Structure and function of laminin: anatomy of a multidomain glycoprotein

Laminin is a large (900 kDa) mosaic protein composed of many distinct domains with different structures and functions. Globular and rodlike domains are arranged in an extended four-armed, cruciform shape that is well suited for mediating between distant sites on cells and other components of the extracellular matrix. The alpha-helical coiled-coil domain of the long arm is involved in the specific assembly of the three chains (A, B1, B2, and possible variants) of laminin and is the only domain composed of multiple chains. It is terminated by a large globular domain composed of five homologous subdomains formed by the COOH-terminal part of the A chain. Sites for receptor-mediated cell attachment and promotion of neurite outgrowth reside in the terminal region of the long arm. A second cell attachment site, a cell signaling site with mitogenic action, binding sites for the closely associated glycoprotein nidogen/entactin, and regions involved in calcium-dependent aggregation are localized in the short arms. These domains, which to a large extent are composed of Cys-rich repeats with limited homology to EGF, are the most highly conserved regions in laminins of different origin. At present, most structural and functional data have been collected for a laminin expressed by a mouse tumor, which can be readily isolated in native form and dissected into functional fragments by limited proteolysis. Increasing information on laminins from different species and tissues demonstrates considerable variations of structure. Isoforms of laminin assembled from different chains are focally and transiently expressed and may serve distinct functions at early stages of development even before being laid down as major components of basement membranes.     

Dissection of laminin by cathepsin G into its long-arm and short-arm structures and localization of regions involved in calcium dependent stabilization and self-association

Native laminin-nidogen complex isolated from mouse Engelbreth-Holm-Swarm tumor was treated with purified cathepsin G or leucocyte elastase, two neutral serine proteases which play a role in inflammatory processes accompanied by degradation of basement membranes. Both enzymes were found to be more active than porcine pancreatic elastase. In the absence of Ca2+, laminin fragments produced by leucocyte elastase resembled those formed by the pancreatic enzyme but at physiological concentrations of Ca2+ cleavage by cathepsin G was much more selective. Initially laminin (900 kDa) was cleaved at two major sites only with similar rates leading to three fragments. Fragment C1-4 (about 550 kDa) comprises the intact three short arms of the molecule and fragment C8-9 (about 350 kDa) contains the entire triple-coiled region by which its three chains are assembled and the major part of the terminal globular domain of the long arm. The remaining C-terminal region of this domain was recovered as fragment C3 of about 50 kDa. Stabilization against proteolytic attack was restricted to the region of fragment C1-4 and only this fragment exhibited strong Ca2+ dependent self-association similar to that of intact laminin or of its complex with nidogen. The associative properties of fragment C1-4 were dramatically diminished upon removal of the tip of one of the short arms comprising fragment 4. In addition, this provides a clear assignment of the important laminin function to a distinct domain in one of its short arms. The new fragment C8-9 may be employed for exploring the properties and possible functions of the upper long-arm region which so far has not been available as a fragment.     

Laminin chain assembly by triple and double stranded coiled-coil structures.

We have previously provided evidence that laminin assembly occurs by the specific interaction of the alpha-helical domains of the A, B1, and B2 chains, located within the long arm of the molecule (Hunter, I., Schulthess, T., Bruch, M., Beck, K., and Engel, J. (1990) Eur. J. Biochem. 188, 205-211). Recent evidence for noncoordinate synthesis of the laminin chains, and in particular, the absence of the 400-kDa A chain from laminins produced by a number of cell types, has led us to examine the molecular mechanism of laminin assembly using the isolated A and B1-B2 chains of laminin fragment E8. E8A shows little tendency to self-associate, and when renatured from urea forms globular structures with little detectable alpha-helix. In contrast, E8B1-B2 renatures to form rod-like molecules, 30 nm in length. The rod-like structure, high alpha-helix content, and sharp thermal transition indicate that they are double stranded coiled coils. When mixed in equimolar amounts, E8A and E8B1-B2 renature to form molecules which are biochemically and ultrastructurally indistinguishable from native E8. If E8A and E8B1-B2 are renatured separately and mixed at a 1:1 molar ratio, they also form E8 molecules. These results suggest a mechanism of laminin assembly which involves the formation of a double coiled-coil B1-B2 intermediate with which the A chain subsequently interacts to form a triple coiled-coil laminin molecule. In addition, our results indicate that isoforms consisting of the B1 and B2 chains only would form stable "laminin-like" structures.     

