DESCRIPTION

Technical Field of the Invention

This invention relates to a gene encoding a hyalu- ronan-binding protein that is restricted to the central nervous system, the polypeptide encoded by the gene, and methods for using the polypeptide.

Background of the Invention

The central nervous system extracellular matrix consists of a heterogenous mixture of glycoconjugates, many of which are proteoglycans (Jaworski, D. . , et al . , J. Cell Biol . 125 : 495-509 (1994), the full text of which is hereby incorporated herein in its entirety by refer- ence) . Proteoglycans are complex acromolecules that consist of a core protein modified with one or more types of glycosaminoglycan chains.

Many functional properties of proteoglycans have been ascribed to glycosaminoglycans (iJi .). Glycosa- minoglycans have been reported to exhibit both adhesive and repulsive properties and, as such, have been suggest¬ ed to mediate neuronal migration and axon guidance. Glycosaminoglycans are believed to regulate the local cellular environment primarily by serving as selective filters, facilitating permeability and retention of low molecular weight solutes, including growth factors, while excluding other macromolecules.

Hyaluronan (also called hyaluronic acid or hyal- uronate, and herein abbreviated HA) is particularly suit¬ ed to this function because of its charge density and hydroscopic nature. HA is a negatively charged high- molecular-weight linear polysaccharide built from repeat¬ ing diεaccharide units (Laurent, I . . , and Fraser, J.R.E., FASEB (Fed . Am . Soc. Exp. Biol . ) 6 : 2397-2404 (1992)). Hyaluronan is ubiquitously distributed in the extracellular matrices of all tissues, including brain, and is believed to have several functions, including the organization of water and extracellular proteins (ibid . ) . During development, HA plays a role in the regulation of morphogenesis and differentiation of neural tissues.

Because HA is ubiquitously present in extracellu- lar space, cell type specific functions attributed to HA may be mediated through its interaction with HA-binding proteins, which not only bind HA but can also contain potential binding sites for other molecules. Several HA- binding proteins in the brain have been reported, a sub- set of which have a high degree of sequence similarity to one another, including versican (Zimmermann, D.R. , and Ruoslahti, E., EMBO (Eur. Mol . Biol . Organ . ) J. 8 : 2975- 2981 (1989)), link protein (Doege, K. , et al . , Proc. Natl . Acad. Sci . USA 83 : 3761-3765 (1986)), neurocan (Rauch, U. , et al . , J. Biol . Chem. 267 : 19536-19547 (1992)), glial hyaluronate binding protein (GHAP, Perides, G. , et al . , J. Biol . Chem . 264 : 5981-5987 (1989)), and CD44 (Culty, M. , et al . , J. Cell Biol . Ill : 2765-2774 (1990)). These have been called the proteogly- can tandem repeat (PTR) family of HA-binding protein.

The spatial distribution and temporal expression of neural extracellular matrix proteoglycans and HA-bind¬ ing proteins indicate that they may be involved in many events in the development and function of the mammalian central nervous system (Jaworski, et al . , cited above)

and in the modulation of cell-cell and cell-matrix inter¬ actions. While some HA-binding proteins represent gener¬ al components of the extracellular matrix, others have a restricted pattern of expression on subsets of neurons. In addition, while some extracellular matrix molecules are transiently expressed during embryogenesis, others are first expressed late in the postnatal period, coin¬ cident with the decline in developmental synaptic plas¬ ticity.

It would be desirable to isolate an HA-binding protein specific to a particular tissue or organ, espe¬ cially where expression of the protein varied with patho¬ logical states so that it could be used as a marker for diagnostic purposes.

Summary of the Invention

It is an object of the invention to provide a gene encoding a mammalian hyaluronan-binding protein and to elucidate the relationship of the structure of the pro¬ tein encoded by the gene to other polypeptides, especial- ly other hyaluronan-binding proteins.

It is another and more specific object of the invention to provide a gene encoding a mammalian hyaluro¬ nan-binding protein that is restricted to central nervous system tissue and the polypeptide encoded by the gene.

These and other objects are accomplished by the present invention which provides purified and isolated DNA fragments comprising DNA sequences encoding mammalian brain enriched hyaluronan binding protein (herein denoted BEHAB) , the polypeptide structures they encode, and the relationship of the structures to other polypeptides.

Also provided are RNA sequences corresponding to the DNA sequences of the genes, biologically functional plasmids

or vectors comprising the DNA or RNA sequences, and pro- caryotic or eucaryotic host cells transformed or trans- fected with the plas ids or vectors in a manner allowing the host cell to express the polypeptides.

DNA sequences encoding rat and cat BEHAB are cloned, characterized, and sequenced, and the putative amino acid sequences of the polypeptides encoded by the open reading frame are determined (SEQ ID NOs 1 and 2) and human BEHAB partially sequenced (SEQ ID NO 7) . The sequence exhibits long stretches of identity between species, suggesting that the encoded protein is function¬ ally important. Unlike other hyaluronan-binding pro¬ teins, the expression of BEHAB DNA is restricted to the central nervous system, and markedly increases in glioma. Thus, the protein can be employed as a diagnostic marker for the detection of brain tumors and other neuropatho- logical states, and the invention encompasses methods of detection of BEHAB in biological samples.

Brief Description of the Figure

Figure 1 sets out sequence alignments of portions of rat BEHAB (SEQ ID NO 1) , portions of cat BEHAB (SEQ ID NO 2) , rat aggrecan (SEQ ID NO 3), rat neurocan (SEQ ID NO 4) , human versican (SEQ ID NO 5) , and rat link protein (SEQ ID NO 6) . To illustrate homologous sequences, the figure employs standard one-letter nomenclature for the amino acids: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, lie; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gin; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; and Y, Tyr. Identical amino acids are shown in black, and amino acid similarity is shown using gray counter-shad¬ ing. The PTR proteins contain three functional domains: an immunoglobulin fold (A) , and two domains thought to be involved in hyaluronan binding, PTR1 (B) and PTR2 (C) .

Detailed Description of the Invention

This invention is based upon the identification of a new hyaluronan-binding protein, denoted BEHAB for Brain Enriched Hyaluronan Binding protein, that is restricted to the brain.

By "hyaluronan-binding" protein is meant a protein that binds hyaluronan, a viscous mucopolysaccharide having the structure [D-glucuronic acid (l-β-3)N-acetyl- D-glucosamine(l-β-4) ] _n (Laurent and Fraser, cited above). As described in the Examples that follow, the hyaluronan- binding proteins of this invention are restricted to central nervous system tissues, found in both white and gray matter, and are not detected in liver, kidney, spleen, lung, muscle or other tissues. Expression is elevated in human brain glioma, but is not detected in non-brain tumors, including breast, lung, and colon. The BEHAB gene encodes a neural specific protein that binds hyaluronan but lacks a transme brane domain.

The expression of BEHAB mRNA is developmentally regulated; expression is first detected in the late embryonic period and peaks during the first two postnatal weeks. In the embryo, BEHAB is expressed at highest levels in mitotically active cells. The size and se¬ quence of BEHAB are consistent with the possibility that it could serve a function like link protein, stabilizing interactions between hyaluronan and brain proteoglycans.

Sequence analyses of rat and cat BEHAB (SEQ ID NOs 1 and 2 and Figure 1) show a substantial degree of amino acid identity to other members of the PTR family, which includes rat aggrecan, SEQ ID NO 3 (48%) ; rat neurocan, SEQ ID NO 4 (48%) ; human versican, SEQ ID NO 5 (46%) ; and rat link protein, SEQ ID NO 6 (42%) . The NH ₂ -terminal do-

main of this family is defined by two structural motifs, (a) an immunoglobulin (Ig) fold (denoted A in Figure 1) and (b) two PTR folds (PTR1 and PTR2, denoted B and C, respectively, in Figure 1) . The PTR folds have been suggested to mediate binding to HA. The Ig domain con¬ tains two clusters of conserved amino acids around the cysteine residues which generate the disulfide bond of the loop. The consensus sequence YxCxVxH in the COOH- terminal cluster is present in all immunoglobulin and major histocompatability complex proteins, and is also present in BEHAB (Figure 1) . The most conserved region of the PTR family's HA-binding protein domain is the sequence CDAGWL(A/S) D(Q/G) (T/S)VRYPI found in PTR1 and PTR2. Two copies of this sequence are also found in BEHAB. The degree of identity of BEHAB between rat and cat is high (84% overall) , with the greatest conservation in PTR1. The identity in PTR1 is 95% over the entire domain and 100% over 44 amino acids of the domain. PTR2 shows the next highest homology (86%) , followed by the Ig domain (84%) . The relative degree of homology between the PTR1, PTR2, and Ig domains observed in rat and cat is also observed between BEHAB and other members of the PTR family. Human human BEHAB is also highly conserved in the PTR1 domain.

