CA2467757A1 - B1k chain of laminin and methods of use - Google Patents

B1k chain of laminin and methods of use Download PDF

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CA2467757A1
CA2467757A1 CA002467757A CA2467757A CA2467757A1 CA 2467757 A1 CA2467757 A1 CA 2467757A1 CA 002467757 A CA002467757 A CA 002467757A CA 2467757 A CA2467757 A CA 2467757A CA 2467757 A1 CA2467757 A1 CA 2467757A1
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Robert E. Burgeson
David Wolfe Wagman
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General Hospital Corp
Oregon Health Science University
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Abstract

Recombinant laminin B1k and fragments thereof are produced. The recombinant laminin B1k is a subunit of kalinin a basement membrane protein useful for promoting adhesion of keratinocytes to a substrate and treatment of disorders of the dermis.

Description

Blk CHAIN OF LAMININ AND METHODS OF I1SE
This invention was made with government support. The U.S, government may have certain rights in this invention.
Back~~round of the Invention The present invention relates to adhesion proteins and to methods of using them, e.g., to promote the adhesion of cells to a substrate, e.g., to human dermis. In particular, overlapping cDNA clones encoding the entire Iaminin Blk chain and recombinant proteins expressed therefrom are disclosed.
The structure of the prototype laminin, a glycoprotein component of most, if not all, basement membranes has been well described in a number of species. its overall appearance, as visualized by rotary shawdowing, is cross-shaped with a single long arm arising from the coiled-coil interaction of three separate polypeptide chains and three short arms, each originating from the individual polypeptide chains. The three chains are: A , typified by the Ae chain of EHS laminin (400-kD); B l, typified by the B le chain of EHS
laminin (220-kD);
and B2, typified by the B2e chain of EHS laminin (210-kD) chains. The primary structure for each of the three prototypic polypeptide chains in humans has been elucidated by overlapping cDNAs.
Additional polypeptides that are related to the laminin chains have been identified. A
rat B 1 chain homologue, s-laminin (B 1 s), has been identified. A human A
chain homologue, merosin (Am), has been described and is the same as a homologue A chain found in mouse and bovine heart. Both chains can combine with the laminin A, B 1 or B2 chains to form the variant trimeric proteins jAe, B 1 s, B2e], [Am, B 1 e, B2e] and [Am, B 1 s].
A second B 1 variant (the sequence cf which is a chain based on partial cDNA sequences) from avian eye has been reported and overlapping cDNAs for a human variant B2 chain called laminin B2t have also been described.
Kalinin is an epithelium-specific laminin variant that is the major, if not the only component of the anchoring filament. (The anchoring filament is a characteristic uItrastructural component of the dermal-epidermal junction of skin believed to mediate the adhesion of the epithelium to the basement membrane.) The kalinin molecule contains three disulfide bond-linked polypeptide chains consisting of a 200-kD kalinin .4 chain (Ak), a 155-kD kalinin B2 chain (B2t), and a 140-kD kalinin B 1 chain (B 1 k). Rotary shadowing of the molecule results in a 107-nm rod with globular domains at each end.
KaIinin is an epithelial-specific cell attachment factor utilized by skin keratinocytes for strengthening their attachment to the underlying dermis. Antibodies to the Ak chain cause the detachment of subconfluent karatinocytes from their growth substrate and deepithelization of intact skin.
~ummarv of the Invention In general, the invention features a purified DNA including a sequence encoding a Blk chain of laminin.
In preferred embodiments: the DNA encodes the B l.k protein of (SEQ ID N0:2);
the encoded B 1k peptide is at least 80, more preferably 90, and most preferably 95 or 98%
homologous with the sequence of (SEQ ID N0:2); the DNA encodes a biologically active B lk.
In another aspect, the invention features a recombinant Blk. , In preferred embodiments: the recombinant B 1 k protein has the 'sequence of (SEQ
ID N0:2); the recombinant B l k peptide is at least 80, more preferably 90, and most preferably 95 or 98% homologous with the sequence of (SEQ ID N0:2); the recombinant B 1 k has biological activity. .
1$ . The invention also includes a vector including a DNA sequence encoding a Blk protein; a cell containing the vector; a method for manufacture of B 1 k including culturing the cell in a medium to express B 1 k.
In another aspect, the invention features a purified DNA including (or consisting essentially of) a sequence encoding a fragment of a B 1 k laminin chain.
In preferred embodiments: the sequence encodes domain VI of B 1 k, or a kalinin A
chain-binding fragment thereof; the sequence encodes a peptide with a biological activity of domain VI of native B 1 k, e.g., the ability to bind to a kalinin A chain; the sequence encodes any of domain VI, V, N, III, II, a., or I of B 1 k.
In other preferred embodiments: the sequence of the encoded Blk fragment is essentially the same as that of a naturally occurring Blk sequence; the DNA
sequence which encodes the Blk fragment is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA
encoding a naturally occurring B 1 k, e.g., B 1 k encoding DNA from SEQ ID NO:1; the sequence which encodes a B1k fragment hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring B I k sequence e.g., the amino acid sequence of SEQ ID NO:1;
the amino acid sequence of the encoded Blk fragment is less than 30, more preferably less than 40, more preferably less than 50, and most preferably less than 60, 80, 100, or 200 amino acid residues in length; the encoded Blk amino acid sequence is at least SO% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most 3~ preferably 95% as long as a naturally occurring B 1 k; the amino acid sequence of the encoded B 1 k fragment is at least 80%, more preferably at Least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99%
homologous with a naturally occurring B 1 k sequence, e.b., the sequence of SEQ ID NO:1; the fragment has r 2a biological activity. For example, in one aspect, the invention provides a purified DNA useful as a probe or primer comprising a sequence which hybridizes under stringent conditions to a nucleic acid encoding a.Blk laminin chain of Figure 2, or encoding a fragment of the Blk laminin chain of Figure 2 which has the ability to bind a kalinin. A chain.

In other preferred embodiments the fragment includes more than one Blk domain and: the domains in the encoded peptide are arranged in the same relative linear order as found in a naturally Blk; the linear order of the encoded domains is different from that found in a naturally occurnng B 1 k; the domains in the encoded peptide differ in one or more of composition (i.e., which domains are present), linear order, or number (i:e., how many domains are present or how many times a given domain is present) from a naturally occurring B 1 k.
In another aspect, the invention features, a DNA, preferably a purified DNA, which includes (or consists essentially of) a sequence encoding a fragment of B lk of 20 or more amino acids in length, the peptide having at least 90% homology with an amino acid sequence which is the same, or essentially the same, as a naturally occurring B 1 k peptide, e:g., the amino acid sequence of SEQ ID N0:2. In preferred embodiments the purified DNA
encodes: a peptide which is at least 30, more preferably at least 40, more preferably at least 50, and most preferably at least 60, 80, 100, or 200 , amino acid residues in length; the encoded peptide is at least SO% more preferably at least 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Blk; a peptide which is at least 80, more preferably at least 85, yet more preferably at least 90, yet more preferably at least 95, and most preferably at least 98 or 99%
homologous with an amino acid sequence which is the same, or essentially the same, as a naturally occurring B 1 k peptide, e.g., the amino acid sequence of SEQ ID NO
2; the peptide has biological activity.
The invention also includes a DNA sequence encoding a B 1 k fragment; a cell containing the purified DNA; a method for manufacture of a B 1 k fragment comprising culturing the cell in a medium to express the Blk fragment.
In another aspect, the invention features a peptide which is a fragment of a B
1 k laminin chain.
In preferred embodiments: the peptide includes (or consists essentially of) domain VI
of B1k or a kalinin A chain-binding fragment thereof; the peptide has a biological activity of domain VI of native Blk, e.g., the ability to bind to a kalinin A chain; the peptide includes any of domain VI, V, IV, III, II, a, or I of B 1 k; the fragment has biological activity.
In other preferred embodiments: the sequence of the peptide is essentially the same as that of a naturally occurring B 1 k sequence; the DNA sequence which encodes the B 1 k peptide is at least 85%, more preferably at least 90%, yet more preferably at least 95%, and most preferably at least 98 or 99% homologous with DNA encoding a naturally occurring 3 ~ B I k, e.g., B 1 k encoding DNA from SEQ ID NO: l ; the sequence which encodes the B 1 k peptide hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring B 1 k sequence e.g., the amino acid sequence of SEQ ID N0:2; the amino acid sequence of the peptide is less than 30, more preferably less than 40, more preferably less than 50, and most preferably less than 60, 80, 100, or 200 amino acid residues in length; the peptide's amino acid sequence is at least 50% more preferably 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring Blk; the amino acid sequence of the peptide is at least 80%, more preferably at least 85%, yet more preferably at least 90%, yet more preferably at least 95%, and a most preferably at least 98 or 99% homologous with a naturally occurring Blk sequence, e.g., the sequence of SEQ ID N0:2.
In other preferred .embodiments the peptide includes more than one B I k domain and:
the domains in the peptide are arranged in the same relative linear order as found in a naturally B 1 k; the linear order of the domains is different from that found in a naturally occurring Blk; the domains in the peptide differ in one or more of composition (i.e., which domains are present), linear order, or number (i.e., how many domains are present or how many times a. given domain is present) from a naturally occurring B 1 k; the peptide has biological activity:.
In another aspect, the invention features a transgenic animal, e.g., a rodent, having a B 1 k transgene, e.g.; a transgene which misexpresses the B 1 k chain of laminin.
In another aspect, the invention features a method of increasing the permeability of the skin including inhibiting an interaction between B1k and a second molecule, e.g., a kalinin A chain.
In preferred embodiments, the interaction is inhibited by: administering an antibody against a site on kalinin A with which Blk interacts; administering an antibody against a site on BIk, e.g., a site in domain VI, which interacts with the second molecule;
administering a fragment of B 1 k, e.g., a fragment containing domain VI which competes, e.g.;
competitively or non-competitively with B I k for a site on the second molecule.
In another aspect, the invention features a method of promoting the adhesion of a molecule, e.g., kalinin A or kalinin A- containing molecule, e.g., kalinin or laminin or a cell, e.g.; a keratinocyte, to a substrate including providing the substrate coupled, linked, or adhered, to a fragment of B I k which includes domain VI, contacting the molecule or cell, with the B 1 k domain VI.
In preferred embodiments, the method further includes forming a covalent bond, e:g., a sulfhydral bond, between the molecule or cell and the B 1 k domain VI.
In another aspect, the invention features a peptide useful for promoting the adhesion of a first molecule or cell, e.g., a keratinocyte, to a second molecule or cell, e.g., a keratinocyte, including a first B 1 k domain linked to a second B I k domain.
(The first domain, e.g., domain VI, binds to the first molecule or cell and the second domain, e.g., domain VI, binds to the second molecule or cell), In another aspect, the invention features a method of coupling a first molecule or cell to a second molecule or cell including providing a molecule having a first B 1 k domain and a second B 1 k domain, linking the f rst molecule or cell to the first domain, and linking the 4a second molecule or cell to the second domain. For example, the present invention provides a method comprising the steps of: providing a coupling molecule having a first Blk domain and a second Blk domain; linking a first molecule.to said first Blk domain; and linking a second molecule to said second Blk domain of the coupling molecule.

S
In preferred embodiments: ~ the first and/or second molecule is an adhesion molecule, e.g., laminin, kalinin, or collagen; the first andlor second Blk domain is domain VI or a kalinin A chain-binding fragment thereof of B 1 k; the first and/or second cell in a keratinocyte. .
The invention also includes substantially pure preparation of an antibody, preferably a monoclonal antibody directed against a Blk protein or a fragment of a Blk protein, e.g., a fragment which contains only one domain of B l k; a therapeutic composition including an B lk protein or fragment thereof and a pharmaceutically acceptable carrier; a therapeutic composition which includes a purified DNA of the invention and a pharmaceutically acceptable carrier.
In another aspect, the~invention features a~method for treating an animal, e.g., a human, a mouse, a transgenic animal,~or an animal model for a disorder, e.g., a disorder of the dermis, e.g., epidermal bulosis, including administering a therapeutically-effective amount of a B 1 k or fragment thereof to the animal.
In another aspect, the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder; e.g., a disorder of .
the dermis; e.g., epidermal bulosis, including administering to the animal cells selected, e.g:, selected in vitro, for the expression of a product of the B l k gene, e.g., cells transformed with B 1 k or B 1 k fragment-encoding DNA.
In preferred embodiments: the cells are, taken from the animal to which they are administered; the cells are taken from an animal which is MHC matched with the animal to which they are administered; the cells are taken from an animal which is syngeneic with the animal to which they are administered; the cells are taken from an animal~which is of the same species as is the animal to which they are administered.
In another aspect, the invention features a method for treating an animal;
e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder, e.g., ~ a disorder of the dermis, e.g, epidermal bulosis, including administering to the animal a nucleic acid encoding a B 1 k or fragment thereof and expressing the nucleic acid.
in another aspect, the invention features a method of evaluating the effect of a treatment, e.g.; a treatment designed to promote adhesion of a keradnocyte to its substrate including carrying out the treatment and evaluating the effect of the treatment on the expression of the B 1 k gene.
In preferred embodiments the treatment is administered: to an animal, e.g.,~a human, a mouse, a transgenic animal, or an animal model for a dermal disorder, e.g., epidermal bulosis, or to a cell, e.g., a cultured cell.

