotypic SUBSTITUTE SHEET (RULE 26) interactions, particularly in traps as evidenced by the cellular expression of BIgR in CHO
cells. Further, the inventors believe that heterotypic interactions for BIgR
either between closely related homologues or other adhesive cell surface proteins occurred (see Figure 11 ).
BIgR may even serve as a receptor for viral entry. In the brain, Ig cell adhesion molecules play roles in growth cone guidance, neurite outgrowth and synaptogenesis. BIgR
may participate in any of these processes.
As discussed in Example 1, the short intracellular domain of BIgR shares high homology with glycophorin C, drosophila neurexin IV and Caspr2. Homology is highest between the novel putative adhesion protein IgSF4 (see Table 3, below). These proteins all conserve a juxtamembrane binding site for members of the protein 4.1 family and all possess a PDZ binding motif at the extreme C-terminus (with the exception of Caspr). A
direct interaction between caspr and protein 4.1 in the brain has been demonstrated.
By analogy, a complex between BIgR intracellular domain and protein 4.1 is predicted. Most recently, brain specific expression of novel members of the protein 4.1 family has been documented. It has been suggested that band 4.1 in the brain is involved in the formation and maintenance of the synapse as a membrane skeletal component at presynaptic terminals in the cerebellum. It is conceivable that BIgR may be a component of such a complex. Further, mice lacking brain 4.1 have deficits in movement, coordination, balance and learning. Thus BIgR
may impinge on these pathways.
Band 4.1 belongs to a growing superfamily of proteins that possess a FERM
domain (NF2/ERM/4.1). A conserved property of the domain is its capacity to bind to the membrane-proximal region of the C-terminal cytoplasmic tail of proteins with a single transmembrane segment. It functions to connect cell surface transmembrane proteins to cytoskeletal molecules. BIgR may link to any of these superfamily members.

SUBSTITUTE SHEET (RULE 26) Table 3 Intracellular domainAcc. No. % identit % similarit Glycophorin C AAA52574 51 67 Caspr2 NP 054860 46 63 Neurexin droso hila AAF49951 46 57 Cas r NP 003623 18 22 IgSF4 AAC32740 77 84 I SF4-similar AAC32740 43 59 Proteins containing PDZ domains are predominantly localized to the plasma membrane and are recruited to specialized sites of cell-cell contact. PDZ
domains are found in diverse membrane-associated proteins including members of the membrane-associated guanylate kinase (MAGUK) family, several protein phosphatases and kinases, neuronal nitric oxide synthase, and several dystrophin-associated proteins. BIgR may interact with PDZ
domains of proteins in any of these categories.
Members of the MAGUK protein family use multiple domains to cluster ion channels, receptors, adhesion molecules and cytosolic signaling proteins at synapses and cellular junctions. They play a fundamental organization role at both the pre-and postsynaptic plasma membranes. Thus, it is possible that BIgR is localized to these structures and functions, either directly or indirectly, in the efficient transmission of signals from the presynaptic terminal. In support of a PDZ domain interaction for BIgR is the established interactions of Neurexin IV with Discs Lost (DLT), mutations in which lead to aberrant localization of Neurexin IV and concomitant loss of epithelial cell polarity.
Another example is provided for by glycophorin C interactions with p55 in the ternary complex with protein 4.1.
Whilst neurexins are found in presynaptic sites and mediate interactions between neurons, Caspr2 localizes to juxtaparanodal regions of myelinated nerves and is thought to mediate neuron-glia interactions. Therefore, it is highly likely that BIgR is recruited to these sites and participates in the axo-glial intercellular junction. Once again, the intercellular localization of BIgR when expressed in CHO cells supports this possibility. It has been SUBSTITUTE SHEET (RULE 26) proposed that the axo-filial junctions function in the establishment and maintenance of axolemmal protein domains of the nodal and paranodal regions. In fact, Caspr2 is found to associate with K+ channels in this region, an interaction dependent upon it C-terminal PDZ
binding domain motif. It may be hypothesized that BIgR plays a similar role in channel clustering.
Finally, if BIgR were found to be transcribed in endothelial cells, the inventors believe that it would play a role in maintenance of the blood brain barrier, similar to the function of drosophila neurexin IV in septate junctions. It may also participate in inflammatory reactions of the brain such as occur during stroke and multiple sclerosis.
The present invention is illustrated by way of the foregoing description and examples.
The foregoing description is intended as a non-limiting illustration, since many variations will become apparent to those skilled in the art in view thereof. It is intended that all such variations within the scope and spirit of the appended claims be embraced thereby.
Changes can be made to the composition, operation and arrangement of the method of the present invention described herein without departing from the concept and scope of the invention as defined in the following claims.