This invention provides purified and isolated DNA fragments comprising DNA sequences encoding mammalian brain enriched hyaluronan binding protein, and purified and isolated DNA fragments comprising DNA sequences which hybridize under stringent conditions with sequences en- coding the protein. Also provided are RNA sequences corresponding to the DNA sequences.

In one embodiment, the invention provides a puri¬ fied and isolated DNA fragment derived from rat brain tissue comprising the nucleotides numbered 251 to 1363 of SEQ ID NO 1, and DNA sequences that hybridize under

stringent conditions with the sequence. In another em¬ bodiment, the invention provides the purified and isolat¬ ed DNA fragment derived from cat brain tissue comprising the nucleotides numbered 270 to 1403 of SEQ ID NO 2, and DNA sequences that hybridize under stringent conditions with the sequence. In a third embodiment, the invention provides a purified and isolated DNA fragment derived from human brain tissue comprising nucleotides of SEQ ID NO 1 , and DNA sequences that hybridize under stringent conditions with the sequence.

Encompassed by this invention are cloned sequences defining BEHAB of this invention, which can then be used to transform or transfect a host cell for protein expres¬ sion using standard means. Also encompassed by this invention are DNA sequences homologous or closely related to complementary DNA described herein, namely DNA se¬ quences which hybridize to BEHAB cDNA, particularly under stringent conditions that result in pairing only between nucleic acid fragments that have a high frequency of complementary base sequences, and RNA corresponding thereto. In addition to the BEHAB-encoding sequences, DNA encompassed by this invention may contain additional sequences, depending upon vector construction sequences, that facilitate expression of the gene. Also encompassed are sequences encoding synthetic BEHAB proteins exhib¬ iting activity and structure similar to isolated or cloned BEHAB. These are referred to herein as "biolog¬ ical equivalents".

Because of the degeneracy of the genetic code, a variety of codon change combinations can be selected to form DNA that encodes hyaluronan-binding protein of this invention, so that any nucleotide deletion(s) , addi¬ tion(s) , or point mutation(s) that result in a DNA encod¬ ing the protein are encompassed by this invention. Since certain codons are more efficient for polypeptide expres-

sion in certain types of organisms, the selection of gene alterations to yield DNA material that codes for the protein of this invention are preferably those that yield the most efficient expression in the type of organism which is to serve as the host of the recombinant vector. Altered codon selection may also depend upon vector con¬ struction considerations.

DNA starting material which is employed to form DNA coding for BEHAB proteins of this invention may be natural, recombinant or synthetic. Thus, DNA starting material isolated from tissue or tissue culture, constru¬ cted from oligonucleotides using conventional methods, obtained commercially, or prepared by isolating RNA cod¬ ing for BEHAB, and using this RNA to synthesize single- stranded cDNA which is used as a template to synthesize the corresponding double stranded DNA, can be employed to prepare DNA of this invention.

DNA encoding the proteins of this invention, or RNA corresponding thereto, are then inserted into a vec- tor, e.g., but not limited to, a p series plasmid such as pBR, pUC, pUB or pET, and the recombinant vector used to transform a microbial host organism. Example host organ¬ isms useful in the invention include, but are not limited to, bacterial (e.g., E. coli or B. subtilis) , yeast (e. g. , S. cerevisiae) or mammalian (e. g. , mouse fibro- blast or other tumor cell line) . This invention thus also provides novel, biologically functional viral and circular plasmid RNA and DNA vectors incorporating RNA and DNA sequences describing BEHAB generated by standard means. Culture of host organisms stably transformed or transfected with such vectors under conditions facilita- tive of large scale expression of the exogenous, vector- borne DNA or RNA sequences and isolation of the desired polypeptides from the growth medium, cellular lysates, or cellular membrane fractions yields the desired products.

The present invention thus provides for the total and/or partial manufacture of DNA sequences coding for BEHAB, and including such advantageous characteristics as incorporation of codons preferred for expression by se- lected non-mammalian hosts, provision of sites of cleav¬ age by restriction endonuclease enzymes, and provision of additional initial, terminal or intermediate DNA sequenc¬ es which facilitate construction of readily expressed vectors. Correspondingly, the present invention provides for manufacture (and development by site specific muta- genesis of cDNA and genomic DNA) of DNA sequences coding for icrobial expression of BEHAB analogues which differ from the forms specifically described herein in terms of identity or location of one or more amino acid residues (i . e . , deletion analogues containing less than all of the residues specified for the protein, and/or substitution analogues wherein one or more residues are added to a terminal or a medial portion of the polypeptide) , and which share the biological properties of BEHAB described herein.

DNA (and RNA) sequences of this invention code for all sequences useful in securing expression in procary- otic or eucaryotic host cells of polypeptide products having at least a part of the primary structural confor- mation, and one or more of the biological properties of BEHAB which are comprehended by: (a) the DNA sequences encoding BEHAB as described herein, or complementary strands; (b) DNA sequences which hybridize (under hy¬ bridization conditions) to DNA sequences defined in (a) or fragments thereof; and (c) DNA sequences which, but for the degeneracy of the genetic code, would hybridize to the DNA sequences defined in (a) and (b) above. Spe¬ cifically comprehended are genomic DNA sequences encoding allelic variant forms of BEHABs included therein, and sequences encoding RNA, fragments thereof, and analogues wherein RNA or DNA sequences may incorporate codons fa-

cilitating transcription or RNA replication of messenger RNA in non-vertebrate hosts.

The invention also provides the BEHAB proteins encoded by the above described DNA and/or RNA, obtained by isolation or recombinant means. In one embodiment, for example, the invention provides a polypeptide having an amino acid sequence depicted in residues numbered 1 to 371 of SEQ ID NO 1 or a biological equivalent thereof. In another embodiment, the invention provides a polypep- tide having the amino acid sequence depicted in residues numbered 1 to 378 of SEQ ID NO 2 or a biological equiva¬ lent thereof. In a third embodiment, the invention pro¬ vides a polypeptide set out in SEQ ID NO 7 or a biologi¬ cal equivalent thereof.

Isolation and purification of proteins provided by the invention are by conventional means including, for example, preparative chromatographic separations such as affinity, ion-exchange, exclusion, partition, liquid and/or gas-liquid chromatography; zone, paper, thin lay- er, cellulose acetate membrane, agar gel, starch gel, and/or acrylamide gel electrophoresis; immunological separations, including those using monoclonal and/or polyclonal antibody preparations; and combinations of these with each other and with other separation tech- niques such as centrifugation and dialysis, and the like.

It is an advantage of the invention that the iso¬ lation and purification of BEHAB provides a polypeptide marker for diagnostic purposes. Since BEHAB is neural- specific, it can be used as a diagnostic agent for brain or other central nervous system tumors or other neuro- pathological states. Expression of BEHAB is markedly increased in human brain glioma. Thus, this invention provides novel diagnostic methods employing biochemical markers for BEHAB, such as specific and sensitive immuno-

assays for the detection of BEHAB and patterns of its distribution in samples, to provide not only an indica¬ tion of ongoing pathological processes in central nervous system tissue, but also differential diagnoses of patho- logical processes involving specific areas of the central nervous system.

In the practice of the invention, the presence or absence of BEHAB, and/or relative concentrations of BE¬ HAB, are assayed in biological samples obtained from animals or human beings. Typical samples include, but are not limited to, cerebrospinal fluid, serum, urine or tissue homogenates such as those obtained from biopsies. Serum and cerebrospinal fluid are particularly preferred.

For diagnostic purposes, any method may be em- ployed to assay for BEHAB protein. Assay methods in¬ clude, but are not limited to. Western blots. Northern blots. Northern dot blots, enzyme-linked immunosorbent assays, radioimmunoassays, or mixtures of these.