5a Thus, in one aspect the invention provides a use, for treating an animal having a disorder of the dermis, of a therapeutically-effective amount of B1k or a fragment thereof, or of a cell selected for the expression of a product of the B1k gene or a fragment thereof, or of a nucleic acid encoding a B1k or a fragment thereof..
In another aspect, the invention provides a use, for preparing a medicament for treating an animal having a disorder of the dermis, of a therapeutically-effective amount of B1k or a fragment thereof, or of a cell selected for the expression of a product of the B1k gene or a fragment thereof, or of a nucleic acid encoding a B1k or a fragment thereof.
In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the B1k gene, e.g., a disorder of the dermis, e.g., epidermal bulosis, including examining the subject for the expression of the B1k gene, non-wild type expression or mis-expression being indicative of risk.
In another aspect, the invention provides an isolated antibody that binds to a polypeptide than consists of an amino acid sequence selected from the group consisting of: (a) domain VI of Blk, (b) domain III of Blk, (c) domain II of Blk, (d) domain I of Blk, (e) domain alpha of Blk, (f) any one of EGF domains 2, 3, 11, 12 or 13 of Blk, and (g) any combination of (a) - (f) .
In another aspect, the invention provides a composition comprising an antibody as described ak>ove and a pharmaceutically acceptable carrier.

5b In another aspect, the invention provides a method for making a domain-specific antibody, the method comprising: immunizing an experimental animal with an immunogen comprising an isolated polypeptide that consists of an amino acid sequence selected from the group consisting of: (a) domain VI of Blk, (b) domain III/V of Blk, (c) domain II of Blk, (d) domain I of Blk, (e) domain alpha of Blk, (f) any one of EGF domains 1, 2, 3, 11, 12 or_ 13 of Blk, (g) immunogenic fragments of any of the foregoing, and (h) any combination of (a)-(g); and preparing, from the experimental animal, an antibody that binds to the immunogen.

In another aspect, the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the B lk gene, e.g., a disorder of the dermis, e.g., epidermal bulosis, including providing a nucleic acid sample from the subject and determining if the structure of an B 1 k gene allele of the subject differs from wild S type.
In preferred embodiments: the determination includes determining if an Blk gene allele of the subject has a gross chromosomal rearrangement; the determination includes sequencing the subject's Blk gene.
In another aspect, the invention features, a method of evaluating an animal or cell model for a disorder, e.g., a disorder of the dermis, e.g., epidermal bulosis, including determining if the B 1 k gene in the animal or cell model is expressed at a predetermined level or if the Blk gene is mis-expressed. In preferred embodiments: the predetermined level is lower than the level in a wild type or normal animal; the predetermined level is higher than the level in a wild type or normal animal; or the pattern of isoform expression is altered from wildtype.
In another aspect, the invention features a transgenic rodent, e.g., a mouse, having a transgene which includes an B 1 k gene or B 1 k protein encoding DNA. In preferred embodiments: the B lk gene or DNA includes a deletion, e.g. a deletion of all or part of B lk, e.g., a deletion of all or part of a domain e.g., domain VI, or is otherwise mis-expressed.
Purified DNA is DNA that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the S' end and one at the 3' end) in the naturally occurnng genome of the organism from which the DNA of the invention is derived. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus;
or into the genomic DNA of a prokaryote or eukaryote, or which exists as a separate molecule (e.g., a cDNA or a genomic DNA fragment produced by PCR or restriction endonuclease treatment) independent of other DNA sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
Homologous refers to the degree of similarity in sequence between two polypeptide molecules or between two nucleic acid molecules. When a position in both of the two compared sequences is occupied by the same base or amino acid monomeric subunit, e.g., if a position in each of two DNA molecules is occupied by adenine, then the molecules are homologous at that position. The homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences. For example, 6 of 10, of the positions in two sequences are matched or homologous then the two sequences are 60% homologous. By way of example, the DNA sequences ATTGCC and TATGGC share 50% homology.

A transgene is defined as a piece of DNA which is inverted by artifice into a cell and becomes a part of the genome of the animas which develops in whole or part from that cell.
Such a transgene may be partly or entirely heterologous to the transgenic animal.
A transgenic animal, e.g., a transgenic mouse, is an animal having cells that contain a transgene, which transgene was introduced into the animal, or an ancestor of the animal, at a prenatal, e.g., an embryonic stage.
A substantially pure preparation of a peptide is a preparation which is substantially free of the peptides with which it naturally occurs in a cell. A substantially pure preparation of a non-naturally occurring peptide is one which is at least 10% 'by weight of the peptide of interest.
Mis-expression, as used herein, refers to a non-wild type pattern of gene expression.
It includes: expression at non-wild type levels, i:e., over or under expression; a pattern of expression that differs from wild type in terms of the time or stage at which the gene is expressed, e.g., increased or decreased expression (as compared with wild type) at a predetermined developmental period or stage; a pattern of expression that differs from wild-type in terms of the tissue specificity of expressions, e.g., increased or decreased expression (as compared with wild-type) in a predetermined cell type or tissue type; a pattern of expression that differs from wild type in terms of the length, amino acid sequence, post-translational modification, or a biological activity of a B 1 k gene product;
a patterns of expression that differs from wild-type in terms of the effect of an environmental stimulus or extracellular stimulus on expression of the gene, e.g., a pattern of increased or decreased expression (as compared with wild type) in the presence of an increase or decrease in the strength of the stimulus; or a pattern of isoform expression which differs from wild-type.
A protein or peptide has B 1 k biological activity if it has one or more of the following 2~ properties: the ability to covalently bind via disulfide bond formation with a kalinin B2 chain and a kalinin A chain to form a trimeric protein, kalinin; the ability to bind the kalinin A
chain through a covalent disulfide bond formation with domain VI of the B 1 k chain; the ability to specifically bind type IV collagen; if a B 1 k domain present on a B 1 k protein or fragment has a biological .property that the domain has when present in the native B lk molecule, e.g., the ability to bind or associate in a specific way with another molecule, e.g., another larninin or kalinin chain or the ability to form a characteristic native rotary shadowy structure characteristic of native B lk.
The molecules of the invention are useful for promoting adhesion of adhesion molecules or keratinocytes to a substrate, e.g., human dermis. The molecules of the invention are also useful for research in cell adhesion. The role of the DNA sequence encoding a peptide having B 1 k activity and its products can be studied in cells, e.g., cultured cells, transformed with the aforementioned DNA sequence, or fragments thereof, or in transgenic animals. The peptides fragments of the invention allow preparation of antibodies, i.e., monoclonal antibodies, directed against a specific domain.

Other features and advantages of the invention will be apparent from the following description and from the claims.
~e~",ai~ed Description Drawings The drawings are first briefly described.
Fig. 1 is a map of the B lk region showing (heavy line) start (ATG) and stop codons (TGA), restriction sites (single letter abbreviations), and domains. The light lines below the map show overlapping cDNA clones encoding the entire kalinin B 1 chain:
Fig. 2 is a map of the nucleotide sequence of the laminin B 1 k chain (SEQ ID
NO: 1 and the predicted amino acid sequence (SEQ ID NO: 2) of the B lk peptide chain. Triangles indicate potential N-linked glycosylation sites. Stars indicate potential glycosaminoglycan attachment sites.
Fig. 3 is a domain-by-domain comparison of the amino acid sequences of the laminin B 1 k chain (SEQ ID NO: 3) and the human B 1 (SEQ ID NO: 4) chain (B 1 e).
Fig. 4 is: A) a comparison of the domain sizes and percent identity for the various domains of the laminin B 1 a and laminin B 1 k chains; and B) a depiction of the numbering scheme for the Iaminin B 1 k domains. The domains are numbered according to their similarity to the comparable domains in the laminin B 1 a chain. Some of the laminin B 1 a chain domains are missing in the Iaminin Blk chain and those that remain are truncated in comparison to the laminin B 1 a chain.
Fig. 5 is a comparison of the amino acid sequences of domain VI for Blk (SEQ
ID
NO: 5), B 1 a (SEQ ID NO: 6), and B 1 s (SEQ ID NO: 7). The underlined regions are areas where the sequence identity between B 1 a and B I s is above average, but the sequence identity with B 1 is considerably less than average. The arrow shows an additional cysteine contained by B 1 k at residue number 50.
Fig. 6 is a comparison of peptide sequences of rat laminin B 1 s (SEQ ID NO:
8), human laminin B 1 s (SEQ ID N0: 9) and human laminin B 1 k (SEQ ID N0: 10).
Also shown is a comparison of the amino acid sequences of human laminin B2t peptides determined by deduction from cDNA (SEQ ID NO: 1 l and SEQ ID N0: 13) (top line) and from sequencing of purified peptide (bottom line) (SEQ ID NO: 12 and SEQ I.D NO: 14).
Fig. 7 is a comparison of the cloned cDNA sequence to the B 1 and B2 chains of laminin (LAMB 1 a and LAMB2e), the B2 chain of kalinin (LAMB2t) and the B I
chain of s-laminin (LAMBIs).

cDNA Clones for the Kalinin B1 (Laminin Blk) Chain The screening of the squamous cell carcinoma cell cDNA expression library with a polyclonal antibody which recognizes human kalinin yielded several positive clones. The fusion proteins from positive clones were adsorbed to nitrocellulose and exposed to the polyclonal antiserum used for the initial screening. Antibodies binding the fusion proteins were individually collected and used for Western blot analysis of partially purified kalinin.
Clones were identified that expressed fusion proteins that bound antibodies specific for the 140-kD and the 155/105-kD chain. (The B2 chain is processed from a 155 to a 105 kD
form.) Selected clones were sequenced and the predicted amino acid sequences encoded by the cDNAs showed extensive homologies with the B1 and B2 laminin chains. The encoded sequences fro the B 1 k and B2t chains were confirmed by direct amino acid sequencing of the 140-kD and 155/105-kD kalinin chains.
The nucleotide sequences of the 155/105-kD chain were 99.9% identical to the recently published B2t chain and 100-kD chain of nicein. Protein sequencing of two tryptic peptides from the chain exactly matched derived amino acid sequences, confirming that laminin B2t, the 100-kD nicein chain and the 1551105-kD kalinin chain are identical.
Clones encoding the kalinin 140-kD kalinin B 1 chain were selected for further characterization (Ka126, Ka145, Ka148, Ka168, Ka182, and Ka185, Fig. 1 ).
These clones contained 1.5-kb, 0.9-kb, 1.3-kb, 1,8-kb, 1.2-kb, and 2.1-kb inserts, respectively, and nucleotide sequencing demonstrated that the derived amino acid sequences showed extensive similarity to human laminin B 1 chain. Rescreening of the cDNA library with Ka145 resulted in the isolation of clones KalS-5 and Kal6-4 (Fig. 1). These clones contained 2.3-kb and 1.0-kb inserts, respectively. To obtain the 3' end of the cDNA, a 3' RACE
procedure (BRL) was used on total mRNA from squamous cell carcinoma media. This resulted in the clone Ka192-1 (1.8-kb). The complete nucleotide sequence of the overlapping clones and the predicted amino acid sequence are shown in Fig. 2.
The immunogen for polyclonal antiserum against kalinin purified from human keratinocyte-conditioned culture medium has been previously described (Lunstrum et al., 1986; Rousselle et al., 1991).
Isolation of RNA and cDNA synthesis were performed as follows. Ten Costar T-flasks were seeded with squamous carcinoma cells (SCC) and allowed to grow until sub-confluent. Media was removed and the cells were lysed and total RNA isolated following the guanidium thiocyanate method of Chomczynski and Sacchi, 1987. Poly A+RNA was collected using a Collaborative Research oligo dT Cellulose type 3 column and following company guidelines. Six hundred mg of Poly A+ enriched RNA was sent to Clontech Laboratories (Palo Alto, CA.) for construction of the Lambda gtl 1 cDNA
library using random primers.