The N terminus of laminin A chain is homologous to the B chains

A major proteolytic fragment (E1/E1-4) of the basement membrane protein laminin, comprising the three short arms with some terminal globules missing, was isolated by elastase digestion, and partial protein sequence data were determined for several tryptic peptides. Sequences which corresponded to A-chain structures were used to synthesize oligonucleotides for the construction and screening of a primer-extended cDNA library from mouse PYS-2 cells. A clone of 1.1 kb was obtained and shown by sequencing to correspond to the 5' end of the 10-kb mRNA of the A chain of laminin. The clone contains 77 nucleotides of 5' untranslated sequence and a region coding for 334 amino acids, including a presumptive signal peptide of 24 amino acids. The sequence is 30% homologous to the corresponding N-terminal part of the B1 chain of laminin, suggesting the same structure for both domains. The data present further evidence for a recent structural model which postulates that each of the three laminin polypeptide chains forms a distinct short arm.     

The coiled-coil structure potential of the laminin LCC domain is very fragmented and does not differentiate between natural and non-detected isoforms

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.     

Structure of laminin variants. The 300-kDa chains of murine and bovine heart laminin are related to the human placenta merosin heavy chain and replace the a chain in some laminin variants

A variant of laminin has previously been isolated from murine heart and shown to be distinct from laminin purified from a traditional source, the murine Engelbreth-Holm-Swarm (EHS) tumor (Paulsson, M., and Saladin, K. (1989) J. Biol. Chem. 264, 18726-18732). It contains a novel polypeptide chain designated as 300 kDa, which is not found in laminin from the EHS tumor. In the present study, heart laminin was purified from bovine tissue and shown to be structurally and immunochemically closely related to the murine protein. Further, heart laminins were compared with merosin, a laminin-like protein isolated from human placenta (Ehrig, K., Leivo, I., Argraves, W. S., Ruoslahti, E., and Engvall, E. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3264-3268). The 300-kDa chain of bovine heart laminin cross-reacted with the heavy chain of merosin, showing that these polypeptides are closely related, albeit from different species. Heart laminin is more resistant to proteolysis than laminin derived from the EHS tumor. A large fragment could be prepared by digestion with thermolysin, which consisted of an almost intact long arm structure and variably long, residual short arm structures. Analysis of its structure shows that the 300-kDa heavy chain is disulfide-bonded to the B1 and B2 chains in the center of the laminin cross and forms the long arm together with these chains. It thereby replaces the A chain, well known from tumor sources, in the laminin structure.     

The dermal-epidermal junction of human skin contains a novel laminin variant

We report the identification of a novel laminin variant that appears to be unique to a subset of epithelial basement membranes. The variant contains two chains electrophoretically and immunologically identical to the B1 and B2 chains. Epitopes contained in the laminin A chain are absent from the molecule, and a 190-kD chain substitutes for the A chain. V8 protease analysis and Western blotting studies indicate that the variant 190-kD chain shows structural and immunological similarity to the 200-kD chain of kalinin. Rotary shadowing analysis indicates that the 190-kD chain contributes a large globular structure to the variant long arm, but lacks the short arm contributed to laminin by the A chain. The variant is produced by cultured skin explants, human keratinocytes and a squamous cell carcinoma line, and is present in human amniotic fluid. Polyclonal antibodies raised to kalinin, a recently characterized novel component of anchoring filaments, and mAb BM165 which recognizes a subunit of kalinin (Rousselle et al., 1991) cross react with the variant under nonreducing conditions. Immunohistological surveys of human tissues using the crossreacting antikalinin antiserum indicate that the distribution of this laminin variant is at least restricted to anchoring filament containing basement membranes. We propose the name K-laminin for this variant.     