For example, one embodiment employs an enzyme- linked immunosorbent assay (ELISA) . ELISAs typically utilize an enzyme such as horseradish peroxidase, urease, or alkaline phosphatase conjugated to an antibody or conjugated with a tag that interacts with a correspond¬ ingly tagged antibody. Example tags, where employed, are avidin and biotin. Test sample is incubated in the wells of microtiter plates with conjugated antibody. If the serum contains BEHAB antigen, the conjugated antibodies adhere to it. Subsequent measurement of enzyme activity estimates how much tagged antibody is present and bound to BEHAB. From that, amounts of BEHAB in the original test sample are calculated. Preferred ELISAs employ sub¬ strates known to those skilled in the art to be easily measurable, for example, by viewing color development in comparison with standards or by employing a spectropho-

to eter. These and other variations on ELISA protocols known by those skilled in the art are encompassed by the invention.

Most preferred substrates are chromophoric or yield chromophoric products, so that enzyme activity can be readily measured by the appearance or disappearance of color. Examples of enzyme substrates include p-nitrophe- nyl phosphate for alkaline phosphatase, bromocresol pur¬ pose and urea for urease, p-nitrophenyl-β-galactopyra- noside for β-galaactosidase, and the like. Horseradish peroxidase requires hydrogen peroxide in addition to another substrate that serves as a hydrogen donor includ¬ ing, for example, 2,2'-azino-jis-(3-ethylbenzthiazoline- 6-sulfonic acid) , 5-aminosalicylic acid, o-diaminobenzi- dine, 3,3 '-dimethoxybenzidine, o-phenylenediamine (free base or dihydrochloride) , 3,3' ,5,5'-tetramethylbenzidine (base or dihydrochloride) , and the like chromogens.

An alternate embodiment employs a radioimmunoassay (RIA) . Typical RIAs employ antigens radiolabelled with ¹²⁵ I, ³ H or other isotope that can be easily detected. For example, ¹²⁵ I-labelled BEHAB can be employed. Antibody is titrated with labelled antigen, and the activity and sensitivity of the antiserum is determined. A dilution series of samples to which known amounts of antigen have been added are distributed in wells of microtiter plates. Antibody is added, the well material and/or the superna- tants analyzed for radioactivity after incubation, and compared to a standard curve prepared using pure antigen. Amounts of unlabelled antigen bound are calculated by difference. These and other variations on RIA protocols known by those skilled in the art are encompassed by this invention.

The following exampes are presented to further illlustrate and explain the present invention and should not be taken as limiting in any regard.

Examples

Example 1

Rat and cat cDNA clones encoding BEHAB from the two species are prepared in this example.

To isolate rat cDNA clones encoding HA-binding proteins involved in neural development, an unamplified postnatal day 12 rat brain λgtlO cDNA library is screened with rat aggrecan clone pRCP 4 encoding the HA-binding region (described by Doege, K. , et al . , J. Biol . Chem. 262 : 17757-17767 (1987)). A total of 3.2 X 10 ⁵ recombinants are screened resulting in two positives. The library is rescreened with one of these clones, re¬ sulting in 15 additional clones. 4 x 10* phage (per 150- m plate) are plated with E. coli C600 bacteria, immobi¬ lized onto nitrocellulose filters, and prepared for hy¬ bridization using standard techniques. Filters are pre- washed for 1 hour in 1 M NaCl, 0.1% sodium dodecyl sul- fate (SDS), 20 mM Tris-HCl (pH 8.0) and 1 mM EDTA at 65°C. Filters are then prehybridized for an additional 4 to 6 hours in 50% forma ide, 5 x SCC (1 x SCC = 0.15 M sodium chloride, 0.015 M sodium citrate), 1% SDS, 1 x Denhardt's (0.02% Ficoll, 0.02% bovine serum albumin (BSA, Fraction V), 0.02% polyvinylpyrrolidone) , 50 mM sodium phosphate (pH 6.7), and 100 μg/ml salmon sperm DNA at 37°C. Hybridization is carried out in the identical solution with the inclusion of lθ ⁶ cpm pRCP 4 probe/ml for 24 hours at 37 ^β C. For all experiments, radiolabelled probes ( ³² P-dCTP, Amersha ) are prepared by random priming (Boehringer Mannheim Corp. , Indianapolis IN) gel purified

cDNA inserts, followed by the removal of unincorporated radionucleotides (NICK column, Pharmacia) . One post hybridization wash is in 2 x SSC, 0.1% SDS and one in 0.2 x SSC for 1 hour each are performed at room temperature. Phage DNA is isolated using DE52 (Whatman) and the cDNA insert excised by EcoRI digestion. The insert size of the clones are determined and partial restriction maps are prepared to eliminate redundant clones. The cDNA is gel purified (Gene-Clean ^® , Bio 101), eight clones sub- cloned into pBluescript ^® KS+ (Stratagene, LaJolla, CA) and transformed into DH5α (GIBCO BRL, Gaithersburg, MD) .

To isolate cat cDNA clones, random nona ers (1.4 mg) are used to synthesize first cDNA from 5 μg poly A ⁺ RNA isolated from P39 cat cortex, cDNA synthesis is per- formed according to manufacturer's instructions for the production of nondirectional libraries (Stratagene) and size-fractionated by column chromatography (GIBCO BRL) . 50 ng of cDNA is ligated to 1 μg J_"σoRI cut, phosphatized Lambda Zap ^® II vector and packaged into phage (Gigapack II Gold ^® , Stratagene). This yields 0.5 x 10 ⁶ recombinants when transfected into XLl-Blue ^® (Stratagene) . The unam- plified library is screened with rat clone HI. Hybrid¬ ization is performed in 6 x SSC, 0.1% SDS, 1 x Denhardt's and 100 μg/ml salmon sperm DNA at 65°C. Filters are washed twice in 2 x SSC, 0.1% SDS and twice in 0.2 x SSC at 65°C for 20 minutes. A total of 3.2 x 10 ⁵ recombinants are screened, resulting in 5 positives. cDNA inserts of plaque-purified positive clones are isolated in pBlue¬ script ^® SK ^" by in vivo excision.

Example 2

DNA clones prepared in Example 1 are sequenced and compared with previously reported sequences in this Exam¬ ple.

DNA sequencing is performed by the dideoxy chain termination method using Sequenase ^® (U.S. Biochemical, Cleveland, OH) . Bluescript SK/KS primers or cDNA specif¬ ic 20-mers are used. Sequence is verified from overlap- ping clones or by sequencing both strands of DNA. Se¬ quence compressions are resolved using dITP nucleotides. After labelling, the reactions are incubated at 37"C for 30 minutes in the presence of 1 x reaction buffer, 1 mM dNTPs (pH 7.0) and 0.5 U terminal deoxynucleotidyl trans- ferase to prevent premature termination caused by the use of dITP. Sequence analyses are performed using the Uni¬ versity of Wisconsin Genetics Computer Group programs.

For the rat BEHAB sequence, the composite sequence obtained from the overlapping clones identified after subcloning into pBluescript ^® KS+ as described in the previous Example is used (SEQ ID NO 1; sequence data are recorded in EMBL/GenBank/DDBJ under accession number Z28366) . The complete BEHAB coding sequence is 1,113 base pairs. The nucleotide sequence preceding the first AUG contains a consensus sequence for translation initia¬ tion. In the 3' untranslated region, only that sequence verified from three clones is presented. The deduced amino acid composition of the BEHAB protein is comprised of 371 amino acids and includes a putative signal peptide cleavage site at Ala-22. The resulting mature protein has a predicted molecular mass of 38,447 kD. Analysis of the deduced amino acid sequence indicates the presence of two NX(S/T) consensus sequences for potential N-glycolsa- tion.

Similarly, the composite cat BEHAB sequence is obtained from the overlapping clones obtained in the pBluescript ^® SK ^" excision as described in the above Exam¬ ple. The results are set out in SEQ ID NO 2 (sequence data are recorded in EMBL/GenBank/DDBJ under accession number Z28367) . The complete coding sequence for cat

BEHAB is 1,134 base pairs. The first AUG is preceded by both an in-frame termination codon and the translation initiation consensus sequence. The cat BEHAB sequence encodes 378 amino acids which, like the rat, contains a 22 residue signal peptide. However, cat BEHAB contains 6 additional amino acids at the carboxy terminus, resulting in a predicted molecular mass of 38,955 kD. In the cat, Trp-373 is encoded by TGG, while the corresponding rat sequence of TAG results in the termination. This termi- nation sequence is verified in three rat clones and by sequencing both strands of a cat clone. Cat BEHAB also contains one additional site for potential N-glycosyla- tion not present in the rat.