Library screening was performed as follows. The anti-kalinin polyclonal antibody (pAB} was diluted in 1:10 in l OmM TNT (iris-HCI, pH 8.0; 1 SO mM NaCI; 0.05%
Tween 20; 3% BSA}. E. coli (Y-1090 strain) whole cell lysate was added to the diluted antibody and '' incubated at 4°C for 24 hours on a nutator. The pre-absorbed antibody was centrifuged at S 10,000 rpm for 10 minutes at 4°C and the supernatant collected. The absorbed antibody was then diluted 1,10 (final dilution 1:100) in TEST (50 mM Tris-HCI, pH 7.9; 1 SO
mM NaCI;
O.OS% Tweeii 20) and 1.2% BSA added. The diluted absorbed antibody was used to screen 8.34 X 10S plaques from the unamplified random-primed cDNA Library and horseradish peroxidase (HRP) secondary antibody was used to visualize the positive plaques. A total of 10 89 positive individual plaques were purified in a larger scale and tested again against the antibody.
Epitope determination for phage clones were performed as follows. Far each clone, three I SO X 1 Smm LB-ampicillin plates were plated at a density of 6000 pfu and grown 3 hours at 37°C. The plates were overlaid with IPTG saturated nitrocellulose filters and 1 S incubated overnight at 37°C. Plates were cooled at 4°C for I
S minutes and the filters were removed and washed 3 times in TBST (1S min for each wash). The filters were blocked in 4% BSA in TEST for 1 hour at zoom temperature (RT). Filters were then washed 3 times in TBST. Filters were exposed to the pAB for 3-4 hours at RT followed by 3.washes in TBST.
The antibody was eluted from the filter by soaking each filter in 2Sml of 1M
acetic acid for 20°minutes. The antibody/acetic acid solution for each of the triplicate samples was pooled and 2 drops of a saturated phenol red solution was added. T'he solution was neutralized by the addition of an aqueous solution saturated with Tris-HCI and 0.03% BSA was added. The solution was dialyzed against two changes of 1X TBS at 4°C overnight.
The purified antibody solution was collected from the dialysis membrane and a "pinch" of BSA was 2S added. The solution was frozen at -20°C until needed.
Mini-western blots of purified kalinin were made and exposed to purified antibody from each of the clones for 60-hours at 4°C. Blots were then washed three times xn TBST for 1 S minutes each. Secondary HRP conjugated antibody was used to illuminate the particular band of kalinin chain corresponding to the clone.
Northern blots were performed as follows. Poly A+RNA was isolated from cell culture of 2 T165 flasks of 70-80% confluent squamous.carcinoma cells using Invitrogen's Fast Track RNA isolation systems and exactly following the manufacturer's recommendations. The final RNA pellet was resuspended in SO ml elution buffer.
Twenty mb of Poly A+RNA was used for the gel and subsequent blot using the procedure outlined by 3S Fourney et al. Clone KaIS-S was radioactively labeled with the Amersham Random labeling system. The blot was placed against X-ray film for 2 hours at -80°C.
3' Rapid Amplification of cDNA Ends (RACE) was performed as follows. A 3' RACE kit was purchased from GIBCO BRL and I mg poly A+RNA in I3 ml DEPC-treated water was made into cDNA by reverse transcriptase according to manufacturer's *trade-mark recommendations. The first strand DNA was amplified by PCR following the manufacturer's protocol using the provided antisense poly (T) primer called AP and a specific sense primer for the kalinin B 1 chain called D92 (GCT TCA ATG GTC TCC TTA CTA TGT A) (SEQ
ID
NO: 15):
The Laminin Blk Chain Encodes a Distinct Laminin-like Polypeptide Analysis of the sequence showed that the first possible translated methionine (first amino acid residue, Fig. 2) is followed by a stretch of hydrophobic amino acid residues which are typical for a signal peptide. From the formula far a signal peptide (von Heijne, 1983 and 1986), the signal peptide would he cleaved following Alal7. The 17 residue long signal peptide is followed by an open reading frame of 1148 amino acid residues with a deduced molecular weight of 126,464 daltons: There are 3 putative N-linked glycosylation sites having the predicted residue sequence Asn-X-Ser/Thr, 3 potential O-linked glycosylation 1 S sites having the predicted cluster of three or more consecutive Ser and Thr residues and 2 potential glycosaminoglycan attachment consensus sequences, Ser-Gly-X-Gly. In addition there are 120 nucleotides of 5' untranslated sequences and 31 S nucleotides of 3° untranslated sequences for a total of 3931 bases. Northern blot analysis showed a single message of 4.0-kb when probed with the cDNA clone KalS-5.
Protein Sequencing was performed generally as according to Aebersold et al., 1987.
Kalinin purified from amnion (Marinkovich et al., 1992a) was run on a polyacrylamide gel in the presence of 2-mercaptoethanol and blotted on a nitrocellulose membrane (Biorad). The 140-kD band was excised and digested by the protease Lys-C. The digested product was separated by HPLC and one fragment was sequenced on an Applied Biosystem sequencer.
Computer analysis of the mature polypeptide demonstrated that the laminin Blk chain is most similar to the human laminin B 1 chain (Lama 1 E). A comparison of the laminin B 1 k polypeptide to this chain is presented in Fig. 3.
Pyroglutamate aminopeptidase reaction was performed generally as according to Andrews et al., 1991. Briefly, kalinin purified from amnion was run on a polyacrylamide gel in presence of 2-mercaptoethanol and blotted on a PVDF membrane in 25mM Tris, 192mM
glycine, 0.05% SDS and 10% methanol for 4 hours. The 140-kD band was excised, blocked in PVP-40 in O.1M acetic acid at 37°C for 30 minutes, washed ten times in water and digested by pyro-glutamate aminopeptidase (Boehringer Mannheim) (62.5 mg/mg of protein in SOmM sodium phosphate, IOrnM EDTA, SmM DTT, 5% glycerol, pH 8.0) for 12 hours at 3~ 4°C. An additional 62.5 mg of pyroglutamate aminopeptidase/mg of protein was added and digestion was done for 12 hours at 37°C. The blot was washed ten times in water, dried under vacuum and subjected to sequencing on an Applied Biosystem sequencer.

Domain Structure of the Laminin Bllc Chain Since the laminin B 1 k chain has similarity to the laminin B 1 a chain, its domains were assigned numbers according to their similarity to the comparable domains in laminin (Fig.
4A). Some of the laminin B 1 a chain domains are missing in the laminin B 1 k chairs and those that remain are all truncated in comparison to the laminin B le chain.
Specifically, the carboxy-terminal 1/3 of domain V, all of domain IV, and the amino-terminal 2/3 of domain III are missing in the laminin B 1 k chain. Fig. 4A shows a comparison of the domain sizes and percent identity for the various domains of the laminin Ble and laminin Blk chains. The most amino-terminal domain, domain VI (residues 1-231), is a 231-amino acid residue region containing 9 cystine residues. This domain is likely to form a globular structure similar to domain VI in the laminin B I a chain. Domain III/V (residues 232-559) contains six cysteine-rich EGF modules with three of them similar to comparable modules in domain III (EGF 1, 2, and 3) and three of them similar to comparable modules in domain V (EGF 11, 12, 13) of the laminin Ble chain. The laminin Blk chain has no globular domain IV as is found the in the laminin B 1 a chain. Domain II (residues 560-766), as in the laminin B 1 a chain, begins with two closely spaced cysteins and is predicted to be an a-helical domain containing heptad repeats typical for coiled-coil proteins. Domain I (residues 798-1148) also contains heptad repeats typical for coiled-coil proteins. Just as in laminin B 1 e, this domain contains a single cysteine residue one residue away from the carobxyl-terminal end. Also similar to the laminin B 1 a chain is a cysteine-rich (6 cysteine residues) a domain that interrupts the helical structures of domains I and II.
Since domain VI is the only globular domain retained by the Blk chain, and since the homologous domain in laminin and s-laminin are believed to mediate self assembly, the sequences of domain VI for B 1 k, B 1 a and B 1's were compared (Fig. 5). The amino acid identity of domain vi for B 1 a and B 1 s shows 70% sequence conservation (Fig. 5). The number and location of cysteinyl residues is identical. Comparisons of the B l k sequence with these two chains shows 49.8% overall sequence identity. As shown in Fig, 5, B 1 a and B 1 s contain several regions within domain VI where the sequence identity is above average.
Three of these regions share considerably less than average sequence identity with the B1k chain (Fig. 5, underlined). The B 1 k chain contains an additional cysteine at amino acid residue number S O (Fig. 5, arrow). This region is also highly divergent from the B 1 a and B 1 s chains with an 18% amino acid residue identity to the B 1 a chain (excluding the obligatory cysteine) whereas, the same region is 70% identical between B 1 a and B 1 s.
These 3 5 comparisons suggest that B 1 k shares an overall structural similarity with B 1 a and B 1 s, but the chains are likely to be functionally divergent.

