# Patent

## Title
B1k chain of laminin and methods of use

## Abstract

Recombinant laminin B1k and fragments thereof.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 09/161,872, filed Sep. 28, 1998 now abandoned, which is a continuation of U.S. application Ser. No. 08/735,893, filed Oct. 23, 1996, now U.S. Pat. No. 5,914,317, which is a divisional of U.S. Ser. No. 08/144,121, filed on Oct. 27, 1993, now U.S. Pat. No. 5,610,031, the disclosures of which are incorporated herein by reference in their entirety.


This invention was made with government support. The U.S. government may have certain rights in this invention.


BACKGROUND 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 laminin B1k 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); B1, typified by the B1e 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 B1 chain homologue, s-laminin (B1s), 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, B1 or B2 chains to form the variant trimeric proteins [Ae, B1s, B2e], [Am, B1e, B2e] and [Am, B1s]. A second B1 variant (the sequence of 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 ultrastructural 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 A chain (Ak), a 155-kD kalinin B2 chain (B2t), and a 140-kD kalinin B1 chain (B1k). Rotary shadowing of the molecule results in a 107-nm rod with globular domains at each end.
Kalinin 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.
SUMMARY OF THE INVENTION
In general, the invention features a purified DNA including a sequence encoding a B1k chain of laminin.
In preferred embodiments: the DNA encodes the B1k protein of (SEQ ID NO:2); the encoded B1k peptide is at least 80, more preferably 90, and most preferably 95 or 98% homologous with the sequence of (SEQ ID NO:2); the DNA encodes a biologically active B1k.
In another aspect, the invention features a recombinant B1k.
In preferred embodiments: the recombinant B1k protein has the sequence of (SEQ ID NO:2); the recombinant B1k peptide is at least 80, more preferably 90, and most preferably 95 or 98% homologous with the sequence of (SEQ ID NO:2); the recombinant B1k has biological activity.
The invention also includes a vector including a DNA sequence encoding a B1k protein; a cell containing the vector; a method for manufacture of B1k including culturing the cell in a medium to express B1k.
In another aspect, the invention features a purified DNA including (or consisting essentially of) a sequence encoding a fragment of a B1k laminin chain.
In preferred embodiments: the sequence encodes domain VI of B1k, or a kalinin A chain-binding fragment thereof, the sequence encodes a peptide with a biological activity of domain VI of native B1k, e.g., the ability to bind to a kalinin A chain; the sequence encodes any of domain VI, V, IV, III, II, α, or I of B1k.
In other preferred embodiments: the sequence of the encoded B1k fragment is essentially the same as that of a naturally occurring B1k sequence; the DNA sequence which encodes the B1k 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 B1k, e.g., B1k 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 B1k sequence e.g., the amino acid sequence of SEQ ID NO:1; the amino acid sequence of the encoded B1k 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 B1k 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 B1k; the amino acid sequence of the encoded B1k 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 B1k sequence, e.g., the sequence of SEQ ID NO:1; the fragment has biological activity.
In other preferred embodiments the fragment includes more than one B1k domain and: the domains in the encoded peptide are arranged in the same relative linear order as found in a naturally B1k; the linear order of the encoded domains is different from that found in a naturally occurring B1k; 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 B1k.
In another aspect, the invention features, a DNA, preferably a purified DNA, which includes (or consists essentially of) a sequence encoding a fragment of B1k 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 B1k peptide, e.g., the amino acid sequence of SEQ ID NO: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 50% more preferably at least 60%, more preferably 70%, more preferably 80%, more preferably 90%, and most preferably 95% as long as a naturally occurring B1k; 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 B1k 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 B1k fragment; a cell containing the purified DNA; a method for manufacture of a B1k fragment comprising culturing the cell in a medium to express the B1k fragment.