Database analyses at both the nucleic acid and amino acid levels indicate that BEHAB is a previously unreported member of the PTR family of HA-binding pro¬ teins. BEHAB has a substantial degree of amino acid identity to the other members of the PTR family, which includes rat aggregan, SEQ ID NO 3 (48%) ; rat neurocan, SEQ ID NO 4 (48%) ; human versican, SEQ ID NO 5 (46%) ; and rat link protein, SEQ ID NO 6 (42%) . See Figure 1. The NH ₂ -terminal domain of this family is defined by two structural motifs, (a) an immunoglobulin (Ig) fold and (b) two PTR folds (PTR1 and PTR2) . The PTR folds have been suggested to mediate binding to HA. The Ig domain contains two clusters of conserved amino acids around the cysteine residues which generate the disulfide bond of the loop. The consensus sequence YxCxVxH in the COOH- ter inal cluster is present in all immunoglobulin and major histocompatability complex proteins, and is also present in BEHAB (Figure 1) . The most conserved region of the PTR family's HA-binding protein domain is the sequence CDAGWL(A/S)D(Q/G) (T/S) RYPI found in PTR1 and PTR2. Two copies of this sequence are also found in BEHAB. The degree of identity of BEHAB between rat and cat is high (84% overall) , with the greatest conservation

in PTRl. The identity in PTRl is 95% over the entire domain and 100% over 44 amino acids of the domain. PTR2 shows the next highest homology (86%) , followed by the Ig domain (84%) . The relative degree of homology between the PTRl, PTR2, and Ig domains observed in rat and cat is also observed between BEHAB and other members of the PTR family (Table I and Figure 1) .

Table I. Percent Identity of rat BEHAB to Other Members of the PTR Family of HA-Binding Proteins

Protein ig PTRl PTR2

Cat BEHAB 84% 95% 86%

Aggrecan 40% 60% 51%

Neurocan 37% 56% 57%

Versican 36% 59% 48%

Rat Link 34% 48% 53%

CD44 22%

Sequence homology is similarly observed for human BEHAB (SEQ ID NO 7) . To determine the human BEHAB se¬ quence, total RNA is extracted from a sample of human brain and reverse transcriptase polymerase chain reac¬ tions (PCR) performed using degenerate oligonucleotide primers corresponding to the ends of the PTRl domain in rat and cat. PCR products are subcloned into the TA vector and sequenced by the dideoxy chain termination method described above.

Example 3

In this Example, tissue distribution of BEHAB mRNA is determined by Northern blot analysis and the spatial distribution, by in situ hybridization on central nervous system tissue sections.

For Northern analysis, 25 μg total RNA is dena¬ tured in 2.2 M formaldehyde, 50% formamide, 1 x MOPS (3— (N-morpholino)propanesulfonic acid) buffer at 65°C for 15 minutes. The RNA is electrophoresed on a 1.0% agarose- formaldehyde gel with 1 x MOPS buffer at 50V with buffer recirculation. The gel is briefly neutralized in trans¬ fer buffer (20 x SSC) and RNA blotted to Zetaprobe ^® (Bio- Rad Labs., Hercules CA) by capillary transfer. Filters are rinsed briefly in 2 x SSC, and RNA is immobilized both by UV cross-linking and baking in vacuuo (80°C for 1 hour). Hybridization in 7% SDS, 1% BSA, 0.5 M phosphate buffer (PB, pH 6.8), 1 mM EDTA and 0.5-2.5 x 10 ⁶ cpm rat HI probe/ml are carried out for at least 8 hours at 65°C. Filters are washed twice in 5% SDS, 0.5% BSA, 40 mM PB, 1 mM EDTA and twice in 1% SDS, 40 mM PB, 1 mM EDTA at 65°C, and exposed to film (Hyperfilm, Amersham) at -70°C. Mo¬ lecular sizes are determined relative to RNA molecular weight standards (GIBCO BRL) and 28S and 18S riboso al RNA observed during UV illumination. The ubiquitously expressed, non-developmentally regulated gene cyclophilin is used to determine equal loading of lanes. Densitome- try is performed using the NIH Image program. The two clones recognize the same size mRNA transcript.

Tissue distribution of rat BEHAB mRNA using this procedure shows a single 3.9-kb mRNA transcript detected in adult rat cortex, spinal cord and cerebellum. This transcript is not detected in liver, kidney, spleen, lung or muscle, even with long film exposures. Observed amounts of human BEHAB mRNA is markedly (i . e . , at least about four-fold) higher in brain glioma tissue in compar¬ ison to what is seen in normal brain tissue using the procedure. Moreover, BEHAB is not detected in non-brain tumor tissues, including breast, lung, or colon tumors.

These observations are confirmed by in situ hy- bridization to whole embryos, which show that BEHAB ex-

pression is restricted to the central nervous system. In situ hybridization is performed on 12 to 14 micron thic frozen sections thaw-mounted onto gelatin-coated slides and postfixed in 0.1 M sodium phosphate buffered 4% para- formaldehyde (pH 7.4). Sections are rinsed in 1 x PBS

(137 mM NaCl, 2.7 mM KC1, 10 mM Na ₂ P0 _4/ 1.8 mM KH ₂ P0 2 X SSC and acetylated with 0.5% acetic anhydride in 0.1 M triethanolamine (pH 8.0). Sections are then rinsed in 2 x SSC, 1 x PBS, dehydrated in ethanol and delipidated in chloroform. Sections are prehybridized in 2 x SSC, 50% formamide at 50"C for 1 hour, and then hybridized in 0.75 M NaCl, 50% formamide, 1 x Denhardt's, 10% dextran sul- fate, 30 mM DTT, 10 mM Tris-HCl (pH 7.5), 1 mM EDTA, 100 μg/ l salmon sperm DNA, 0.5 mg/ml yeast tRNA and 10 ⁶ cpm probe per slide at 50°C for 12 to 15 hours. ( ³⁵ S)-CTP

(New England Nuclear, Boston MA) labelled cRNA probes are synthesized using T3 (GIBCO BRL), SP6, and T7 RNA poly- merases (New England Biolabs inc. , Beverly, MA) . After hybridization, sections are washed in 2 x SSC, 50% form- amide, 0.1% BME (β-mercaptoethanol) at 50"C for 1 hour and treated with 20 μg/ml RNase A in 0.5 M NaCl, 10 mM Tris-HCl (pH 8.0) at 37 ^e C for 30 minutes. Sections are then washed in 2 x SSC, 50% formamide, 0.1% BME at 58°C for 30 minutes and 0.1 x SSC, 0.1% BME at 63°C for 30 minutes and dehydrated. For initial localization of probe, the slides are exposed to film (Hyperfil , Amer- sham) for 4 days. Autoradiograms are used as negatives for prints. For higher resolution, the slides are dipped in NTB-2 emulsion (Kodak) , developed after 5 days and counterstained with cresyl violet. Neurofilament-middle (NF) antisense and rat clone sense probes are used as positive and negative controls, respectively.

The spatial distribution of BEHAB mRNA within the nervous system is determined at higher resolution by in situ hybridization on tissue sections from P21 rat fore- brain, brainstem, spinal cord, and cerebellum. Near

adjacent sections are probed with an antisense cRNA probe of a rat clone and positive and negative controls. Using these procedures, BEHAB expression is found to be widely distributed in the brain, in both gray and white matter. The cortex exhibits diffuse hybridization with no laminar specification. Hybridization is detected in white matter tracts, including the corpus callosum, the fimbria of the hippocampus, and the anterior commissure. In the hippo¬ campus, the most intense hybridization is present over neurons; it is highest in the CA1 subfield. The pattern of NF hybridization in the hippocampus is essentially reciprocal to that of BEHAB; the NF probe hybridizes most intensely in subfields CA2, CA3, and in the dentate gyrus. BEHAB hybridization is also seen throughout the inferior colliculus and less intensely in the superior colliculus. In addition to the hippocampus, BEHAB hy¬ bridization in gray matter is most intense in the sub- stantia nigra. The rat sense probe generates almost no signal in most of the brain, but a low level of hybrid- ization is seen in the hippocampus and dentate gyrus.