The Laminin Blk Chain Is a Truncated Chain As described above, overlapping cDNA clones encoding the entire 140-kD laminin B 1 k chain were characterized. The 3.9-kb sequenced corresponds well with the 4.0-kb message size predicted by northern blot analysis. 3' and 5' RACE procedures and were not able to extend the sequence further on either end.
The identity of the cDNAs were confirmed by sequencing a 19-residue long tryptic peptide from the purified I40-kD laminin Blk chain (double-underlined in Fig.
2).
Additional protein sequencing of the amino-terminal end of the polypeptide chain confirmed that the end was blocked and therefore most likely began with the residue Gln.
After unblocking the end we determined the partial sequence Q-A-C-X-R (X is an indeterminate residue) which corresponds well with our predicted signal peptide cleavage site (start of domain VI, Fig. 2).
The estimated protein size from the cDNAs is 126,464 daltans. This is in contrast to protein data which predicts a protein of about 140,000 daltons. The most likely explanation for this discrepancy is that the chain is glycosylated. There are three potential N-linked glycosylation sites which are underlined in Fig. 2. There am two potential glycosaminoglycan attachment sites marked with stars and three potential N-linked glycosylation sites marked by triangle in Fig. 2. It is interesting to note that the three potential O-linked glycosylation sites are all located in the amino-terminal globular domain, domain VI, which rotary shadowed images predicts to project from the long arm, an ideal position to interact with other molecules such as carbohydrates. In addition, one N-linked glycosylation site is present in the a domain which may extend away from the long arm of the chain and interact with other molecules. The function of the a domain is not known.
The Laminin Blk Chain Is Similar To The Laminin Ble and Laminin Bls Chains Fig. 7 shows a comparison of our cDNA sequence to the B 1 and B2 chains of laminin (LAMB 1 E and LAMB2E), the B2 chain of kalinin (LAMB2'T) and the B 1 chain of s-laminin (LAMB 1 S). Since the kalinin B I chain is clearly related to these other laminin subunits, the convention of Engel et al., 1991 was followed and the Kalinin B I chain will be named Laminin B 1 k. As is apparent from Fig. 7, the human laminin B 1 k chain is most similar to the human laminin B 1 a (34.1 % identity) and rat laminin B 1 s (3 7. I %
identity) chains. Initially it seemed possible that the laminin B I k chain might be the human equivalent of the laminin B 1 s chain since the amino acid residue identity was high when considering comparing two different species. There are two pieces of evidence that show that the laminin B 1 k chain is distinct from the laminin B 1 s chain. The first is the size of the laminin B
1 k chain polypeptide which was previously reported to be 140-kD. The laminin B 1 s chain in rat is about 190-kD which is only slightly smaller than the 200-kD laminin B 1 a chain. Since there is good conservation of protein size between species (from human to drosophila) for all three of the laminin chains (Laminin Ae, B 1 e, and B2e), one expects the same will hold true between species for the.laminin B l s chain as well and it is predicted that this chain will be 190-200-kD in size. Additional evidence that the laminin B lk chain is distinct from the laminin B 1 s chain is the fact that a human Cryptic peptide sequence was found that is not found in the laminin B 1 k chain, but has 95.8% identity to the rat laminin B
1 s chain.
Since the human sequence of the laminin B 1 s chain is not available, the B 1 k sequence was compared to the most well described similar molecule, the laminin B 1 a chain. The maj or difference between the laminin B 1 a and Iaminin B 1 k: chains is their size. The Iaminin B l k chain has a truncated structure and, therefore, a lower molecular mass than the 200-kD
laminin B 1 a chain. This smaller size is mainly due to the absence of the globular domain which corresponds to domain IV in the laminin B 1 a chain and to the fact that the corresponding domains III and V are fused into a single domain that is about half the size of the two domain together. There may also be differences in glycosylation between the two polypeptides.
As was found for the laminin B2t chain, the short arms of laminin B 1 k and laminin B 1 a have the greatest sequence homology than the long arms (Fig. 4, upper panel: compare domains III-VI, 40-50% identity, to domains I-II, 20-30% identity).
Domain Structure Of The Short Arm Of The Laminin Blk Chain The greatest functional significance of the short arm is found in the amino-terminal domain VI. In laminin B 1 e, domain VI has been reported to aid in the self assembly of the laminin molecules in vitro. The presence of this domain in the Iaminin B 1 k chain suggests that this domain could help to organize the extracellular matrix by associating with either other kalinin or laminin molecules. Since this domain is missing in laminin B2t, if the laminin protein associates with other molecules, then this domain is especially crucial in ,laminin Blk. One possible ligand for this domain is the recently described K-laminin molecule which contains the laminin B 1 a and B2e chains and a novel A chain.
A second candidate for the interaction is type IV collagen which has been reported to bind to the short arms of the laminin B chains.
The comparison of the B 1 k sequence to B 1 a and B 1 s within the VI domain are particularly interesting. The highly divergent amino acid residue identity in certain areas (Fig. 5, underlined) strongly suggests that domain VI of Blk is functionally different from the other known laminin B 1 chains. B lk domain VI also contains an odd number of cysteine residues (Figure 6, arrow), suggesting that one of these is unpaired and available to stabilize interactions of domain VI with another entity. These observations support the hypothesis that kalinin is unlikely to self assemble through interactions of the VI domains, but rather, the VI
domain specifically interacts with the A chain of K-laminin. In tissues, kalinin is disulfide bonded to K-laminin, but not to other laminins that do not contain the K-laminin A chain.
Rotary-shadowed images of the adduct suggest that the short arm region of kalinin associates at the crotch of the K-laminin short arms. Since the B 1 k chain is the only kalinin chain that remains unprocessed in the mature kalinin molecule, the association with K-laminin appears 5 to be mediated by the B 1 k chain. The significant diversion in sequence homology between the VI domains of B 1 k versus B I a and B I s, and the presence of a potentially unpaired cysteine residue are consistent with the concept that the Blk VI domain binds the short arm of the K-laminin A chain enabling alignment of an unpaired cysteine in each molecule and subsequent disulfide bond formation.
10 Domain IV is missing in the laminin B 1 k chain and while no functions have been reported for the comparable domain in the laminin B 1 a chain, some investigators reported small peptide sequences in this area can bind to heparin. Since the entire domain is absent in kalinin these sequences are missing.
Two cell-surface binding peptide sequences (PDSGR and YIGSR) have been reported 15 in the EGF repeat number 9 in domain III of the laminin B 1 a chain. These peptide sequences are not present since the EGF repeats numbered d-10 are alll missing in domain III of the laminin B 1 k chain.
Domain Structure Of The Long Arm Of The Kalinin Bl Chain The long arm of the laminin B lk chain contains numerous heptad-repeats similar to those found in the two B chains of laminin: The laminin B 1 a and B2e chains have been found to associate with one another and are in fact disulfide-banded. Clearly the three chains of kalinin are disulfide-bonded since they can only be separated by gel electrophoresis only after reduction by ~i-mercaptoethanol. The 155-kD kalinin chain is known to correspond to the previously reported truncated laminin B2t chain by the cDNAs discussed herein as well as to sequenced tryptic peptides (Fig. 7). The laminin Blk chain appears to interact with the laminin B2t chain by forming an a-helix as is found between the laminin~ B 1 a and B2e chains and in fact computer analysis predicts that laminin Blk can form an a-helical coiled-coil 3 0 structure just as laminin B 1 e. The laminin B 1 k and the laminin B2t chain each have a single cysteine in their carboxy-terminal regions that are candidates for disulfide-bonding. The laminin Blk chain also has the short cysteine-rich a domain that divides domains I and II and is predicted to stick out from the long-arm and perform as yet unknown functions.
Finally, adhesion of ciliary ganglion neurons has been attributed to the specific sequence LRE in the laminin B 1 s chain. This sequence is not found in the laminin B lk chain and this function would therefore be missing.

Other Embodiments Nucleic acid encoding all or part of the Blk chain can be used to transform cells. For example, the B 1 k gene, e.g., a mis-expressing or mutant form of the B 1 k gene, e.g., a deletion, or DNA encoding a B 1 k chain can be used to transform a cell and to produce a cell in which the cell's genomic B 1 k gene has been replaced by the transformed gene, producing, e.g., a cell deleted for the B lk gene. Such cells can be used with cells capable of being grown in culture, e.g., cultured stem cells, to investigate the fuunction of the B lk gene.
Analogously, nucleic acid encoding all or part ofthe Blk gene, e.g., a mis-expressing or mutant form of the gene, e.g., a deletion, can be used to transform a cell which subsequently gives rise to a transgenic animal. ' This approach can be used to create, e.g., a transgenic animal in which the Blk gene is, e.g., inactivated, e.g., by a deletion.
Homozygous transgenic animals can be made by crosses between the offspring of a founder transgenic animal. Cell or tissue cultures can be derived from a transgenic animal and the in vivo effects of the Iaminin B 1 k chain can subsequently be studied.
The invention includes any fragment of B 1 k, or any recombinantly produced B
1 k or fragment thereof which is substantially homologous to a B 1 k protein, e.g., the B 1 k protein shown in Fig.2, or other isoforms. Also included are: allelic variations;
natural mutants;
induced mutants, e.g., in vitro deletions; proteins encoded by DNA that hybridizes under high or low (e.g., .washing at 2xSSC at 40° C with a probe length of at least 40 nucleotides) stringency conditions to a nucleic acid naturally occurring (for other definitions of high and low stringency see Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, 6.3. i - 6.3.6, ) ; and polypeptides or proteins specifically bound by antisera to a B 1 k protein, especially by antisera to the active site or binding domain of a B 1 k protein. The term also includes chimeric polypeptides that include a B lk protein.
DNA and peptide sequences of the invention can be, e.g., mouse, primate, e.g., human, or non-naturally occurring sequences.
The invention also includes any biologically active fragment or analog of a Blk protein. By "biologically active" is meant possessing any in vivo or in vitro activity which is characteristic of Blk, e.g., B lk activity as described above. Because the Blk protein exhibits a range of physiological properties and because such properties may be attributable to different portions of the B lk protein molecule, a useful Blk protein fragment or Blk pi°oicin analog is one which exhibits a biological activity in any one (or more) of a variety of B 1 Ic 3~ protein assays, for example, the ability to bind the laminin Ak chain, as described above. A
B 1 k protein fragment or analog possesses, most preferably 90%, preferably 40%, or at Ieasi 10%, of the activity of a naturally occurnng B 1 k isoform, e.g., of the B 1 k protein shown in Fig. 2, in any in vivo or in vitro B 1 k assay.

Preferred analogs include B l k peptides or recombinant B l k proteins or peptides (or biologically active fragments thereof] whose sequences differ from the wild-type sequence by one or more conservative amino acid substitutions or by one or more non-conservative amino acid substitutions, deletions, or insertions which do not~abolish biological activity.
Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics, e.g., substitutions within the following groups:
valine, glycine;
glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid;
asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Other conservative substitutions can be taken from the table below.
CONSERVATIVE AMINO ACID REPLACEMENTS
For Amino Code Replace with any of Acid Alanine A D-Ala, Gly, beta-Ala, L-Cys, D-Cys Arginine R D-Arg, Lys, D-Lys, homo-Arg, D-homo-Arg, Met, Ile, D-Met, D-Ile, Orn, D-Orn Asparagine N D-Asn, Asp, D-Asp, Glu, D-Glu, Gin, D-Gln Aspartic Acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys, Met, D-Met, T'hr, D-Thr Glutamine Q D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala, Pro, D-Pro, ~i-Ala Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Leu, D-Leu, Met, D-Met Lysine K D-Lys, Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val PhenyIalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, D-His, Trp, D-Trp, Trans-3,4, or 5-phenylproline, cis-3,4, or 5-phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D-or L-1-oxazolidine-4-carboxylic acid Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), L-Cys, D-Cys Threonine T D-Thr, Ser, D-Ser, alto-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, D-His Valine V D-Val, Leu, D-I,eu, Ile, D-Ile, Met, D-Met Other useful modifications include those which increase peptide stability;
such analogs may contain, for example, one or more non-peptide bonds (which replace peptide bonds) or D-amino acids in the peptide sequence.
Analogs can differ from a naturally occurring Blk protein in amino acid sequence or can modified in ways that do not affect sequence, or both. Analogs of the invention will generally exhibit at least 70%, more preferably 80%, more preferably 90%, and most preferably 95% or even, 99%, homology with a segment of 20 amino acid residues, preferably more than 40 amino acid residues or more preferably the entire sequence of naturally occurnng B l k protein sequence.
Alterations in primary sequence include genetic variations, both natural and induced.
Also included are analogs that include residues other than naturally occurring L-amino acids, e.g., D-amino acids or non-naturally occurring or synthetic amino acids, e.g.,~3 or y amino acids. Alternatively, increased stability may be conferred by cyclizing the peptide molecule.
Nonsequence modification include in vivo or in vitro chemical derivatization or polypeptides, e.g., acetylation, methylation, phosphorylation, carboxylation, or glycosylation;
glycosylation can be modif ed, e.g., by modifying the glycosylation patterns of a polypeptide during its synthesis and processing or in further processing steps, e.g., by exposing the polypeptide to glycosylation-affecting enzymes derived from cells that normally provide such processing, e.g., mammalian glycosylation enzymes; phosphorylation can be modified by exposing the polypeptide to phosphorylation-altering enzymes, e.g., kinases or phosphatases.
Fragments of B 1 k proteins or peptides can be made by methods known to those skilled in the art, e.g., by expressing B lk DNA which has been manipulated in vitro to encode the desired fragment; e:g., by restriction digestion or other manipulation of a B 1 k DNA e.g., the sequence in Fig. 2. Analogs can be made by methods known to those skilled in the art, , e.g., by in vitro DNA sequence modifications of the sequence of a B lk DNA e.g., the sequence in Fig. 2. For example, in vitro mutagenesis can be used to convert the DNA
S sequence of Figure 2 into a sequence which encodes an analog in which one or more amino acid residues has undergone a replacement, e.g., a conservative replacement as described in the table of conservative amino acid substitutions provided herein. Fragments or analogs can be tested by methods known to those skilled in the art for the presence of B 1 k activity.
Also included are B Ik protein polypeptides containing residues that are not required for biological activity of the peptide, such as residues that are not required for the biological activity of the polypeptide, or that result from alternative mRNA splicing or alternative protein processing events.
The invention also includes nucleic acids encoding the polypeptides of the invention.
In order to obtain a B 1 k protein, or fragment thereof, one can insert B 1 k-encoding DNA into an expression vector, introduce the vector into a cell suitable for expression of the desired protein, and recover and purify the desired protein by prior art methods. Antibodies to B I k proteins can be made by immunizing an animal, e.g., a rabbit or mouse, and recovering anti-Blk antibodies by prior art methods.
Other embodiments are within the following claims.