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<213> murine <220>
<221> CDS
<222> (25)..(1212) <400> 3 ccggggaggt ggccaggaag cgcg atg ggg gcc cct tcc gcc ctg ccc ctg 51 Met Gly Ala Pro Ser Ala Leu Pro Leu ctc ctg ctc ctc gcc tgc tcc tgg gcg ccc ggc ggg gcc aat ctt tcc 99 Leu Leu Leu Leu Ala Cys Ser Trp Ala Pro Gly Gly Ala Asn Leu Ser cag gac gat agc cag ccc tgg aca tct gat gaa aca gtt gtg get ggt 147 Gln Asp Asp Ser Gln Pro Trp Thr Ser Asp Glu Thr Val Val Ala Gly ggc aca gtg gtt ctc aag tgt caa gta aaa gac cat gaa gac tca tct 195 Gly Thr Val Val Leu Lys Cys Gln Val Lys Asp His Glu Asp Ser Ser ctg cag tgg tct aac cct get cag cag acc cta tac ttc ggg gag aag 243 Leu Gln Trp Ser Asn Pro Ala Gln Gln Thr Leu Tyr Phe Gly Glu Lys aga gcc ctt cga gat aat cgg att cag ctg gtt agc tct act ccc cat 291 Arg Ala Leu Arg Asp Asn Arg Ile Gln Leu Val Ser Ser Thr Pro His gag ctc agc atc agc atc agc aat gtg gcg ctg gcc gat gag ggg gag 339 Glu Leu Ser Ile Ser Ile Ser Asn Val Ala Leu Ala Asp Glu Gly Glu tac acg tgc tcc atc ttc act atg cct gtg cga acc gcc aag tcc ctt 387 Tyr Thr Cys Ser Ile Phe Thr Met Pro Val Arg Thr Ala Lys Ser Leu gtc act gtg ctc gga atc cca cag aaa ccc ata atc act ggt tat aag 435 Val Thr Val Leu Gly Ile Pro Gln Lys Pro Ile Ile Thr Gly Tyr Lys tca tca ttg cgg gaa aag gag aca gcc act cta aat tgt cag tct tct 483 Ser Ser Leu Arg Glu Lys Glu Thr Ala Thr Leu Asn Cys Gln Ser Ser ggg agc aaa cct gca gcc cag ctc acc tgg agg aaa ggt gac caa gaa 531 Gly Ser Lys Pro Ala Ala Gln Leu Thr Trp Arg Lys Gly Asp Gln Glu ctc cac ggg gac caa aca cga atc cag gaa gat ccc aac ggg aaa acc 579 Leu His Gly Asp Gln Thr Arg Ile Gln Glu Asp Pro Asn Gly Lys Thr SU6STiTUTE SHEET (RULE 25) Page 6 of 10 ttc act gtg agc agc tca gtg tca ttc cag gtt acc cgg gag gat gat 627 Phe Thr Val Ser Ser Ser Val Ser Phe Gln Val Thr Arg Glu Asp Asp gga gca aac atc gtg tgc tct gtg aac cat gaa tct ctg aag gga gcc 675 Gly Ala Asn Ile Val Cys Ser Val Asn His Glu Ser Leu Lys Gly Ala gac aga tcc act tct cag cgc att gaa gtg tta tac aca cca aca gcc 723 Asp Arg Ser Thr Ser Gln Arg Ile Glu Val Leu Tyr Thr Pro Thr Ala atg att agg cca gaa cct get cat cct cga gaa ggc cag aag ctg ttg 771 Met Ile Arg Pro Glu Pro Ala His Pro Arg Glu Gly Gln Lys Leu Leu tta cat tgt gag ggg cgt ggc aat cca gtc ccc cag cag tac gtg tgg 819 Leu His Cys Glu Gly Arg Gly Asn Pro Val Pro Gln Gln Tyr Val Trp gta aag gaa ggc agt gag cca ccc ctc aag atg acc caa gag agt get 867 