In the brainste , BEHAB is expressed throughout the reticular formation. Several brainstem nuclei also express BEHAB, including the superior olivary nucleus, the vestibular nuclei, the abducens nucleus and the dor- sal column nuclei. A similar hybridization pattern is observed with NF, while no hybridization signal is de¬ tected with the sense probe.

BEHAB expression in the spinal cord is greater in the gray matter than in white matter. In the gray mat- ter, BEHAB expression is slightly greater in the ventral than in the dorsal horn. BEHAB hybridization is lacking in the substantia gelatinosa. In the ventral horn, hy¬ bridization is seen over motor neurons. In the spinal cord white matter, the size of labelled cells and their distribution indicates that BEHAB is expressed by glial

cells. Like BEHAB, NF expression is greater in the ven¬ tral horn than in the dorsal horn; however, unlike BE¬ HAB, NF is not detected in the spinal white matter. As observed in the brainstem, no hybridization signal is detected in the spinal cord with the sense probe.

In the cerebellum, BEHAB expression is greatest in the deep cerebellar nuclei. In the cerebellar cortex, labeling is detected in all three cortical layers. In the molecular layer, the distribution of silver grains parallels the distribution of basket and stellate cells. In the Purkinje cell layer, labeling is clustered over Purkinje cells and, in the granule cell layer, it is clustered over Golgi II cells. The white matter of the cerebellar cortex also shows hybridization signal. NF is primarily expressed by Purkinje cells and by cells of the deep cerebellar nuclei. The sense probe generates a low level of diffuse hybridization signal throughout the granule cell layer.

To determine the temporal regulation of BEHAB mRNA expression. Northern blot analysis is performed using total RNA from embryonic and postnatal rat cortex and spinal cord. The non-developmentally regulated gene cyclophilin is used as a control probe to verify equal loading. Unlike actin and tubulin, which exhibit varia- tion of abundance with development, cyclophilin maintains a constant relative abundance throughout the central nervous system with development. The Northern blots are analyzed by densitometry, and band intensity of BEHAB is standardized by calculating a ratio of the abundance of BEHAB to cyclophilin at each developmental age.

In the cortex, BEHAB recognizes a single 3.9-kb mRNA transcript. BEHAB expression is detected at embry¬ onic day 17 and gradually increases to attain adult lev¬ els by postnatal day 21. In the spinal cord, BEHAB also

recognizes a 3.9-kb mRNA transcript. At all ages except the adult, BEHAB expression is greater in the spinal cord than in the cortex. Like the cortex, BEHAB is present in the spinal cord at embryonic day 17 and gradually in- creases with age until reaching a maximal level at post¬ natal day 14. Unlike the cortex, BEHAB expression in the spinal cord then declines slightly.

The expression of BEHAB in the embryo, like in the postnatal animal, is restricted to the central nervous system. BEHAB expression is absent in dorsal root gan¬ glia, a peripheral nervous system structure. Tissues in the embryo that express high levels of closely related genes such as cartilage (which expresses aggrecan) also show no hybridization signal for BEHAB. The distribution of BEHAB expression in the embryonic central nervous sys¬ tem differs slightly from the postnatal brain. The high¬ est levels of BEHAB expression are found in regions that contain itotically active cells, such as the ventricular zone of the medulla, midbrain, and spinal cord. Expres- sion of BEHAB is heterogenous in the developing brain.

The above description is for the purpose of teaching the person of ordinary skill in the art how to practice the present invention, and it is not intended to detail all those obvious modifications and variations of it which will become apparent to the skilled worker upon reading the description. It is intended, however, that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates the contrary.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANTS: Susan Hockfield

Diane M. Jaworski (ii) TITLE OF INVENTION: BEHAB, A Brain Hyaluronan-

Binding Protein (iii) NUMBER OF SEQUENCES: 7 (iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: St. Onge Steward Johnston & Reens

(B) STREET: 986 Bedford Street

(C) CITY: Stamford

(D) STATE: CT

(E) COUNTRY: United States

(F) ZIP: 06905 (V) COMPUTER READABLE FORM

(A) MEDIUM TYPE: 3.5" 1.44 Mb diskette

(B) COMPUTER: IBM PC

(C) OPERATING SYSTEM: MS DOS

(D) SOFTWARE: Word Processor (Viii) ATTORNEY INFORMATION

(A) NAME: Mary M. Krinsky

(B) REGISTRATION NUMBER: 32423

(C) DOCKET NUMBER: 1751-P0004 (ix) TELECOMMUNICATION INFORMATION

(A) TELEPHONE NUMBER: 203-324-6155

(B) TELEFAX NUMBER: 203-327-1096

(2) INFORMATION FOR SEQ ID NO: 1

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1520 bases encoding 371 amino acids

(B) TYPE: nucleic acid and amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE

(A) DESCRIPTION: DNA encoding a protein (v) FRAGMENT TYPE: entire sequence (vi) IMMEDIATE SOURCE: rat brain (ix) FEATURE

(A) NAME: rat BEHAB (xi) SEQUENCE DESCRIPTION: SEQ ID NO 1:

CG AGACCCGCGC AGAGAAGGGA GCGGGTCCCG TGACCGCGCA 42

GAGCCCCCCA CGCGGCCAAA GGCCGGGGAC GCGGGGAAGG CGGGGCGCGT 92

GGGAAGAAAC CCCCTTTTGT GCGGCTCCCG GCGAGCTGGC GCCCCCGTCT 142

GCGTCCCGCG CGCCCGGCCC TGCTCGCGCC CGCGCATTGC CGCAGTCTCG 192

GCTGCGTGCG GGACGCGGTG TGTGGAGGGG ACCTCACAAG TTCTTCCAAG 242

TTTGCAGC ATG ATC CCA TTG CTT CTG TCC CTG CTG GCA GCT CTG 286 Met lie Pro Leu Leu Leu Ser Leu Leu Ala Ala Leu

5 10

GTC CTG ACC CAA GCC CCT GCA GCC CTC GCT GAT GAC CTG AAA 328 Val Leu Thr Gin Ala Pro Ala Ala Leu Ala Asp Asp Leu Lys 15 20 25

GAA GAC AGC TCA GAG GAT CGA GCC TTT CGG GTG CGC ATC GGT 370 Glu Asp Ser Ser Glu Asp Arg Ala Phe Arg Val Arg lie Gly 30 35 40

GCC GCG CAG CTG CGG GGT GTG CTG GGC GGT TGG GTG GCC ATC 412 Ala Ala Gin Leu Arg Gly Val Leu Gly Gly Trp Val Ala lie

45 50

CCA TGC CAC GTC CAC CAC CTG AGG CCG CCG CCC AGC CGC CGG 454 Pro Cys His Val His His Leu Arg Pro Pro Pro Ser Arg Arg 55 60 65

GCC GCG CCG GGC TTT CCC CGA GTC AAA TGG ACC TTC CTG TCC 496 Ala Ala Pro Gly Phe Pro Arg Val Lys Trp Thr Phe Leu Ser 70 75 80

GGG GAC CGG GAG GTG GAG GTG CTG GTG GCG CGC GGG CTG CGC 538 Gly Asp Arg Glu Val Glu Val Leu Val Ala Arg Gly Leu Arg 85 90 95

GTC AAG GTA AAC GAA GCC TAT CGG TTC CGC GTG GCG CTG CCT 580 Val Lys Val Asn Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro 100 105 110

GCC TAC CCC GCA TCG CTC ACA GAT GTG TCT TTA GTA TTG AGC 622 Ala Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Val Leu Ser

115 120

GAA CTG CGG CCC AAT GAT TCC GGG GTC TAT CGC TGC GAG GTC 664 Glu Leu Arg Pro Asn Asp Ser Gly Val Tyr Arg Cys Glu Val 125 130 135

CAG CAC GGT ATC GAC GAC AGC AGT GAT GCT GTG GAA GTC AAG 706 Gin His Gly lie Asp Asp Ser Ser Asp Ala Val Glu Val Lys 140 145 150