SEQUENCE LISTING
S
(1) GENERAL INFORMATION:
(i) APPLICANTS:

(A) NAME: The General Hospital Corporation (B) STREET: 55 Fruit Street (C) CITY: Boston 10 (D} STATE: Massachusetts (E} COUNTRY: USA

(F) POSTAL CODE (ZIP): 02114 (G) TELEPHONE: (617) 726-8608 (H} TELEFAX: d617) 726-1668 (A) NAME: The State of Oregon Acting By and Through the State Board of Higher Education on Behalf of Oregon Health Sciences University (B) STREET: 3181 SW San Jackson Park Road, Mail Code L335 20 (C) CITY: Portland (D) STATE: Oregon (E) COUNTRY: USA

(F) POSTAh CODE (ZIP): 97201-3098 (G) TELEPHONE:

2S (H) TELEFAX:

(ii) TITLE OF INVENTION: Blk CHAIN OF LAMININ AND METHODS OF USE
(iii) NUMBER OF SEQUENCES: 14 (iv) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
3S (D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (v) CURRENT APPLICATION DATA:
APPLICATION NO. PCT/US94/
{vi) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: U5 08/144,121 (B) FILING DATE: 27-OCT-1993 (C) CLASSIFICATION:
4S (vii} ATTORNEY/AGENT INFORMATION:
(A) NAME: Myers, Paul L.
(B) REGISTRATION NUMBER: 35,965 (C) REFERENCE/DOCKET NUMBER: {MGH-0780.0} MGP-021PC
SO {viii) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (617) 227-7400 {B) TELEFAX: (617) 227-5941 (2) INFORMATION FOR SEQ ID NO: l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 3931 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 122..3617 (ix) FEATURE:
(A) NAME/KEY: mat~eptide (B) LOCATION: 173..3617 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

TCCTCTTCCT GGGAGCTCAC.TCAGGAAAGG TCCTTTCTGG GGATCACCCC ATTGGCTGAA 120 Met Arg Pro Phe Phe Leu Leu Cys Phe Ala Leu Pro Gly Leu Leu 3~ -17 -15 -10 -5 His Ala Gln. Gln Ala Cys Ser Arg Gly Ala Cys Tyr Pro Pro Val Gly Asp Leu Leu Val Gly Arg Thr Arg Phe Leu Arg Ala Ser Ser Thr Cys G1y Leu Thr Lys Pro Glu Thr Tyr Cys Thr Gln Tyr Gly Glu Trp Gln Met Lys Cys Cys Lys Cys Asp Ser Arg Gln Pro His Asn Tyr Tyr Ser His Arg Val Glu Asn Val Ala Ser Ser Ser Gly Pro Met Arg Trp Trp Gln Ser Gln Asn Asp Val Asn Pro Val Ser Leu G1n Leu Asp Leu Asp 5$

Arg Arg Phe Gln Leu Gln Glu Val Met Met Glu Phe Pro Gly Ala His Ala AIa Gly Met Leu Ile Glu Arg Ser 5er Asp Phe Gly Lys Thr Trp Arg Val Tyr Gln Tyr Leu Ala Ala Asp Cys Thr Ser Thr Phe Pro Arg Val Arg Gln Gly Arg Pro Gln Ser Trp Gln Asp Val Arg Cys Gln Ser Leu Pro Gln Arg Pro Asn Ala Arg Leu Asn Gly Gly Lys Val Gln Leu Asn Leu Met Asp Leu Val Ser Gly Ile Pro Ala Thr Gln Ser Gln Lys Ile Gln Glu VaI G1y Glu Ile Thr Asn Leu Arg Val Asn Phe Thr Arg ZS

Leu Ala Pro Val Pro Lys Leu Asp His Pro Pro Ser Ala Tyr Tyr Ala Val Ser Gln Leu Arg Leu Gln Gly Ser Cys Phe Cys His Gly His Ala 3S Asp Arg Cys Ala Pro Lys Pro GIy Ala Ser AIa Gly Ser Thr Ala Val Gln Val His Asp Vai Cys Val Cys Gln His Asn Thr Ala G1y Pro Asn Cys Glu Arg Cys Ala Pro Phe Tyr Asn Asn Arg Pro Trp Arg Pro Ala Glu Gly Gln Asp Ala His Glu Cys Gln Arg Cys Asp Cys Asn Gly His Ser Glu Asn Cys His Phe Asp Pro Ala Val Phe Ala Ala Ser Gln Gly SS Ala Tyr Gly Gly Val Cys Asp Asn Cys Arg Asp His Thr Glu Gly Lys Asn Cys Glu Arg Cys Gln Leu His Tyr Phe Arg Asn Arg Arg Pro Gly Ala Ser Ile Gln Glu Thr Cys Ile Ser Cys Glu Cys Asp Pro Asp Gly Gln Trp Ala Gly Ala Pro Cys Asp Pro Val Thr Gly Gln Cys Val Cys Lys Glu His Val Gln Gly Glu Arg Cys Asp Leu Cys Lys Pro Gly Phe Thr Gly Leu Thr Tyr Ala Asn Pro Gln Gly Cys His Arg Cys Asp Cys Asn Ile Leu Pro Ser Arg Arg Leu Pro Cys Asp Glu Glu Ser Gly Arg Cys Leu Cys Leu Pro Asn Val Gly Gly Pro Lys Cys Asp Gln Cys Ala Pro Tyr His Trp Lys Leu Ala Ser Gly Gln Gly Cys Glu Pro Cys Ala Cys Asp Pro His Asn Ser Leu Ser Pro Gln Cys Asn Gln Phe Thr Gly Gln Cys Pro Cys Arg Glu Gly Phe Gly Gly Leu Met Cys Ser Ala Ala Ala Ile Arg Gln Cys Pro Asp Arg Thr Tyr Gly Asp Val Ala Thr Gly Cys Arg Ala Cys Asp Cys Asp Phe Arg Gly Thr Glu Gly Pro Gly Cys SO Asp Lys Ala Ser Gly Val Leu Cys Arg Pro Gly Leu Thr Gly Pro Arg Cys Asp Gln Cys Gln Arg Gly Tyr Cys Asn Arg Tyr Pro Val Cys Val Ala Cys His Pro Cys Phe Gln Thr Tyr Asp Ala Asp Leu Arg Glu Gln Ala Leu Arg Phe Gly Arg Leu Pro Asn Ala Thr Ala Ser Leu Trp Ser Gly Pro Gly Leu Glu Asp Arg Gly Leu Ala Ser Arg Ile Leu Asp Ala ' Lys Ser Lys Ile Glu Gln Ile Arg Ala Val Leu Ser Ser Pro Ala Val 1$

Thr Glu Gln Glu Val Ala Gln Val Ala Ser Ala Ile Leu Ser Leu Arg Arg Thr Leu Gln Gly Leu Gln Leu Asp Leu Pro Leu Glu Glu Glu Thr 2$ Leu Ser Leu Pro Arg Asp Leu Glu Ser Leu Asp Arg Ser Phe Asn Gly Leu Leu Thr Met Tyr Gln Arg Lys Arg Glu Gln Phe Glu Lys Ile Ser Ser Ala Asp Pro Ser Gly Ala Phe Arg Met Leu Ser Thr Ala Tyr Glu 3$

Gln Ser Ala Gln Ala Ala Gln Gln Val Ser Asp Ser Ser Arg Leu Leu CTC GAC CGG GCA GTG
AGA GAG
AGG

Asp Gln Arg Ser ArgGlu Glu Leu Arg Gln Leu Asp Arg Ala Arg Val GGA GGA ACC CCC CTT GCC

4$ Ala Gly Gly Gly GlySer Lys Val Leu Arg Gly Gly Thr Pro Leu Ala ATG TCG CCT ACA ACC AAC

Leu Glu Ser Leu AspLeu Pro Phe Lys Leu Met Ser Pro Thr Thr Asn $O 755 760 76S

AAC AGG ATG ACC ATA TGC

Cys Gly Ser Gln AlaCys Pro Ser Pro Gly Asn Arg Met Thr Ile Cys $$

TGT CAA AAT GCC GCG CGC

Glu Leu Pro Asp GlyThr Cys Ser Cys Arg Cys Gln Asn Ala Ala Arg Gly Val Leu Pro Arg Ala Gly Gly Ala Phe Leu Met Ala Gly_Gln Val Ala Glu Gln Leu-Arg Ala Ser Met Pro Ala Pro Ala Thr Arg Gln Met Ile Arg Ala Ala Glu Glu Ser Ala Ser Gln Ile Gln Ser Ser Ala Gln Arg Leu Glu Thr Gln Val Ser Ala Ser Arg Ser Gln Met Glu Glu Asp 2~ Val Arg Arg Thr Arg Leu Leu Ile Gln Gln Val Arg Asp Phe Leu Thr Asp Pro Asp Thr Asp Ala Ala Thr Ile Gln Glu Val Arg Arg Ala Val CTG GCC CTG TGG CTG CCC ACA GAC TCA GCT ACT GTT.CTG CAG AAG ATG 2902 Leu Ala Leu Trp Leu Pro Thr Asp Ser Ala Thr Val Leu Gln Lys Met Asn Glu Ile Gln Ala Ile Ala Ala Arg Leu Pro Asn Val Asp Leu Val Leu Ser Gln Thr Lys Gln Asp Ile Gly Gly Ala Arg Arg Leu Gln Ala Glu Ala Glu Glu Ala Arg Ser Arg Ala His Ala Val Glu Gly Gln Val GAG GAT GTG GTT GGG AAC CTG CGG CAG GGG ACA GTG GCF. CTG CAG GAA 3094 Glu Asp Val Val Gly Asn Leu Arg Gln Gly Thr Val Ala Leu Gln Glu ~S 960 965 970 Ala Gln Asp Thr Met Gln Gly Thr Ser Arg Ser Leu Arg Leu Ile Gln ' 975 980 985 990 - SU

Asp Arg Val Ala Glu Val Gln Gln Val Leu Gly Gln Gln Lys Leu Val Thr Ser Met Thr Lys Gln Leu G1y Asp Phe Trp Thr Arg Met Glu Glu Leu Gln Ala Arg Gln Gly Glu Ala Val Gln Gln Arg Gln Ala Ala His GCG AGC GCA GAG

$ Gln Leu Glu Gly Ala Glu Gln Leu Ser Ala Gln Gly , Ala Ser Ala Glu AGA AAG GAG TTG

Phe Glu Ile Lys Gln Tyr Ala Leu Lys Asp Arg Gly Arg Lys Glu Leu 1~ 1055 1060 1065 1070 GCC CGG

1$ Gln Ser Ser Met Leu Gly Glu Gln Gly Ile Gln Ser Val Lys Ala Arg ACC ATG

Thr Glu Ala Glu Glu Leu Phe Gly Glu Glu Met Met Asp Arg Thr Met CGG GCA

Met Lys Asp Met Glu Leu Glu Leu Leu Ala Gly His His Ala Arg Ala 2$

AAG CGT

Ala Leu Ser Asp Leu Thr Gly Leu Glu Val Glu Gln Ile Arg Lys Arg TAT GCC

Asp His Ile Asn Gly Arg Val Leu Tyr Thr Cys Lys Tyr Ala 4$

(2) INFORMATION FOR SEQ ID N0:2:

(i) SEQUENCE CHARACTERISTICS:

$~ (A) LENGTH: 1165 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein $$

(xi) SEQUENCE DESCRIPTION: SEQ ID N0:2:

Met Arg Pro Phe Phe Leu Leu Cys Phe Pro Gly Leu Leu His Ala Leu Ala Gln Gln Ala Cys Ser Arg Gly Ala Cys Tyr Pro Pro Val Gly Asp ' $ Leu Leu Val Gly Arg Thr Arg Phe Leu Arg Ala Ser Ser Thr Cys Gly Leu Thr Lys Pro Glu Thr Tyr Cys Thr Gln Tyr Gly Glu Trp Gln Met Lys Cys Cys Lys Cys Asp Ser Arg Gln Pro His Asn Tyr Tyr Ser His 1$
Arg val Glu Asn Val Ala Ser Ser Ser Gly Pro Met Arg Trp Trp Gln Ser Gln Asn Asp Val Asn Pro Val Ser Leu Gln Leu Asp Leu Asp Arg Arg Phe Gln Leu Gln Glu Val Met Met Glu Phe Pro Gly Ala His Ala 2$ Ala Gly Met Leu Ile Glu Arg Ser Ser Asp Phe Gly Lys Thr Trp Arg Val Tyr Gln Tyr Leu Ala Ala Asp Cys Thr Ser Thr Phe Pro Arg Val Arg Gln Gly Arg Pro.Gln Ser Trp Gln Asp Val Arg Cys Gln Ser Leu Pro Gln Arg Pro Asn Ala Arg Leu Asn Gly Gly Lys Val Gln Leu Asn 3$ 160 165 170 17.5 Leu Met Asp Leu Val Ser Gly Ile Pro Ala Thr Gln Ser Gln Lys Ile Gln Glu Val Gly Glu Ile Thr Asn Leu Arg Val Asn Phe Thr Arg Leu Ala Pro Val Pro Lys Leu Asp His Pro Pro Ser Ala Tyr Tyr Ala Val 4$
Ser Gln Leu Arg Leu Gln Gly Ser Cys Phe Cys His Gly His Ala Asp Arg Cys Ala Pro Lys Pro Gly Ala Ser Ala Gly Ser Thr Ala Val Gln $0 240 245 250 255 Val His Asp Val Cys Val Cys Gin His Asn Thr Ala Gly Pro Asn Cys $$ Glu Arg Cys Ala Pro Phe Tyr Asn Asn Arg Pro Trp Arg Pro Ala Glu Gly Gln Asp Ala His Glu Cys Gln Arg Cys Asp Cys Asn Gly His Ser Glu Asn Cys His Phe Asp Pro Ala Val Phe Ala Ala Ser Gln Gly Ala S Tyr Gly Gly Val Cys Asp Asn Cys Arg Asp His Thr Glu Gly Lys Asn Cys Glu Arg Cys Gln Leu His Tyr Phe Arg Asn Arg Arg Pro Gly Ala 1~
Ser Ile Gln Glu Thr Cys Ile Ser Cys Glu Cys Asp Pro Asp Gly Gln 15 Trp Ala Gly Ala Pro Cys Asp Pro Val Thr Gly Gln Cys Val Cys Lys Glu His Val Gln Gly Glu Arg Cys Asp Leu Cys Lys Pro Gly Phe Thr Gly Leu Thr Tyr Ala Asn Pro Gln Gly Cys His Arg Cys Asp Cys Asn Ile Leu Pro Ser Arg Arg Leu Pro Cys Asp Glu Glu Ser Gly Arg Cys Leu Cys Leu Pro Asn Val Gly Gly Pro Lys Cys Asp Gln Cys Ala Pro 30 Tyr His Trp Lys Leu Ala Ser Gly Gln Gly Cys Glu Pro Cys Ala Cys Asp Pro His Asn Ser Leu Ser Pro Gln Cys Asn Gln Phe Thr Gly Gln Cys Pro Cys Arg Glu Gly Phe Gly Gly Leu Met Cys Ser Ala Ala Ala Ile Arg Gln Cys Prc Asp Arg Thr Tyr Gly Asp Val Ala Thr Gly Cys 4~ 500 505 510 Arg Ala Cys Asp Cys Asp Phe Arg Gly Thr Glu Gly Pro Gly Cys Asp Lys Ala Ser Gly Val Leu Cys Arg Pro Gly Leu Thr Gly Pro Arg Cys Asp Gln Cys Gln Arg Gly Tyr Cys Asn Arg Tyr Pro Val Cys val Ala Cys His Pro Cys Phe Gln Thr Tyr Asp Ala Asp Leu Arg Glu Gln Ala Leu Arg Phe Gly Arg Leu Pro Asn Ala Thr Ala Ser Leu Trp Ser Gly Pro Gly Leu Glu Asp Arg Gly Leu Ala Ser Arg Ile Leu Asp Ala Lys Sar Lys Ile Glu Gln Ile Arg Ala Val Leu Ser Ser Pro Ala Val Thr Glu Gln Glu Val Ala Gln Val Ala Ser Ala Ile Leu Ser Leu Arg Arg Thr Leu Gln Gly Leu Gln Leu Asp Leu Pro Leu Glu Glu Glu Thr Leu 1~
Ser Leu Pro Arg Asp Leu Glu Ser Leu Asp Arg Ser Phe Asn Gly Leu Leu Thr Met Tyr Gln Arg Lys Arg Glu Gln Phe Glu Lys Ile Ser Ser Ala Asp Pro Ser Gly Ala Phe Arg Met Leu Ser Thr Ala Tyr Glu Gln Ser Ala Gln Ala Ala Gln Gln Val Ser Asp Ser Ser Arg Leu Leu Asp Gln Leu Arg Asp Ser Arg Arg Glu Ala Glu Arg Leu Val Arg Gln Ala Gly Gly Gly Gly Gly Thr Gly Ser Pro Lys Leu Val Ala Leu Arg Leu Glu Met Ser Ser Leu Pro Asp Leu Thr Pro Thr Phe Asn Lys Leu Cys Gly Asn Ser Arg Gln Met Ala Cys Thr Pro Ile Ser Cys Pro Gly Glu 3$ Leu Cys Pro Gln Asp Asn Gly Thr Ala Cys Ala Ser Arg Cys Arg Gly Val Leu Pro Arg Ala Gly Gly Ala Phe Leu Met Ala Gly Gln Val Ala Glu Gln Leu Arg Ala Ser Met Pro Ala Pro Ala Thr Arg Gln Met Ile Arg Ala Ala Glu Glu Ser Ala Ser Gln Ile Gln Ser Ser Ala Gln Arg Leu Glu Thr Gln Val Ser Ala Ser Arg Ser Gln Met Glu Glu Asp Val Arg Arg Thr Arg Leu Leu Ile Gln Gln Val Arg Asp Phe Leu Thr Asp Pro Asp Thr Asp Ala Ala Thr Ile Gln Glu Val Arg Arg Ala Val Leu SS
Ala Leu Trp Leu Pro Thr Asp Ser Ala Thr Val Leu Gln Lys Met Asn Glu Ile Gln Ala Ile Ala Ala Arg Leu Pro Asn Val Asp Leu Val Leu Ser Gln Thr Lys Gln Asp Ile Gly Gly Ala Arg Arg Leu Gln Ala Glu Ala Glu Glu Ala Arg Ser Arg Ala His Ala Val Glu Gly Gln Val Glu 10 Asp Val Val Gly Asn Leu Arg Gln Gly Thr Val Ala Leu Gln Glu Ala Gln Asp Thr Met Gln Gly Thr Ser Arg Ser Leu Arg Leu Ile Gln Asp IS
Arg Val Ala Glu Val Gln Gln Val Leu Gly G1n Gln Lys Leu Val Thr Ser Met Thr Lys Gln Leu Gly Asp Phe Trp Thr Arg Met Glu Glu Leu Arg His Gln Ala Arg Gln Gln Gly Ala Glu Ala Val Gln Ala Gln Gln 25 Leu Ala Glu Gly Ala Ser Glu Gln Ala Leu Ser Ala Gln Glu Gly Phe Glu Arg Ile Lys Gln Lys Tyr Ala Glu Leu Lys Asp Arg Leu Gly Gln Ser Ser Met Leu G1y Glu Gln Gly Ala Arg Ile Gln Ser Val Lys Thr Glu Ala Glu Glu Leu Phe Gly Glu Thr Met Glu Met Met Asp Arg Met 3$ 1090 1095 1100 Lys Asp Met Glu Leu Glu Leu Leu Arg Ala Ala Gly His His Ala Ala Leu Ser Asp Leu Thr Gly Leu Glu Lys Arg Val Glu Gln Ile Arg Asp His Ile Asn Gly Arg Val Leu Tyr Tyr Ala Thr Cys Lys (2} INFORMATION FOR SEQ ID N0:3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 1147 amino acids (B) TYpE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide S$ (v} FRAGMENT TYPE: internal (ix) FEATURE:
(A} NAME/KEY: Domain (B) LOCATION: 1..231 (ix) FEATURE:
(A) NAME/KEY: Domain $ (B) LOCATION: 232..411 (ix) FEATURE:

(A) NAME/KEY: Domain (B) LOCATION: 412..765 (ix) FEATURE:

(A) NAME/KEY: Domain 1$ (B) LOCATION: 766..1147 (xi) SEQUENCE SCRIPTION: EQ :3:
DE S ID

0 Gln Gln Ala Ser ArgGlyAla CysTyr ProProVal GlyAspLeu Cys Leu Val Gly Thr ArgPheLeu ArgAla SerSerThr CysGlyLeu Arg 2$

Thr Lys Pro Thr TyrCysThr GlnTyr GlyGluTrp GlnMetLys Glu Cys Cys Lys Asp SerArgGln ProHis AsnTyrTyr SerHisArg Cys Val Glu Asn Ala SerSerSer GlyPro MetArgTrp TrpG1nSer Val 35 Gln Asn Asp Asn ProValSer LeuGln LeuAspLeu AspArgArg Val Phe Gln Leu Glu ValMetMet GluPhe ProGlyAla HisAlaAla Gln Gly Met Leu Glu ArgSerSer AspPhe GlyLysThr TrpArgVal Ile Tyr Gln Tyr Ala AlaAspCys ThrSer ThrPhePro ArgValArg Leu 4$ 130 135 140 Gln Gly Arg Gln SerTrpGln AspVal ArgCysGln SerLeuPro Pro $0 Gln Arg Pro Ala ArgLeuAsn GlyGly LysValGln LeuAsnLeu Asn Met Asp Leu Ser GlyIlePro AlaThr GlnSerGln LysIleGln Val $$

Glu Val Gly Ile ThrAsnLeu ArgVal AsnPheThr ArgLeuAla Glu Pro Val Pro Leu AspHisPro ProSer AlaTyrTyr AlaValSer Lys Gln Leu Arg Leu Gln Gly Ser Cys Phe Cys His Gly His Ala Asp Arg Cys Ala Pro Lys Pro Gly Ala Ser Ala Gly Ser Thr Ala Val Gln Val His Asp Val Cys Val Cys Gln His Asn Thr Ala Gly Pro Asn Cys Glu Arg Cys Ala Pro Phe Tyr Asn Asn Arg Pro Trp Arg Pro Ala Glu Gly 1$ 275 280 285 Gln Asp Ala His Glu Cys Gln Arg Cys Asp Cys Asn Gly His Ser Glu Asn Cys His Phe Asp Pro Ala Val Phe Ala Ala Ser Gln Gly Ala Tyr Gly Gly Val Cys Asp Asn Cys Arg Asp His Thr Glu Gly Lys Asn Cys Glu Arg Cys Gln Leu His Tyr Phe Arg Asn Arg Arg Pro Gly Ala Ser Ile Gln Glu Thr Cys Ile Ser Cys Glu Cys Asp Pro Asp Gly Gln Trp Ala Gly Ala Pro Cys Asp Pro Val Thr Gly Gln Cys Val Cys Lys Glu His val Gln Gly Glu Arg Cys Asp Leu Cys Lys Pro Gly Phe Thr Gly Leu Thr Tyr Ala Asn Pro Gln Gly Cys His Arg Cys Asp Cys Asn Ile Leu Pro Ser Arg,Arg Leu Pro Cys Asp Glu Glu Ser Gly Arg Cys Leu Cys Leu Pro Asn Val Gly Gly Pro Lys Cys Asp Gln Cys Ala Pro Tyr His Trp Lys Leu Ala Ser Gly Gln Gly Cys Glu Pro Cys Ala Cys Asp Pro His Asn Ser Leu Ser Pro Gln Cys Asn Gln Phe Thr Gly Gln Cys Pro Cys Arg Glu Gly Phe Gly Gly Leu Met Cys Ser Ala Ala Ala Ile Arg Gin Cys Pro Asp Arg Thr Tyr G1y Asp Val Ala Thr Gl.y Cys Arg Ala Cys Asp Cys Asp Phe Arg Gly Thr Glu Gly Pro Gly Cys Asp Lys Ala Ser Gly Val Leu Cys Arg Pro Gly Leu Thr Gly Pro Arg Cys Asp Gln CysArg Gly Cys Asn Tyr ProValCys ValAla CysHis Tyr Arg Pro CysPhe GlnThrTyr AspAlaAsp LeuArgGlu GlnAla LeuArg Phe Gly LeuProAsn AlaThrAla SerLeuTrp SerGly ProGly Arg 1$