Val Lys Glu Gly Ser Glu Pro Pro Leu Lys Met Thr Gln Glu Ser Ala ctc atc ttc ccc ttt ttg aat aag agt gac agt ggc act tat ggc tgt 915 Leu Ile Phe Pro Phe Leu Asn Lys Ser Asp Ser Gly Thr Tyr Gly Cys aca gcc aca agc aac atg ggc agc tat aca gcc tac ttc acc ctc aat 963 Thr Ala Thr Ser Asn Met Gly Ser Tyr Thr Ala Tyr Phe Thr Leu Asn gtc aac gac ccc agt cca gtg ccc tcg tcc tcc agt acc tac cac gcc 1011 Val Asn Asp Pro Ser Pro Val Pro Ser Ser Ser Ser Thr Tyr His Ala atc att gga ggg att gtg get ttc att gtc ttc ctg ctg ctc att ctg 1059 Ile Ile Gly Gly Ile Val Ala Phe Ile Val Phe Leu Leu Leu Ile Leu ctc att ttc ctt gga cac tat ttg atc cgg cac aaa gga acc tac ctg 1107 Leu Ile Phe Leu Gly His Tyr Leu Ile Arg His Lys Gly Thr Tyr Leu aca cac gaa gcg aag ggc tcc gac gat get cca gat gcg gat acg gcc 1155 Thr His Glu Ala Lys Gly Ser Asp Asp Ala Pro Asp Ala Asp Thr Ala atc atc aac gca gaa ggc ggg cag tca ggc ggg gat gac aag aag gaa 1203 Ile Ile Asn Ala Glu Gly Gly Gln Ser Gly Gly Asp Asp Lys Lys Glu SUBSTITUTE SHEET (RULE 26) Pagc 7 of 10 tat ttc atc tag 1215 Tyr Phe Ile <210> 4 <211> 396 <212> PRT
<213> murine <400> 4 Met Gly Ala Pro Ser Ala Leu Pro Leu Leu Leu Leu Leu Ala Cys Ser Trp Ala Pro Gly Gly Ala Asn Leu Ser Gln Asp Asp Ser Gln Pro Trp Thr Ser Asp Glu Thr Val Val Ala Gly Gly Thr Val Val Leu Lys Cys Gln Val Lys Asp His Glu Asp Ser Ser Leu Gln Trp Ser Asn Pro Ala Gln Gln Thr Leu Tyr Phe Gly Glu Lys Arg Ala Leu Arg Asp Asn Arg Ile Gln Leu Val Ser Ser Thr Pro His Glu Leu Ser Ile Ser Ile Ser Asn Val Ala Leu Ala Asp Glu Gly Glu Tyr Thr Cys Ser Ile Phe Thr Met Pro Val Arg Thr Ala Lys Ser Leu Val Thr Val Leu Gly Ile Pro Gln Lys Pro Ile Ile Thr Gly Tyr Lys Ser Ser Leu Arg Glu Lys Glu Thr Ala Thr Leu Asn Cys Gln Ser Ser Gly Ser Lys Pro Ala Ala Gln Leu Thr Trp Arg Lys Gly Asp Gln Glu Leu His Gly Asp Gln Thr Arg Ile Gln Glu Asp Pro Asn Gly Lys Thr Phe Thr Val Ser Ser Ser Val Ser Phe Gln Val Thr Arg Glu Asp Asp Gly Ala Asn Ile Val Cys Ser Val Asn His Glu Ser Leu Lys Gly Ala Asp Arg Ser Thr Ser Gln Arg Ile Glu Val Leu Tyr Thr Pro Thr Ala Met Ile Arg Pro Glu Pro Ala SUBSTITUTE SHEET (RULE 26) Pagc 8 of 10 His Pro Arg Glu Gly Gln Lys Leu Leu Leu His Cys Glu Gly Arg Gly Asn Pro Val Pro Gln Gln Tyr Val Trp Val Lys Glu Gly Ser Glu Pro Pro Leu Lys Met Thr Gln Glu Ser Ala Leu Ile Phe Pro Phe Leu Asn Lys Ser Asp Ser Gly Thr Tyr Gly Cys Thr Ala Thr Ser Asn Met Gly Ser Tyr Thr Ala Tyr Phe Thr Leu Asn Val Asn Asp Pro Ser