GTC AAA GGG GTC GTC TTC CTC TAC CGA GAG GGC TCT GCC CGC 748 Val Lys Gly Val Val Phe Leu Tyr Arg Glu Gly Ser Ala Arg 155 160 165

TAT GCT TTC TCC TTC GCT GGA GCC CAG GAA GCC TGT GCT CGC 790 Tyr Ala Phe Ser Phe Ala Gly Ala Gin Glu Ala Cys Ala Arg 170 175 180

ATC GGA GCC CGA ATT GCC ACC CCT GAG CAG CTG TAT GCT GCC 832 lie Gly Ala Arg lie Ala Thr Pro Glu Gin Leu Tyr Ala Ala

185 190

TAC CTC GGC GGC TAT GAA CAG TGT GAT GCT GGC TGG CTG TCC 874 Tyr Leu Gly Gly Tyr Glu Gin Cys Asp Ala Gly Trp Leu Ser 195 200 205

GAC CAA ACC GTG AGG TAC CCC ATC CAG AAC CCA CGA GAA GCC 916 Asp Gin Thr Val Arg Tyr Pro lie Gin Asn Pro Arg Glu Ala 210 215 220

TGT TAT GGA GAC ATG GAT GGC TAC CCT GGA GTG CGG AAT TAC 958 Cys Tyr Gly Asp Met Asp Gly Tyr Pro Gly Val Arg Asn Tyr 225 230 235

GGA GTG GTG GGT CCT GAT GAT CTC TAC GAT GTC TAC TGT TAT 1000 Gly Val Val Gly Pro Asp Asp Leu Tyr Asp Val Tyr Cys Tyr 240 245 250

GCC GAA GAC CTA AAT GGA GAA CTG TTC CTA GGT GCC CCT CCC 1042 Ala Glu Asp Leu Asn Gly Glu Leu Phe Leu Gly Ala Pro Pro

255 260

GGC AAG CTG ACG TGG GAG GAG GCT CGG GAC TAC TGT CTG GAA 1084 Gly Lys Leu Thr Trp Glu Glu Ala Arg Asp Tyr Cys Leu Glu 265 270 275

NOT TAKEN INTO CONSIDERATION

FOR THE PURPOSES OF INTERNATIONAL PROCESSING

(A) NAME: cat brain BEHAB (Xi) SEQUENCE DESCRIPTION: SEQ ID NO 2 :

CGGCACGAG CTCGTGCCGA 19

ATTCGGCACA GAGGGACCGA GCGTGGACCC GGAGGAGAGC CCGGAGGAGA 69

GCCCGGAGGA GGCGCAAACT TGGCGGTGCG CACCCTAGCC CCGGCCCTCG 119

GCCTGCCGGA AGAAAACAAA GGCCCTGAGA GCTTAAGGAA CTTGCAGCAA 169

GTTGACTAGC GCCCAGGTCT TGGTTCCGAG GAGGAATCCT GGTGGGGAGA 219

CAGGATCAGA AGCGAGGGTG TTAACAGTGA GTCCTTCCAG CAGCCTGAGC 269

ATG GCC CCA CTG TTC CTG CCC CTG CTG ATA GCC CTG GCC CTG 311 Met Ala Pro Leu Phe Leu Pro Leu Leu lie Ala Leu Ala Leu

5 10

GCC CCG GGC CCC ACG GCC TCA GCT GAT GTC CTG GAA GGG GAC 353 Ala Pro Gly Pro Thr Ala Ser Ala Asp Val Leu Glu Gly Asp 15 20 25

AGC TCA GAG GAC CGG GCC TTC CGC GTG CGC ATC TCG GGC AAC 395 Ser Ser Glu Asp Arg Ala Phe Arg Val Arg lie Ser Gly Asn 30 35 40

GCG CCG CTG CAG GGC GTG CTG GGC GGC GCC CTC ACC ATC TCG 437 Ala Pro Leu Gin Gly Val Leu Gly Gly Ala Leu Thr lie Ser 45 50 55

TGC CAC GTT CAC TAC CTG CGG CCG CCG CCG GGC CGC CGG GCC 479

Cys His Val His Tyr Leu Arg Pro Pro Pro Gly Arg Arg Ala 60. 65 70

GTG CTG GGC TCC CCG CGG GTC AAG TGG ACC TTC CTG TCC GGG 521 Val Leu Gly Ser Pro Arg Val Lys Trp Thr Phe Leu Ser Gly

75 80

GGC CGG GAG GCC GAG GTG CTG GTG GCG CGG GGG CTG CGC GTC 563 Gly Arg Glu Ala Glu Val Leu Val Ala Arg Gly Leu Arg Val 85 90 95

AAG GTG AGC GAG GCC TAC CGG TTC CGC GTG GCG CTG CCC GCC 605 Lys Val Ser Glu Ala Tyr Arg Phe Arg Val Ala Leu Pro Ala 100 105 110

TAC CCG GCG TCC CTC ACC GAC GTC TCC CTG GCA CTG AGC GAG 647 Tyr Pro Ala Ser Leu Thr Asp Val Ser Leu Ala Leu Ser Glu 115 120 125

CTG CGG CCC AAC GAC TCT GGC ATC TAC CGC TGC GAG GTC CAG 689 Leu Arg Pro Asn Asp Ser Gly lie Tyr Arg Cys Glu Val Gin 130 135 140

CAC GGC ATA GAC GAC AGC AGC GAC GCC GTG GAG GTC AAG GTC 731 His Gly lie Asp Asp Ser Ser Asp Ala Val Glu Val Lys Val

145 150

AAA GGG GTC GTC TTT CTC TAC CGG GAG GGC TCT GCC CGC TAC 773 Lys Gly Val Val Phe Leu Tyr Arg Glu Gly Ser Ala Arg Tyr 155 160 165

GCT TTC TCC TTC GCC CGG GCC CAG GAG GCC TGT GCC CGC ATC 815 Ala Phe Ser Phe Ala Arg Ala Gin Glu Ala Cys Ala Arg lie 170 175 180

GGA GCC CGC ATC GCC ACC CCG GAG CAG CTC TAC GCT GCC TAC 857 Gly Ala Arg lie Ala Thr Pro Glu Gin Leu Tyr Ala Ala Tyr 185 190 195

CTC GGG GGC TAT GAG CAG TGC GAT GCT GGC TGG CTG TCC GAC 899 Leu Gly Gly Tyr Glu Gin Cys Asp Ala Gly Trp Leu Ser Asp 200 205 210

CAA ACC GTG AGG TAT CCC ATC CAG ACC CCA CGG GAG GCC TGT 941 Gin Thr Val Arg Tyr Pro lie Gin Thr Pro Arg Glu Ala Cys

215 220

TAT GGA GAC ATG GAT GGC TTC CCT GGG GTC CGG AAC TAT GGC 983 Tyr Gly Asp Met Asp Gly Phe Pro Gly Val Arg Asn Tyr Gly 225 230 235

CTG GTG GAC CCG GAT GAC CTG TAC GAT ATC TAC TGC TAT GCT 1025 Leu Val Asp Pro Asp Asp Leu Tyr Asp lie Tyr Cys Tyr Ala 240 245 250

GAA GAC CTA AAT GGA GAG CTG TTC CTG GGC GCC CCT CCA GAC 1067 Glu Asp Leu Asn Gly Glu Leu Phe Leu Gly Ala Pro Pro Asp 255 260 265

AAC GTG ACG CTG GAG GAG GCT ACG GCA TAC TGC CGT GAG CGG 1109 Asn Val Thr Leu Glu Glu Ala Thr Ala Tyr Cys Arg Glu Arg 270 275 280

GGT GCA GAG ATT GCT ACC ACG GGC CAG CTG TAT GCA GCC TGG 1151 Gly Ala Glu lie Ala Thr Thr Gly Gin Leu Tyr Ala Ala Trp

285 290

GAT GGC GGC CTG GAC CGC TGC AGC CCC GGC TGG CTG GCC GAT 1193 Asp Gly Gly Leu Asp Arg Cys Ser Pro Gly Trp Leu Ala Asp 295 300 305