Leu GluAsp ArgGlyLeu AlaSerArg IleLeuAsp AlaLys SerLys Ile GluGln IleArgAla ValLeuSer SerProAla ValThr GluGln Glu ValAla GlnValAla SerAlaIle LeuSerLeu ArgArg ThrLeu 2$ Gln GlyLeu GlnLeuAsp LeuProLeu GluGluGlu ThrLeu SerLeu Pro ArgAsp LeuGluSer LeuAspArg SerPheAsn G1yLeu LeuThr' Met TyrGln ArgLysArg GluGlnPhe GluLysIle SerSer AlaAsp Pro SerGly AlaPheArg MetLeuSer ThrAlaTyr GluGln SerAla 3$ 690 695 700 Gln AlaAla GlnGlnVal SerAspSer SerArgLeu LeuAsp GlnLeu 40 Arg AspSer ArgArgGlu AlaGluArg LeuValArg GlnAla GlyGly Gly GlyGly ThrGlySer ProLysLeu ValAlaLeu ArgLeu GluMet 4$

Ser SerLeu ProAspLeu ThrProThr PheAsnLys LeuCys GlyAsn Ser ArgGln MetAlaCys ThrProIle SerCysPro GlyGlu LeuCys $0 770 775 780 Pro GlnAsp AsnGlyThr AlaCysAla SerArgCys ArgGly ValLeu $$ Pro ArgAla GlyGlyAla PheLeuMet AlaGlyGln ValAla GluGln Leu ArgAla SerMetPro AlaProAla ThrArgGln MetIle ArgAla Ala Glu Glu Ser Ala Ser Gln Ile Gln Ser Ser Ala Gln Arg Leu Glu Thr Gln Val Ser Ala Ser Arg Ser Gln Met'Glu Glu Asp Val Arg Arg , Thr Arg Leu Leu Ile Gln Gln Val Arg Asp Phe Leu Thr Asp Pro Asp Thr Asp Ala Ala Thr Ile Gln Glu Val Arg Arg Ala Val Lieu Ala Leu 1$ Trp Leu Pro Thr Asp Ser Ala Thr Val Leu Gln Lys Met Asn Glu Ile Gln Ala Ile Ala Ala Arg Leu Pro Asn Val Asp Leu Val Leu Ser Gln Thr Lys Gln Asp Ile Gly Gly Ala Arg Arg Leu Gln Ala Glu Ala Glu Glu Ala Arg Ser Arg Ala His Ala Val Glu Gly Gln Val Glu Asp Val 2$ 945 950 95S 960 Val Gly Asn Leu Arg Gln Gly Thr Val Ala Leu Gln Glu Ala Gln Asp Thr Met Gln Gly Thr Ser Arg Ser Leu Arg Leu Ile Gln Asp Arg Val 3$
Ala Glu Val Gln Gln Val Leu Gly Gln Gln Lys Leu Val Thr Ser Met Thr Lys Gln Leu Gly Asp Phe Trp Thr Arg Met Glu Glu Leu Arg His Gln Ala Arg Gln Gln Gly Ala Glu Ala Val Gln Ala Gln Gln Leu Ala Glu Gly Ala Ser Glu Gln Ala Leu Ser Ala Gln Glu Gly Phe Glu Arg 4$ Ile Lys Gln Lys Tyr Ala Glu Leu Lys Asp Arg Leu Gly Gln Ser Ser Met Leu Gly Glu Gln Gly Ala Arg Ile Gln Ser Val Lys Thr Glu Ala $~
Glu Glu Leu Phe Gly Glu Thr Met Glu Met Met Asp Arg Met Lys Asp Met Glu Leu Glu Leu Leu Arg Ala Ala Gly His His Ala Ala Leu Ser $$ 1105 1110 1115 1120 Asp Leu Thr Gly Leu GIu Lys Arg Val Glu Gln Ile Arg Asp His Ile Asn Gly Arg Val Leu Tyr SerThr CysLys Tyr S (2) INFORMATION
FOR
SEQ
ID N0:4:

(i) SEQUENCE CHARACTERISTICS:

(A} LENGTH: 1196 acids amino (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (ix) FEATURE:

(A) NAME/KEY: Damain (B) LOCATION: 1..250 (ix} FEATURE:

(A) NAME/KEY: Domain (B) LOCATION: 251..437 2S (ix) FEATURE:

(A) NAME/KEY: Domain (B) LOCATION: 438..807 , (ix} FEATURE:

(A} NAME/KEY: Domain (B} LOCATION: 808..840 (ix) FEATURE:

(A) NAME/KEY: Domain 3S (B) LOCATION: 841..1196 (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:4:

Gln Glu Pro Glu Phe Ser GlyCys AlaGlu Gly Ser Cys Tyr Pro Tyr Ala Thr Gly Asp Leu Leu GlyArg AlaGln Lys Leu Ser Val Thr Ile Ser Thr Thr Cys Gly Leu LysPro GluPro Tyr Cys Ile Val Ser His ' His Leu Gln Glu Asp Lys CysPhe IleCys Asn Ser Gln Asp Pro Lys S~ 50 55 60 Tyr His Glu Thr Leu Asn AspSer HisLeu Ile Glu Asn Val Val Pro $S Thr Thr Phe Ala Pro Asn LeuLys IleTrp Trp Gln Ser Glu Asn Arg Gly Val Glu Asn Val Thr GlnLeu AspLeu Glu Ala Glu Phe His Ile Phe Thr His Leu Ile Met Thr Phe Lys Thr Phe Arg Pro Ala Ala Met $ 115 120 125 , Leu Ile Glu Arg Ser Ser Asp Phe Gly Lys Thr Trp Gly Val Tyr Arg Tyr Phe Ala Tyr Asp Cys Glu Ala Ser ~Phe Pro Gly Ile Ser Thr Gly ' Pro Met Lys Lys Val Asp Asp Ile Ile Cys Asp Ser Arg Tyr Ser Asp 1$
Ile Glu Pro Ser Thr Glu Gly Glu Val Ile Phe Arg Ala Leu Asp Pro Ala Phe Lys Ile Glu Asp Pro Tyr Ser Pro Arg Ile Gln Asn Leu Leu Lys Ile Thr Asn Leu Arg Ile Lys Phe Val Lys Leu His Thr Leu Gly 2$ Asp Asn Leu Leu Asp Ser Arg Met Glu Ile Arg Glu Lys Tyr Tyr Tyr Ala Val Tyr Asp Met Val Val Arg Gly Asn'Cys Phe Cys Tyr Gly His Ala Ser Glu Cys Ala Pro Val Asp Gly Phe Asn Glu Glu Val Glu Gly Met Val His Gly His Cys Cys Arg Asn Lys Gly Leu Asn Met His Thr Cys Glu Leu Cys Met Asp Tyr His Leu Trp Arg.Pro Ala Phe Asp Pro 4U Glu Gly Arg Asn Ser Asn Cys Lys Cys Cys Asn Glu His Ala Lys Asn Ser Ile Ser Cys His Phe Asp Met Ala Val Tyr Leu Ala Thr Gly Asn 4$
Val Ser Gly Gly Val Cys Asp Asp Cys Gln His Asn Thr Met Gly Arg Asn Cys Glu Gln Cys Lys Pro Phe Tyr Tyr Gln His Pro Glu Arg Asp Ile Arg Asp Pro Asn Phe Cys Glu Arg Cys Thr Cys Asp Pro Ala Gly 370 375 380 , $$ Ser Gln Asa Glu Gly Ile Cys Asp Ser Tyr Thr Asp Phe Ser Thr Gly Leu Ile Ala Gly Gln Cys Arg Cys Lys Leu Asn Val Glu Gly Glu His 405 410 41.5 Cys AspVal CysLysGlu Gly Tyr Phe Asp Leu Ser Ser Glu Asp Pro - $

Phe GlyCys LysSerCys ValCysAsn Tyr Leu Gly Thr Val Gln Glu His CysAsn GlySer CysGlnCys Asp Asp Lys Ala Thr Gly Gln Cys Leu CysLeu ProAsnVal IleGlyGln Asn Asp Arg Cys Ala Pro Cys Asn ThrTrp GlnLeuAla SerGlyThr Gly Asp Pro Cys Asn Cys Cys Asn AlaAla HisSerPhe GlyProSer Cys Glu Phe Thr Gly Gln Asn Cys GlnCys MetProGly PheGlyGly Arg Cys Ser Glu Cys Gln Thr Glu LeuPhe TrpGlyAsp ProAspVal Glu Arg Ala Cys Asp Cys Cys Asp ProArg GlyIleGlu ThrProGln Cys Gln Ser Thr Gly Gln Asp Cys ValCys ValGluGly ValGluGly Pro Cys Asp Lys Cys Thr Arg Arg GlyTyr SerGlyVal PheProAsp Cys Pro Cys His Gln Cys Thr Phe AlaLeu TrpAspVal IleIleAla Glu Thr Asn Arg Thr His Leu Arg PheLeu GluLysAla LysAlaLeu Lys Ser Gly Val Ile Gly Ile Pro TyrArg GluThrVal AspSerVal Glu Lys Val Ser Glu Ile Arg Lys AspIle LeuAlaGln SerProAla Ala Pro Leu Lys Asn Ile Glu Gly AsnLeu PheGluGlu AlaGluLys Leu Lys Asp Val Thr Glu Ile Met MetAla GlnValGlu ValLysLeu Ser Thr Thr Ser Gln Ser Asp Asn SerThr AlaLysGlu LeuAspSer Leu Thr Glu Ala Glu Ser Gln 690 695 700 .

Leu Asn ThrValLys GluLeu Glu Leu Glu Phe Ile Lys Asp Ala Gln .

Asn Ser Asp Ile Arg Gly Ala Leu Asp Ser Ile Thr Lys Tyr Phe Gln Met Ser Leu Glu Ala Glu G1u Arg Val Asn Ala Ser Thr Thr Glu Pro 740 ' 745 750 Asn Ser Thr Val Glu Gln Ser Ala Leu Met Arg Asp Arg Val Glu Asp 755 760 ?65 ' Val Met Met Glu Arg Glu Ser Gln Phe Lys G1u Lys Gln Glu Glu Gln 770 7?5 780 Ala Arg Leu Leu Asp Glu Leu Ala Gly Lys Leu G1n Ser Leu Asp Leu Ser Ala Ala Ala Glu Met Thr Cys Gly Thr Pro Pro Gly Ala Ser Cys Ser Glu Thr Glu Cys Gly Gly Pro Asn Cys Arg Thr Asp Glu Gly Glu Arg Lys Cys Gly Gly Pro Gly Cys Gly Gly Leu Val Thr Val Ala His Asn Ala Trp Gln Lys Ala Met Asp Leu Asp Gln Asp Va1 Leu. Ser Ala 850 855 860 .
Leu Ala Glu Val Glu Gln Leu Ser Lys Met Val Ser Glu Ala Lys Leu 3~ 865 870 875 880 Arg Ala Asp Glu Ala Lys Gln Ser Ala Glu Asp Ile Leu Leu Lys Thr 3$ Asn Ala Thr Lys Glu Lys Met Asp Lys Ser Asn G1u Glu Leu Arg Asn Leu Ile Lys Gln Ile Arg Asn Phe Leu Thr Gln Asp Ser Ala Asp Leu Asp Ser Ile Glu Ala Val Ala Asn Glu Val Leu Lys Met Glu Met Pro Ser Thr Pro Gln Gln Leu Gln Asn Leu Thr Glu Asp Ile Arg Glu Arg Val Glu Ser Leu Ser Gln Va1 Glu Val Ile Leu Gln His Ser Ala Ala SO Asp Ile Ala Arg Ala Glu Met Leu Leu Glu Glu Ala Lys Arg Ala Ser Lys Ser Ala Thr Asp Val Lys Val Thr Ala Asp Met Val Lys Glu Ala Leu Glu Glu Ala Glu Lys Ala Gln Val Ala Ala Glu Lys Ala Ile Lys Gln Ala Asp Glu Asp Ile Gln Gly Thr Gln Asn Leu Leu Thr Ser Ile Glu Ser Glu Thr Ala A1a Ser Glu Glu Thr Leu Phe Asn Ala Ser Gln ' $ 1045 1050 1055 Arg Ile Ser Glu Leu Glu Arg Asn Val Glu Glu Leu Lys Arg Lys Ala ' 1060 1065 107 0 Ala Gln Asn Ser Gly Glu Ala Glu Tyr Ile Glu Lys Val Val Tyr Thr Val Lys Gln Ser Ala Glu Asp Val Lys Lys Thr Leu Asp Gly Glu Leu I$
Asp Glu Lys Tyr Lys Lys Val Glu Asn Leu Ile Ala Lys Lys Thr Glu Glu Ser Ala Asp Ala Arg Arg Lys Ala Glu Met Leu Gln Asn Glu Ala Lys Thr Leu Leu Ala Gln Ala Asn Ser Lys Leu Gln Leu Leu Lys Asp Leu Glu Arg Lys Tyr Glu Asp Asn Gln Arg Tyr Leu Glu Asp Lys Ala 1155 1160 1165 .
Gln Glu Leu Ala Arg Leu Glu Gly Glu Val Arg Ser Leu Leu Lys Asp Ile Ser Gln Lys Val Ala Val Tyr Ser Thr Cys Leu (2) INFORMATION FOR SEQ ID N0:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH; 231 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:5:
Gln Gln Ala Cys Ser Arg Gly Ala Cys Tyr Pro Pro Val Gly Asp Leu Leu Val Gly Arg Thr Arg Phe Leu Arg Ala Ser Ser Thr Cys Gly Leu SS Thr Lys Pro Glu fihr Tyr Cys Thr Gln Tyr Gly Glu Trp G1n Met Lys Cys Cys Lys Cys Asn Ser Arg Gln Pro His Asn Tyr Tyr Ser His Arg Val Glu Val Ala Ser SerGly ProMetArg TrpTrp Gln,Ser Asn Ser Gln Asn Val Asn Pro, SerLeu GlnLeuAsp LeuAsp ArgArg Asp Val 85 90 95 ' Phe Gln Gln Glu Val MetGlu PheProGly AlaHis AlaAla Leu Met.