In another aspect, the invention features a peptide which is a fragment of a B1k 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 B1k, e.g., the ability to bind to a kalinin A chain; the peptide includes any of domain VI, V, IV, III, II, α, or I of B1k; the fragment has biological activity.
In other preferred embodiments: the sequence of the peptide is essentially the same as that of a naturally occurring B1k sequence; the DNA sequence which encodes the B1k 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 B1k, e.g., B1k encoding DNA from SEQ ID NO:1; the sequence which encodes the B1k peptide hybridizes under high or low stringency to a nucleic acid which encodes a naturally occurring B1k sequence e.g., the amino acid sequence of SEQ ID NO: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 B1k; 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 B1k sequence, e.g., the sequence of SEQ ID NO:2.
In other preferred embodiments the peptide includes more than one B1k domain and: the domains in the peptide are arranged in the same relative linear order as found in a naturally B1k; the linear order of the domains is different from that found in a naturally occurring B1k; 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 B1k; the peptide has biological activity.
In another aspect, the invention features a transgenic animal, e.g., a rodent, having a B1k transgene, e.g., a transgene which misexpresses the B1k 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 B1k interacts; administering an antibody against a site on B1k, e.g., a site in domain VI, which interacts with the second molecule; administering a fragment of B1k, e.g., a fragment containing domain VI which competes, e.g., competitively or non-competitively with B1k 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 B1k which includes domain VI, contacting the molecule or cell, with the B1k 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 B1k 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 B1k domain linked to a second B1k 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 B1k domain and a second B1k domain, linking the first molecule or cell to the first domain, and linking the second molecule or cell to the second domain.
In preferred embodiments: the first and/or second molecule is an adhesion molecule, e.g., laminin, kalinin, or collagen; the first and/or second B1k domain is domain VI or a kalinin A chain-binding fragment thereof of B1k; 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 B1k protein or a fragment of a B1k protein, e.g., a fragment which contains only one domain of B1k; a therapeutic composition including an B1k 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 B1k 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 B1k gene, e.g., cells transformed with B1k or B1k 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 B1k 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 keratinocyte to its substrate including carrying out the treatment and evaluating the effect of the treatment on the expression of the B1k 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.
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 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 providing a nucleic acid sample from the subject and determining if the structure of an B1k gene allele of the subject differs from wild type.
In preferred embodiments: the determination includes determining if an B1k gene allele of the subject has a gross chromosomal rearrangement; the determination includes sequencing the subject's B1k 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 B1k gene in the animal or cell model is expressed at a predetermined level or if the B1k 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 B1k gene or B1k protein encoding DNA. In preferred embodiments: the B1k gene or DNA includes a deletion, e.g. a deletion of all or part of B1k, 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 5′ end and one at the 3′ end) in the naturally occurring 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 animal 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 B1k 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 B1k biological activity if it has one or more of the following 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 B1k chain; the ability to specifically bind type IV collagen; if a B1k domain present on a B1k protein or fragment has a biological property that the domain has when present in the native B1k molecule, e.g., the ability to bind or associate in a specific way with another molecule, e.g., another laminin or kalinin chain or the ability to form a characteristic native rotary shadowy structure characteristic of native B1k.
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 B1k 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.