The Coiled-coil Structure Potential of the Laminin LCC Domain is Very Fragmented and Does Not Differentiate Between Natural and Non-detected Isoforms

Abstract

There are 15 known laminins, which differ in the isoforms of the three chains that assemble into the cross-shape molecules that are observed by electron microscopy. The amino acid sequences of the rod-like portion of the long arm have long been recognized as having a potential for coiled-coil structure formation; however, an experimental determination of its structure is hampered by the complexity of laminin, a multidomain, heterotrimeric, and glycosilated 800 kDa molecule. Here, we have investigated the coiled-coil structure potential of laminin to evaluate its distribution along the long arm, the presence of conserved patterns, and differences between natural and non-natural isoforms. With these aims, we have analysed the sequences of each laminin chain in the context of the three-chain assemblies to yield an overall score of coiled-coil potential for the 15 natural laminins and for the other 30 possible but non-detected ones. The potential has been calculated with two different existing methods to exclude algorithm specific biases and with different chain alignments to evaluate the dependency of the results on uncertainties in the specific alignment along the domain. The analysis shows that the distribution of the potential is discontinuous, highly fragmented along the arm, without a common pattern except for a higher potential at the C-terminus, and that natural and non-natural laminins cannot be distinguished based on their coiled-coil potential, indicating that other factors are responsible for the selection of chain assembly.     

Cell and heparin binding in the distal long arm of laminin: identification of active and cryptic sites with recombinant and hybrid glycoprotein [published erratum appears in J Cell Biol 1993 Dec;123(6 Pt 1):1623]

The long arm of laminin, which binds heparin and cells, consists of three polypeptides (A, B1, and B2) joined in a coiled-coil rod attached to a terminal A chain globule (G). Previously, we found that recombinant globular domain (rG) supported heparin and myoblast binding (Yurchenco, P. D., U. Sung, M. D. Ward, Y. Yamada, and J. J. O'Rear. 1993. J. Biol. Chem. 268:8356-8365). To further analyze long arm functions, we expressed the distal moiety of the mouse laminin A chain extending from the middle of the rod to the carboxyl terminus (rAiG). This larger glycoprotein, secreted by Sf9 insect cells infected with recombinant baculovirus, was intercalated in vitro into the corresponding disulfide-linked B chain segments of laminin fragment E8 (distal long arm rod and proximal globule). The hybrid molecule (B- rAiG) possessed a structure similar to laminin long arm as judged by electron microscopy and limited proteolysis. By joining rAiG with E8-B chains, the affinity of G domain for heparin decreased from that observed with rAiG and rG to one similar to native protein. HT1080 cells adhered to E8, rAiG, and B-rAiG, less well to rG, and not to denatured E8/B-rAiG, the A and B chain moieties of E8, or to a mixture of rG and E8-B chains. Cell adhesion to E8 and B-rAiG, in contrast to rAiG, was inhibited with antibodies specific for alpha 6 and beta 1 integrin chains. Since intercalation (a) restored a conformationally dependent alpha 6 beta 1 integrin recognition site present in native protein, (b) inactivated a cryptic cell binding activity in the A chain, and (c) inhibited a heparin binding site present in proximal G domain, we conclude that biological activities of laminin are different from that of its isolated subunits.     

Drosophila laminin A chain sequence, interspecies comparison, and domain structure of a major carboxyl portion.

Recent studies ascribed some biological actions of cell adhesion and cell outgrowth to the carboxyl-most 1200 amino acids of vertebrate laminin A chains. Here we report a 6.1-kilobase pair nucleotide cDNA sequence encoding 1951 amino acids and the carboxyl end of a Drosophila laminin A chain. It corresponds to the mouse laminin A domains G, I, II, and III, but may represent a different type of laminin A chain. The arrangement of the cysteine-rich repeats of domain III resembles that of B2 chains. However, it has more amino acid identity with a portion of the mouse laminin A chain domain IIIb than with other laminin repeats. Domains I and II are consistent with an interrupted coiled-coil alpha-helical model of the long arm of laminin but are poorly conserved. The G domain contains five subdomains which are individually related to subdomains of vertebrate laminin A chains. The results indicate that laminin G subdomains should be considered individually, rather than merely as parts of a G-globule. A sequence of hydroxyamino acids contributes to a spacer between two of the subdomains. Stretches of hydroxyamino acids may be indicative of junctions between domains of extracellular Drosophila proteins.     