Pro Val Pro Ser Ser Ser Ser Thr Tyr His Ala Ile Ile Gly Gly Ile Val Ala Phe Ile Val Phe Leu Leu Leu Ile Leu Leu Ile Phe Leu Gly His Tyr Leu Ile Arg His Lys Gly Thr Tyr Leu Thr His Glu Ala Lys Gly Ser Asp Asp Ala Pro Asp Ala Asp Thr Ala Ile Ile Asn Ala Glu Gly Gly Gln Ser Gly Gly Asp Asp Lys Lys Glu Tyr Phe Ile <210> 5 <211> 29 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 5 cccagaagac tgacagttta gggtggctg 29 <210> 6 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer SUBSTIfTUTE SHEET (RULE 26) Page 9 of 10 <400> 6 gcgcagtgac gagggacttg gcagttc 27 <210> 7 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 7 ttcaggctcg ccagcgccca g 21 <210> 8 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 8 ctagatgaaa tattccttct tgtcgtc 27 <210> 9 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 9 ccggatatcg cgatgggggc ccca 24 <210> 10 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 10 gatggtaccg tggtaggtgc tggagga 27 <210> 11 <211> 34 SUBSTITUTE SHEET (RULE 26) Page 10 of 10 <212> PRT
<213> Homo sapiens <400> 11 Gly Tyr Trp Gln Glu Gln Val Leu Glu Leu Gly Thr Leu Ala Thr Leu Asp Glu Ala Ile Ser Ser Thr Val Trp Ser Ser Pro Asp Met Leu Ala Ser Gln SUBSTtTUTE SHEET (RULE 26)

Claims (11)

WHAT IS CLAIMED IS:

1. An isolated and purified polynucleotide comprising SEQ ID NO:1.

2. An isolated and purified polynucleotide comprising SEQ ID NO:3.

3. An isolated and purified polypeptide comprising an amino acid sequence of SEQ
ID NO:2 or fragment thereof.

4. An isolated and purified polypeptide comprising an amino acid sequence of SEQ
ID NO:4 or fragment thereof.

5. A recombinant vector comprising a polynucleotide having a nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3 or a fragment thereof, said polynucleotide being operatively linked to a promoter that controls expression of said polynucleotide sequence and a termination segment.

6. The vector of claim 5 wherein the promoter is a LTR, SV40, E. coli, lac, trp, or phage lambda P L promoter.

7. A host cell comprising the recombinant vector of claim 5.

8. The host cell of claim 7 wherein the host cell is a bacterial cell, an animal cell or a plant cell.

9. A transgenic mammal comprising a null mutation of the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

10. A transgenic mammal overexpressing the nucleotide sequence of SEQ ID NO:1 or SEQ ID NO:3.

11. An antibody binding to the polypeptide of claims 3 or 4.