GGC AGC GTG CGC TAC CCC ATC GTC ACG CCC AGC CAG CGC TGC 1235 Gly Ser Val Arg Tyr Pro lie Val Thr Pro Ser Gin Arg Cys 310 315 320

GGT GGG GGC CTG CCT GGC GTC AAG ACT CTC TTC CTC TTC CCC 1277 Gly Gly Gly Leu Pro Gly Val Lys Thr Leu Phe Leu Phe Pro 325 330 335

AAC CAG ACC GGC TTC CCC AAC AAG TAC AGC CGC TTC AAC GTC 1319

Asn Gin Thr Gly Phe Pro Asn Lys Tyr Ser Arg Phe Asn Val 340 345 350

TAC TGC TTC CGA GAC TCT GGC CAG CCC TCC ACC ACC CCT GAG 1361

Tyr Cys Phe Arg Asp Ser Gly Gin Pro Ser Thr Thr Pro Glu

355 360

GCC TCT GAC CAG CCT CTG ACG GGC TGG AGG CCA TTG TCA CAG 1403

Ala Ser Asp Gin Pro Leu Thr Gly Trp Arg Pro Leu Ser Gin 365 370 375

TGACAGAGAC CCTAGAGGAG CTCCACGTGC CGCGGGAAGC TGTGGAGAGC 1453

GAGTCCCGGG GAGCCATCTA CTCCGTCCCC ATTGTGGAGG ATGGGGAGGT 1503

GCAAGGTCCC CCTCCA 1519

(4) INFORMATION FOR SEQ ID NO: 3 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 334 residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE

(A) DESCRIPTION: polypeptide (v) FRAGMENT TYPE: functional domains (ix) FEATURE

(A) NAME: rat aggrecan (X) PUBLICATION INFORMATION

(A) AUTHOR: Doege, K. , Sasaki, M. , Hori- gan, E., Hassell, J.R. , and Yamada, Y.

(B) TITLE: Complete primary structure of the rat cartilage proteoglycan core protein deduced from cDNA clones.

(C) JOURNAL: J. Biol . Chem.

(D) VOLUME: 262

(F) PAGES: 17757-17767

(G) DATE: 1987

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 3:

Glu Glu Val Pro Asp His Asp Asn Ser Leu Ser Val Ser lie Pro

5 10 15

Gin Pro Ser Pro Leu Lys Ala Leu Leu Gly Thr Ser Leu Thr lie

20 25 30

Pro Cys Tyr Phe lie Asp Pro Met His Pro Val Thr Thr Ala Pro

35 40 45

Ser Thr Ala Pro Leu Thr Arg lie Lys Trp Ser Arg Val Ser Lys

50 55 60

Glu Lys Glu Val Val Leu Leu Val Ala Thr Glu Gly Gin Val Arg

65 70 75

Val Asn Ser lie Tyr Gin Asp Lys Val Ser Leu Pro Asn Tyr Pro

80 85 90

Ala lie Pro Ser Asp Ala Thr Leu Glu lie Gin Asn Leu Arg Ser

95 100 105

Asn Asp Ser Gly lie Tyr Arg Cys Glu Val Met His Gly lie Glu

110 115 120

Asp Ser Glu Ala Thr Leu Glu Val lie Val Lys Gly lie Val Phe

125 130 135

His Tyr Arg Ala lie Ser Thr Arg Tyr Thr Leu Asp Phe Asp Arg

140 145 150

Ala Gin Arg Ala Cys Leu Gin Asn Ser Ala lie lie Ala Thr Pro

155 165 170

Glu Gin Leu Gin Ala Ala Tyr Glu Asp Gly Phe His Gin Cys Asp

175 180 185

Ala Gly Trp Leu Ala Asp Gin Thr Val Arg Tyr Pro lie His Thr

190 195 200

Pro Arg Glu Gly Cys Tyr Gly Asp Lys Asp Glu Phe Pro Gly Val

205 210 215

Arg Thr Tyr Gly lie Arg Asp Thr Asn Glu Thr Tyr Asp Val Tyr

220 225 230

Cys Phe Ala Glu Glu Met Glu Gly Glu Phe Tyr Ala Thr Ser Pro

235 240 245

Glu Lys Phe Thr Phe Gin Glu Ala Ala Asn Glu Cys Arg Thr Val

250 255 260

Gly Ala Arg Leu Ala Thr Thr Gly Gin Leu Tyr Leu Ala Trp Gin

265 270 275

Gly Gly Met Asp Met Cys Ser Ala Gly Trp Leu Ala Asp Arg Ser

280 285 290

Val Arg Tyr Pro lie Ser Lys Ala Arg Pro Asn Cys Gly Gly Asn

295 300 305

Leu Leu Gly Val Arg Thr Val Tyr Leu His Ala Asn Gin Thr Gly

310 315 320

Tyr Pro Asp Pro Ser Ser Arg Tyr Asp Ala lie Cys Tyr Thr

325 330

(5) INFORMATION FOR SEQ ID NO: 4 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 333 residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE

(A) DESCRIPTION: polypeptide (v) FRAGMENT TYPE: functional domains (ix) FEATURE

(A) NAME: rat neurocan (X) PUBLICATION INFORMATION

(A) AUTHOR: Rauch, U. , Karthikeyan, L. , Maurel, P., Margolis, R.U. , and Margolis, R.K.

(B) TITLE: Cloning and primary structure of neu¬ rocan, a developmentally regulated, aggregating chondroitin sulfate proteoglycan of brain.

(C) JOURNAL: J. Biol . Chem .

(D) VOLUME: 267

(F) PAGES: 19536-19547

(G) DATE: 1992

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 4:

Asp Thr Gin Asp Thr Thr Thr Thr Glu Lys Gly Leu His Met Leu

5 10 15

Lys Ser Gly Ser Gly Pro lie Gin Ala Ala Leu Ala Glu Leu Val

20 25 30

Ala Leu Pro Cys Phe Phe Thr Leu Gin Pro Arg Gin Ser Pro Leu

35 40 45

Gly Asp lie Pro Arg lie Lys Trp Thr Lys Val Gin Thr Ala Ser

50 55 60

Gly Gin Arg Gin Asp Leu Pro lie Leu Val Ala Lys Asp Asn Val

65 70 75

Val Arg Val Ala Lys Gly Trp Gin Gly Arg Val Ser Leu Pro Ala

80 85 90

Tyr Pro Arg His Arg Ala Asn Ala Thr Leu Leu Leu Gly Pro Leu

95 100 105

Arg Ala Ser Asp Ser Gly Leu Tyr Arg Cys Gin Val Val Lys Gly

110 115 120 lie Glu Asp Glu Gin Asp Leu Val Thr Leu Glu Val Thr Gly Val

125 130 135

Val Phe His Tyr Arg Ala Ala Arg Asp Arg Tyr Ala Leu Thr Phe

140 145 150

Ala Glu Ala Gin Glu Ala Cys His Leu Ser Ser Ala Thr lie Ala

155 160 165

Ala Pro Arg His Leu Asn Ala Ala Phe Glu Asp Gly Phe Asp Asn

170 175 180

Cys Asp Ala Gly Trp Leu Ser Asp Arg Thr Val Arg Tyr Pro lie

185 190 195

Thr Gin Ser Arg Pro Gly Cys Tyr Gly Asp Arg Ser Ser Leu Pro

200 205 210

Gly Val Arg Ser Tyr Gly Arg Arg Asp Pro Gin Glu Leu Tyr Asp

215 220 225

Val Tyr Cys Phe Ala Arg Glu Leu Gly Gly Glu Phe Tyr Val Gly

230 235 240

Pro Ala Arg Arg Leu Thr Leu Ala Gly Ala Arg Ala Leu Cys Gin

245 250 255

Arg Gin Gly Ala Ala Leu Ala Ser Val Gly Gin Leu His Leu Ala

260 265 270

Trp His Glu Gly Leu Asp Gin Cys Asp Pro Gly Trp Leu Ala Asp

275 280 285

Gly Ser Val Arg Tyr Pro lie Gin Thr Pro Arg Arg Arg Cys Gly

290 295 300

Gly Ser Ala Pro Gly Val Arg Thr Val Tyr Arg Phe Ala Asn Arg

305 310 315

Thr Gly Phe Pro Ala Pro Gly Ala Arg Phe Asp Ala Tyr Cys Phe

320 325 330

Arg Ala His

(6) INFORMATION FOR SEQ ID NO: 5 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 328 residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ϋ) MOLECULE TYPE

(A) DESCRIPTION: polypeptide (v) FRAGMENT TYPE: functional domains (ix) FEATURE

(A) NAME: human versican (X) PUBLICATION INFORMATION

(A) AUTHOR: Zimmermann, D.R. , and Ruoslahti, E.