Gly Met Ile Glu Arg SerAsp PheGlyLys ThrTrp ArgVal Leu Ser 1$ Tyr Gln Leu Ala Ala CysThr SerThrPhe ProArg ValArg Tyr Asp Gln Gly Pro Gln Ser GlnAsp ValArgCys GlnSer LeuPra Arg Trp Gln Arg Asn Ala Arg AsnGly GlyLysVal GlnLeu AsnLeu Pro Leu 165 170, 175 Met Asp Val Ser Gly GluAla ThrGlnSer GlnLys IleGln Leu Ile 2$ 180 18s 190 Glu Val Glu Ile Thr LeuArg IleLysPhe ValLys LeuAla Gly Asn 30 Pro Val Lys Leu Asp ProPro SerAlaTyr TyrAla ValSer Pro, His Gln Leu Leu Gln Gly Arg Ser 3$

(2) INFORMATION
FOR
SEQ
ID N0:6:

(i) SEQUENCECHARACTERISTICS:

(A) cids LENGTH:

amino a 4~ {B).TYPE:
amino acid (D) TOPOLOGY:
linear (ii) MOLECULETYPE: peptide 4$ (v) FRAGMENTTYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:6:
$0 Gln Glu Pro Glu Phe Ser Tyr Gly Cys Ala Glu Gly Ser Cys Tyr Pro Ala Thr Gly Asp Leu Leu Ile Gly Arg Ala Gln Lys Leu Ser Val Thr $$ 20 25 30 Ser Thr Cys Gly Leu His Lys Pro Glu Pro Tyr Cys Ile Val Ser His Leu Gln Glu Asp Lys Lys Cys Phe Ile Cys Asn Ser Gln Asp Pro Tyr His Glu Thr Leu Asn Pro Asp Ser His Leu Ile Glu Asn Val Val Thr 65~ 70 75 80 Thr Phe Ala Pro Asn Arg Leu Lys Ile Trp Trp Gln Ser Glu Asn Gly Val Glu Asn Val Thr Ile Gln Leu Asp Leu Glu Ala Glu Phe His Phe Thr His Leu Ile Met Thr Phe Lys Thr Phe Arg Pro Ala Ala Met Leu 1$ 115 120 125 Ile Glu Arg Sex Ser Asp Phe Gly Lys Thr Trp Gly Val Tyr Arg Tyr Phe Ala Tyr Asp Cys Glu Ala Ser Phe Pro Gly Ile Ser Thr Gly Pro Met Lys Lys Val Asp Asp Ile Ile Cys Asp Ser Arg Tyr Ser Asp Ile Glu Pro Ser Thr G1u Gly Glu Val Ile Phe Arg Ala Leu Asp Pro Ala Phe Lys Ile Glu Asp Pro Tyr Ser Pro Arg Ile Gln Asn Leu Leu Lys Ile Thr Asn Leu Arg Ile Lys Phe Val Lys Leu His Tar Leu Gly Asp Asn Leu Leu Asp Ser Arg Met.Glu Ile Arg Glu Lys Tyr Tyr Tyr Ala Val Tyr Asp Met Val Val Arg Gly Asn (2) INFORMATION FOR SEQ ID N0:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 250 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide $0 (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:7:
$~
Gln Val Pro Ser Leu Asp Val Pro Gly Cys Ser Arg Gly Ser Cys Tyr 4~
Pro Ala Thr Gly Asp Leu Leu Val Gly Arg Ala Asp Arg Leu Thr Ala Ser Ser Thr Cys Gly Leu His Ser Pro Gln Pro Tyr Cys Ile Val Ser $ 35 40 45 His Leu Gln Asp Glu Lys Lys Cys Phe Leu Cys ASp Ser Arg Arg Pro ' Phe Ser Ala Arg Asp Asn Pro Asn Ser His Arg Ile Gln Asn Val Val 1$
Thr Ser Phe Ala Pro Gln Arg Arg Thr Ala Trp Trp Gln Ser Glu Asn Gly Val Pro Met Val, Thr Ile Gln Leu Asp Leu Glu Ala Glu Phe His Phe Thr His Leu Ile Met Thr Phe Lys Thr Phe Arg Pro Ala Ala Met Leu VaI Glu Arg Ser AIa Asp Phe Gly Arg Thr Trp Arg Val Tyr Arg 2$ Tyr Phe Ser Tyr Asp Cys Gly Al.a Asp Phe Pro Gly Ile.Pro Leu Ala Pro Pro Arg Arg Trp Asp Asp Val Val Cys Glu Ser Arg Tyr Ser Glu 165 l~0 1~5 Ile Glu Pro Ser Thr Glu Gly Glu Val Ile Tyr Arg Val Leu Asp Pro Ala Ile Pro Ile Pro Asp Pro Tyr Ser Ser Arg Ile Gln Asn Leu Leu 3$ 195 200 205 Lys Ile Thr Asn Leu Arg Val Asn Leu Thr Arg Leu His Thr Leu Gly Asp Asn Leu Leu Asp Pro Arg Arg Glu Ile Arg Glu Lys Tyr Tyr Tyr Ala Leu Tyr Glu Leu Val Ile Arg Gly Asn 4$
(2) INFORMATION FOR SEQ ID N0:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids $0 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide $$
(v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:8:

Glu Ala Leu Lys Leu Lys Arg Gly Asn Ser Leu,Ala Ala Ala Ser Thr Ala Glu Glu Thr Ala Gly Ser Gln Ser~Arg Ala Arg Glu Ala Ala Glu Lys Gln Leu Arg Glu Gln Val Gly (2) INFORMATION
FOR
SEQ
ID N0:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 24 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal 2S (xi) SEQUENCE DESCRIPTION:
SEQ ID N0:9:

Ala Gly Asn Ser Leu Ala Ala Thr Ala Glu Glu Thr Ala Ser Gly Ser Ala Gln Gly Arg Ala Gln Glu Ala (2) INFORMATION
FOR
SEQ
ID NO:10:

3$ (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:10:
Glu Glu Leu Arg His Gln Ala Arg Gln Gln Gly Ala Glu Ala Val Gln ' 1 5 10 15 Ala Gln Gln Leu Ala Glu Gly Ala Ser Glu Gln Ala Leu Ser Ala Gln $$ Glu Gly Phe Glu Arg Ile Lys Gln (2) INFORMATION FOR SEQ ID NO:11:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear S
(ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Thr Gly Asp Cys Tyr Ser Gly Asp Glu Asn Pro Asp Ile Glu Cys Ala iS 1 5 10 15 Asp Cys Pro Ile Gly Phe Tyr Asn Asp Pro His Asp Pro Arg Ser Cys 0 Lys Pro Cys Pro Cys His Asn Gly (2) INFORMATION
FOR
SEQ
ID N0:12:

ZS (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: l7 amino acids (8) TYPE: amino acid , (D) TOPOLOGY: linear 30 (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:12:
Asp Glu Asn Pro Asp Ile Glu Cys Ala Asp Cys Pro Ile Gly Phe Tyr Asn (2) INFORMATION FOR SEQ ID N0:13:

(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 40 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: SEQ ID N0:13:

~~

Lys Val Asp Thr Arg Ala Ala Gly Thr Ile Gln Asp Lys Asn Va1 Thr Leu Asn Thr Leu Asp Gly His Leu Asg Gln Pro Leu Leu Leu Met Ser ' $ 20 25 30 ' Val Asp Glu Glu Gly Leu Val Leu IO (2) INFORMATION
FOR
SEQ
ID N0:14:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 25 amino acids (B) TYPE: amino acid (D} TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (v) FRAGMENT TYPE: internal (xi) SEQUENCE DESCRIPTION: 14:
SEQ ID N0:

2$ Asn Ala Gly Val Thr Ile Thr Leu Thr Leu Asp Gly Gln Asp Asn Leu 1 5. 10 15 Leu His Leu Met Asp Gln Ser Pro Leu

Claims (12)

1. An isolated antibody that binds to a polypeptide that consists of an amino acid sequence selected from the group consisting of:
(a) domain VI of B1k, (b) domain III of B1k, (c) domain II of B1k, (d) domain I of B1k, (e) domain alpha of B1k, (f) any one of EGF domains 2, 3, 11, 12 or 13 of B1k, and (g) any combination of (a) - (f).
2. The antibody of claim 1 wherein the antibody binds to (a).
3. The antibody of claim 1 wherein the antibody binds to (b).
4. The antibody of claim 1 wherein the antibody binds to (c).
5. The antibody of claim 1 wherein the antibody binds to (d).
6. The antibody of claim 1 wherein the antibody binds to (e).
7. The antibody of claim 1 wherein the antibody binds to (f).
8. The antibody of any one of claims 1 to 7 wherein the antibody is a mouse antibody.
9. A composition comprising an antibody according to any one of claims 1 to 8 and a pharmaceutically acceptable carrier.
10. A method for making a domain-specific antibody, the method comprising:
immunizing an experimental animal with an immunogen comprising an isolated polypeptide that consists of an amino acid sequence selected from the group consisting of:
(a) domain VI of B1k, (b) domain III/V of B1k, (c) domain II of B1k, (d) domain I of B1k, (e) domain alpha of B1k, (f) any one of EGF domains 1, 2, 3, 11, 12 or 13 of B1k, (g) immunogenic fragments of any of the foregoing, and (h) any combination of (a) - (g) ; and preparing, from the experimental animal, an antibody that binds to the immunogen.
11. The method of claim 10, wherein the immunogen is a polypeptide consisting of domain VI of B1k or an immunogenic fragment thereof.
12. The method of claim 10, wherein the immunogen is a polypeptide consisting of domain III/V of B1k or an immunogenic fragment thereof.
CA002467757A 1993-10-27 1994-10-26 B1k chain of laminin and methods of use Abandoned CA2467757A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US08/144,121 US5610031A (en) 1993-10-27 1993-10-27 B1k chain of laminin and methods of use
US08/144,121 1993-10-27
CA002173626A CA2173626C (en) 1993-10-27 1994-10-26 B1k chain of laminin and methods of use

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CA002173626A Division CA2173626C (en) 1993-10-27 1994-10-26 B1k chain of laminin and methods of use

Publications (1)

Publication Number Publication Date
CA2467757A1 true CA2467757A1 (en) 1995-05-04

Family

ID=32657344

Family Applications (1)

Application Number Title Priority Date Filing Date
CA002467757A Abandoned CA2467757A1 (en) 1993-10-27 1994-10-26 B1k chain of laminin and methods of use

Country Status (1)

Country Link
CA (1) CA2467757A1 (en)

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