DETAILED DESCRIPTION 
Drawings 
The drawings are first briefly described.
FIG. 1 is a map of the B1k 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 B1 chain.
FIG. 2 is a map of the nucleotide sequence of the laminin B1k chain (SEQ ID NO: 1) and the predicted amino acid sequence (SEQ ID NO: 2) of the B1k peptide chain. Triangles indicate potential O-linked glycosylation sites. Stars indicate potential glycosaminoglycan attachment sites. Potential N-linked glycosylation sites are underlined.
FIG. 3 is a domain-by-domain comparison of the amino acid sequences of the laminin B1k chain (SEQ ID NO: 3) and the human B1 (SEQ ID NO: 4) chain (B1e).
FIG. 4 is: A) a comparison of the domain sizes and percent identity for the various domains of the laminin B1e and laminin B1k chains; and B) a depiction of the numbering scheme for the laminin B1k domains. The domains are numbered according to their similarity to the comparable domains in the laminin B1e chain. Some of the laminin B1e chain domains are missing in the laminin B1k chain and those that remain are truncated in comparison to the laminin B1e chain.
FIG. 5 is a comparison of the amino acid sequences of domain VI for B1k (SEQ ID NO: 5), B1e (SEQ ID NO: 6), and B1s (SEQ ID NO: 7). The underlined regions are areas where the sequence identity between B1e and B1s is above average, but the sequence identity with B1 is considerably less than average. The arrow shows an additional cysteine contained by B1k at residue number 50.
FIG. 6 is a comparison of peptide sequences of rat laminin B1s (SEQ ID NO: 8), human laminin B1s (SEQ ID NO: 9) and human laminin B1k (SEQ ID NO: 10). Also shown is a comparison of the amino acid sequences of human laminin B2t peptides determined by deduction from cDNA (SEQ ID NO: 11 and SEQ ID NO: 13) (top line) and from sequencing of purified peptide (bottom line) (SEQ ID NO: 12 and SEQ ID NO: 14).
FIG. 7 is a comparison of the cloned cDNA sequence to the B1 and B2 chains of laminin (LAMB1e and LAMB2e), the B2 chain of kalinin (LAMB2t) and the B1 chain of s-laminin (LAMB1s).
cDNA Clones for the Kalinin B1 (Laminin B1k) 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 B1k 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 155/105-kD kalinin chain are identical.
Clones encoding the kalinin 140-kD kalinin B1 chain were selected for further characterization (Kal26, Kal45, Kal48, Kal68, Kal82, and Kal85, 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 B1 chain. Rescreening of the cDNA library with Kal45 resulted in the isolation of clones Kal5-5 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 Kal92-1 (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-225 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, Calif.) for construction of the Lambda gt11 cDNA library using random primers.
Library screening was performed as follows. The anti-kalinin polyclonal antibody (pAB) was diluted in 1:10 in 10 mM TNT (Tris-HCl, pH 8.0; 150 mM NaCl; 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 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 TBST (50 mM Tris-HCl, pH 7.9; 150 mM NaCl; 0.05% Tween 20) and 1.2% BSA added. The diluted absorbed antibody was used to screen 8.34×105 plaques from the unamplified random-primed cDNA library and horseradish peroxidase (HRP) secondary antibody was used to visualize the positive plaques. A total of 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. For each clone, three 150×15 mm 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 incubated overnight at 37° C. Plates were cooled at 4° C. for 15 minutes and the filters were removed and washed 3 times in TBST (15 min for each wash). The filters were blocked in 4% BSA in TBST for 1 hour at room 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 25 ml 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. The solution was neutralized by the addition of an aqueous solution saturated with Tris-HCl and 0.03% BSA was added. The solution was dialyzed against two changes of 1× TBS at 4° C. overnight. The purified antibody solution was collected from the dialysis membrane and a “pinch” of BSA was 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 in TBST for 15 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 50 ml elution buffer. Twenty mb of Poly A+RNA was used for the gel and subsequent blot using the procedure outlined by Fourney et al. Clone Kal5-5 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 1 mg poly A+RNA in 13 ml DEPC-treated water was made into cDNA by reverse transcriptase according to manufacturer's 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 B1 chain called D92 (GCT TCA ATG GTC TCC TTA CTA TGT A) (SEQ ID NO: 15).
The Laminin B1k 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 for a signal peptide (von Heijne, 1983 and 1986), the signal peptide would be cleaved following Ala17. 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 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 315 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 Kal5-5.
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 B1k chain is most similar to the human laminin B1 chain (LamB1E). A comparison of the laminin B1k 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 25 mM Tris, 192 mM glycine, 0.05% SDS and 10% methanol for 4 hours. The 140-kD band was excised, blocked in PVP-40 in 0.1M 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 50 mM sodium phosphate, 10 mM EDTA, 5 mM DTT, 5% glycerol, pH 8.0) for 12 hours at 4° C. An additional 62.5 mg of pyroglutamate aminopcptidase/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 B1k Chain