(B) TITLE: Multiple domains of the large fibro- blast proteoglycan, versican.

(C) JOURNAL: EMBO (Eur. Mol . Biol . Organ . ) J.

(D) VOLUME: 8

(F) PAGES: 2975-2981

(G) DATE: 1989

(Xi) SEQUENCE DESCRIPTION: SEQ ID NO 5:

Leu His Lys Val Lys Val Gly Lys Ser Pro Pro Val Arg Gly Ser

5 10 15

Leu Ser Gly Lys Val Ser Leu Pro Cys His Phe Ser Thr Met Pro

20 25 30

Thr Leu Pro Pro Ser Tyr Asn Thr Ser Glu Phe Leu Arg lie Lys

35 40 45

Trp Ser Lys lie Glu Val Asp Lys Asn Gly Lys Asp Leu Lys Glu

50 55 60

Thr Thr Val Leu Val Ala Gin Asn Gly Asn lie Lys lie Gly Gin

65 70 75

Asp Tyr Lys Gly Arg Val Ser Val Pro Thr His Pro Glu Ala Val

80 85 90

Gly Asp Ala Ser Leu Thr Val Val Lys Leu Leu Ala Ser Asp Ala

95 100 105

Gly Leu Tyr Arg Cys Asp Val Met Tyr Gly lie Glu Asp Thr Gin

110 115 120

Asp Thr Val Ser Leu Thr Val Asp Gly Val Val Phe His Tyr Arg

125 130 135

Ala Ala Thr Ser Arg Tyr Thr Leu Asn Phe Glu Ala Ala Gin Lys

140 145 150

Ala Cys Leu Asp Val Gly Ala Val lie Ala Thr Pro Glu Gin Leu

155 160 165

Phe Ala Ala Tyr Glu Asp Gly Phe Glu Gin Cys Asp Ala Gly Trp

170 175 180

Leu Ala Asp Gin Thr Val Arg Tyr Pro lie Arg Ala Pro Arg Val

185 190 195

Gly Cys Tyr Gly Asp Lys Met Gly Lys Ala Gly Val Arg Thr Tyr

200 205 210

Gly Phe Arg Ser Pro Gin Glu Thr Tyr Asp Val Tyr Cys Tyr Val

215 220 225

Asp His Leu Asp Gly Asp Phe His Leu Thr Val Pro Ser Lys Phe

230 235 240

Thr Phe Glu Glu Ala Ala Lys Glu Cys Glu Asn Gin Asp Ala Arg

245 250 255

Leu Ala Thr Val Gly Glu Leu Gin Ala Ala Trp Arg Asn Gly Phe

260 265 270

Asp Gin Cys Asp Tyr Gly Trp Leu Ser Asp Ala Ser Val Arg His

275 280 285

Pro Val Thr Val Ala Arg Ala Gin Cys Gly Gly Gly Leu Leu Gly

290 295 300

Val Arg Thr Leu Tyr Arg Phe Glu Asn Gin Thr Gly Phe Pro Pro

305 310 315

Pro Asp Ser Arg Phe Asp Ala Tyr Cys Phe Lys Arg Arg

320 325

(7) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 326 residues

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE

(A) DESCRIPTION: polypeptide (v) FRAGMENT TYPE: functional domains (ix) FEATURE

(A) NAME: rat link protein (X) PUBLICATION INFORMATION

(A) AUTHOR: Doege, K. , Hassell, J.R. , Ca- terson, B. , and Yamada, Y.

(B) TITLE: Link protein cDNA sequence reveals a tande ly repeated protein sequence.

(C) JOURNAL: Proc . Natl . Acad . Sci . USA

(D) VOLUME: 83

(F) PAGES: 3761-3765

(G) DATE: 1986

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 6:

Asp Arg Val lie His lie Gin Ala Glu Asn Gly Pro Arg Leu Leu

5 10 15

Val Glu Ala Glu Gin Ala Lys Val Phe Ser His Arg Gly Gly Asn

20 25 30

Val Thr Leu Pro Cys Lys Phe Tyr Arg Asp Pro Thr Ala Phe Gly

35 40 45

Ser Gly lie His Lys lie Arg lie Lys Trp Thr Lys Leu Thr Ser

50 55 60

Asp Tyr Leu Arg Glu Val Asp Val Phe Val Ser Met Gly Tyr His

65 70 75

Lys Lys Thr Tyr Gly Gly Tyr Gin Gly Arg Val Phe Leu Lys Gly

80 85 90

Gly Ser Asp Asn Asp Ala Ser Leu lie lie Thr Asp Leu Thr Leu

95 100 105

Glu Asp Tyr Gly Arg Tyr Lys Cys Glu Val lie Glu Gly Leu Glu

110 115 120

Asp Asp Thr Ala Val Val Ala Leu Glu Leu Gin Gly Val Val Phe

125 130 135

Pro Tyr Phe Pro Arg Leu Gly Arg Tyr Asn Leu Asn Phe His Glu

140 145 150

Ala Arg Gin Ala Cys Leu Asp Gin Asp Ala Val lie Ala Ser Phe

155 160 165

Asp Gin Leu Tyr Asp Ala Trp Arg Gly Gly Leu Asp Trp Cys Asn

170 175 180

Ala Gly Trp Leu Ser Asp Gly Ser Val Gin Tyr Pro lie Thr Lys

185 190 195

Pro Arg Glu Pro Cys Gly Gly Gin Asn Thr Val Pro Gly Val Arg

200 205 210

Asn Tyr Gly Phe Trp Asp Lys Asp Ser Arg Tyr Asp Val Phe Cys

215 220 225

Phe Thr Ser Asn Phe Asn Gly Arg Phe Tyr Tyr Leu lie His Pro

230 235 240

Thr Lys Leu Thr Tyr Asp Glu Ala Val Gin Ala Cys Leu Asn Asp

245 250 255

Gly Ala Gin lie Ala Lys Val Gly Gin lie Phe Ala Ala Trp Lys

260 265 270

Leu Leu Gly Tyr Asp Arg Cys Asp Ala Gly Trp Leu Ala Asp Gly

275 280 285

Ser Val Arg Tyr Pro lie Ser Arg Pro Trp Arg Arg Cys Ser Pro

290 295 300

Thr Glu Ala Ala Val Arg Phe Val Gly Phe Pro Asp Lys Lys His

305 310 315

Lys Leu Tyr Gly Val Tyr Cys Phe Arg Ala Tyr

320 325

(8) INFORMATION FOR SEQ ID NO: 7 (i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 156 bases encoding 52 amino acids

(B) TYPE: nucleic acid and amino acid

(C) STRANDEDNESS: double

(D) TOPOLOGY: linear (ii) MOLECULE TYPE

(A) DESCRIPTION: DNA encoding a polypeptide (v) FRAGMENT TYPE: partial sequence, PTRl domain (vi) IMMEDIATE SOURCE: human brain (ix) FEATURE

(A) NAME: human BEHAB ( i) SEQUENCE DESCRIPTION: SEQ ID NO 7:

GAG AGG GCT CTG CGC TAT GCT TTC TCC TTT TCT GGG GCC CAG 42 Glu Arg Ala Leu Arg Tyr Ala Phe Ser Phe Ser Gly Ala Gin

5 10

GAG GCT TGT GCC CGC ATT GGA GCC CAC ATC GCC ACC CCG GAG 84 Glu Ala Cys Ala Arg lie Gly Ala His lie Ala Thr Pro Glu 15 20 25

CAG CTC TAT GCC GCC TAC CTT GGG GGC TAT GAG CAA TGT GAT 126 Gin Leu Tyr Ala Ala Tyr Leu Gly Gly Tyr Glu Gin Cys Asp 30 35 40

GCT GGC TGG CTG TCG GAT CAG ACC GTG AGA 156

Ala Gly Trp Leu Ser Asp Gin Thr Val Arg 45 50