Since the laminin B1k chain has similarity to the laminin B1e chain, its domains were assigned numbers according to their similarity to the comparable domains in laminin (FIG. 4A). Some of the laminin B1e chain domains are missing in the laminin B1k chain and those that remain are all truncated in comparison to the laminin B1e 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 B1k chain. FIG. 4A shows a comparison of the domain sizes and percent identity for the various domains of the laminin B1e and laminin B1k 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 B1e 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 B1e chain. The laminin B1k chain has no globular domain IV as is found the in the laminin B1e chain. Domain II (residues 560-766), as in the laminin B1e chain, begins with two closely spaced cysteins and is predicted to be an α-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 B1e, this domain contains a single cysteine residue one residue away from the carobxyl-terminal end. Also similar to the laminin B1e 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 B1k chain, and since the homologous domain in laminin and s-laminin are believed to mediate self-assembly, the sequences of domain VI for B1k, B1e and B1s were compared (FIG. 5). The amino acid identity of domain VI for B1e and B1s shows 70% sequence conservation (FIG. 5). The number and location of cysteinyl residues is identical. Comparisons of the B1k sequence with these two chains shows 49.8% overall sequence identity. As shown in FIG. 5, B1e and B1s 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 B1k chain contains an additional cysteine at amino acid residue number 50 (FIG. 5, arrow). This region is also highly divergent from the B1e and B1s chains with an 18% amino acid residue identity to the B1e chain (excluding the obligatory cysteine) whereas, the same region is 70% identical between B1e and B1s. These comparisons suggest that B1k shares an overall structural similarity with B1e and B1s, but the chains are likely to be functionally divergent.
The Laminin B1k Chain Is a Truncated Chain
As described above, overlapping cDNA clones encoding the entire 140-kD laminin B1k 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 140-kD laminin B1k 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 daltons. 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 are two potential glycosaminoglycan attachment sites marked with stars and three potential O-linked glycosylation sites marked by triangles 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 α domain which may extend away from the long arm of the chain and interact with other molecules. The function of the α domain is not known.
The Laminin B1k Chain Is Similar to the Laminin B1e and Laminin B1s Chains
FIG. 7 shows a comparison of our cDNA sequence to the B1 and B2 chains of laminin (LAMB1E and LAMB2E), the B2 chain of kalinin (LAMB2T) and the B1 chain of s-laminin (LAMB1 S). Since the kalinin B1 chain is clearly related to these other laminin subunits, the convention of Engel et al., 1991 was followed and the Kalinin B 1 chain will be named Laminin B1k. As is apparent from FIG. 7, the human laminin B1k chain is most similar to the human laminin B1e (34.1% identity) and rat laminin B1s (37.1% identity) chains. Initially it seemed possible that the laminin B1k chain might be the human equivalent of the laminin B1s 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 B1k chain is distinct from the laminin B1s chain. The first is the size of the laminin B1k chain polypeptide which was previously reported to be 140-kD. The laminin B1s chain in rat is about 190-kD which is only slightly smaller than the 200-kD laminin B1e chain. Since there is good conservation of protein size between species (from human to drosophila) for all three of the laminin chains (Laminin Ae, B1e, and B2e), one expects the same will hold true between species for the laminin B1s chain as well and it is predicted that this chain will be 190-200-kD in size. Additional evidence that the laminin B1k chain is distinct from the laminin B1s chain is the fact that a human tryptic peptide sequence was found that is not found in the laminin B1k chain, but has 95.8% identity to the rat laminin B1s chain.
Since the human sequence of the laminin B1s chain is not available, the B1k sequence was compared to the most well described similar molecule, the laminin B1e chain. The major difference between the laminin B1e and laminin B1k chains is their size. The laminin B1k chain has a truncated structure and, therefore, a lower molecular mass than the 200-kD laminin B1e chain. This smaller size is mainly due to the absence of the globular domain which corresponds to domain IV in the laminin B1e 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 B1k and laminin B1e 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 B1k Chain
The greatest functional significance of the short arm is found in the amino-terminal domain VI. In laminin B1e, domain VI has been reported to aid in the self-assembly of the laminin molecules in vitro. The presence of this domain in the laminin B1k 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 B1k. One possible ligand for this domain is the recently described K-laminin molecule which contains the laminin B1e 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 B1k sequence to B1e and B1s 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 B1k is functionally different from the other known laminin B1 chains. B1k domain VI also contains an odd number of cysteine residues (FIG. 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 B1k chain is the only kalinin chain that remains unprocessed in the mature kalinin molecule, the association with K-laminin appears to be mediated by the B1k chain. The significant diversion in sequence homology between the VI domains of B1k versus B1e and B1s, and the presence of a potentially unpaired cysteine residue are consistent with the concept that the B1k 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.
Domain IV is missing in the laminin B1k chain and while no functions have been reported for the comparable domain in the laminin B1e 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 in the EGF repeat number 9 in domain III of the laminin B1e chain. These peptide sequences are not present since the EGF repeats numbered 6-10 are all missing in domain III of the laminin B1k chain.
Domain Structure of the Long Arm of the Kalinin B1 Chain
The long arm of the laminin B1k chain contains numerous heptad-repeats similar to those found in the two B chains of laminin. The laminin B1e and B2e chains have been found to associate with one another and are in fact disulfide-bonded. Clearly the three chains of kalinin are disulfide-bonded since they can only be separated by gel electrophoresis only after reduction by β-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 B1k chain appears to interact with the laminin B2t chain by forming an α-helix as is found between the laminin B1e and B2e chains and in fact computer analysis predicts that laminin B1k can form an α-helical coiled-coil structure just as laminin B1e. The laminin B1k and the laminin B2t chain each have a single cysteine in their carboxy-terminal regions that are candidates for disulfide-bonding. The laminin B1k chain also has the short cysteine-rich α 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 B1s chain. This sequence is not found in the laminin B1k chain and this function would therefore be missing.
Other Embodiments
Nucleic acid encoding all or part of the B1k chain can be used to transform cells. For example, the B1k gene, e.g., a mis-expressing or mutant form of the B1k gene, e.g., a deletion, or DNA encoding a B1k chain can be used to transform a cell and to produce a cell in which the cell's genomic B1k gene has been replaced by the transformed gene, producing, e.g., a cell deleted for the B1k gene. Such cells can be used with cells capable of being grown in culture, e.g., cultured stem cells, to investigate the function of the B1k gene.
Analogously, nucleic acid encoding all or part of the B1k 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 B1k 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 laminin B1k chain can subsequently be studied.
The invention includes any fragment of B1k, or any recombinantly produced B1k or fragment thereof which is substantially homologous to a B1k protein, e.g., the B1k 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 2×SSC 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, 6.3.1-6.3.6, hereby incorporated by reference); and polypeptides or proteins specifically bound by antisera to a B1k protein, especially by antisera to the active site or binding domain of a B1k protein. The term also includes chimeric polypeptides that include a B1k 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 B1k protein. By “biologically active” is meant possessing any in vivo or in vitro activity which is characteristic of B1k, e.g., B1k activity as described above. Because the B1k protein exhibits a range of physiological properties and because such properties may be attributable to different portions of the B1k protein molecule, a useful B1k protein fragment or B1k protein analog is one which exhibits a biological activity in any one (or more) of a variety of B1k protein assays, for example, the ability to bind the laminin Ak chain, as described above. A B1k protein fragment or analog possesses, most preferably 90%, preferably 40%, or at least 10%, of the activity of a naturally occurring B1k isoform, e.g., of the B1k protein shown in FIG. 2, in any in vivo or in vitro B1k assay.
Preferred analogs include B1k peptides or recombinant B1k 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




Acid
Code
Replace with any of





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, Gln,




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, Thr,




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, β-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


Phenylalanine
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, allo-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 B1k 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 occurring B1k 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.,β or γ 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 modified, 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 B1k proteins or peptides can be made by methods known to those skilled in the art, e.g., by expressing B1k DNA which has been manipulated in vitro to encode the desired fragment; e.g., by restriction digestion or other manipulation of a B1k 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 B1k DNA e.g., the sequence in FIG. 2. For example, in vitro mutagenesis can be used to convert the DNA sequence of FIG. 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 B1k activity.
Also included are B1k 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 B1k protein, or fragment thereof, one can insert B1k-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 B1k proteins can be made by immunizing an animal, e.g., a rabbit or mouse, and recovering anti-B1k antibodies by prior art methods.
Other embodiments are within the following claims.

## Claims

1. A purified DNA that encodes SEQ ID NO:2.