The laminin B2 chain has a multidomain structure homologous to the B1 chain

Laminin (Mr = 850,000) is a large basement membrane-specific glycoprotein composed of three chains: A, B1, and B2. Previously, we have reported the primary structure of the B1 chain of mouse laminin deduced from sequencing cDNA clones (Sasaki M., Kato, S., Kohno, K., Martin, G. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 935-939). Here we report the isolation of overlapping cDNA clones spanning 7642 bases which encode the entire B2 chain. The nucleotide sequence of the clones contains an open reading frame of 4821 bases coding for a protein of 1607 amino acids including 33 amino acids of a presumptive signal peptide. The mRNA for the B2 chain contains 2.5 kilobases of 3'-untranslated region. The deduced amino acid sequence indicates that the B2 chain consists of six distinct domains, including two domains with alpha-helical, coiled-coil structures, two domains with cysteine-rich homologous repeats, and two globular domains. These structural features of the B2 chain are similar to those of the B1 chain. In addition, the amino acid sequences of the B2 and B1 chains demonstrate considerable homology, suggesting that the genes for these two chains arose from a common ancestor.     

Electron microscopic structure of agrin and mapping of its binding site in laminin-1.

Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.     

The complete sequence of perlecan, a basement membrane heparan sulfate proteoglycan, reveals extensive similarity with laminin A chain, low density lipoprotein-receptor, and the neural cell adhesion molecule.

A heparan sulfate proteoglycan is a component of all basement membranes. This molecule consists of three heparan sulfate side chains linked to a large core protein of approximately 400 kDa. We have isolated seven overlapping murine cDNA clones that encode the entire mRNA sequence of 12.685 kilobases of this molecule. This sequence has a single open reading frame of 3,707 amino acids that encodes for a protein of 396 kDa. Identical or near identical matchups with nine peptide sequences derived from the core protein of the molecule isolated from the Engelbreth-Holm-Swarm tumor were found with the deduced sequence. Sequence analysis and data base comparison of the deduced sequence show the protein to consist of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment, SGD. Domain II contains four cysteine- and acidic amino acid-rich repeats that are very similar to those found in the LDL receptor and proteins such as GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule. Domain V contains three repeats with similarity to the laminin A chain G domain that are separated by epidermal growth factor-like regions not found in the laminin A chain. As the primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or "beads on a string," we have adopted the name perlecan for this molecule. The variety of domains in perlecan suggest multiple interactions with other molecules.     

Terminal short arm domains of basement membrane laminin are critical for its self-assembly

Laminin self-assembles into large polymers by a cooperative two-step calcium-dependent mechanism (Yurchenco, P. D., E. C. Tsilibary, A. S. Charonis, and H. Furthmayr. 1985. J. Biol. Chem. 260:7636-7644). The domain specificity of this process was investigated using defined proteolytically generated fragments corresponding to the NH2-terminal globule and adjacent stem of the short arm of the B1 chain (E4), a complex of the two short arms of the A and B2 chains attached to the proximal stem of a third short arm (E1'), a similar complex lacking the globular domains (P1'), and the distal half of the long arm attached to the adjacent portion of the large globule (E8). Polymerization, followed by an increase of turbidity at 360 nm in neutral isotonic TBS containing CaCl2 at 35 degrees C, was quantitatively inhibited in a concentration-dependent manner with laminin fragments E4 and E1' but not with fragments E8 and P1'. Affinity retardation chromatography was used for further characterization of the binding of laminin domains. The migration of fragment E4, but not of fragments E8 and P1', was retarded in a temperature- and calcium-dependent fashion on a laminin affinity column but not on a similar BSA column. These data are evidence that laminin fragments E4 and E1' possess essential terminal binding domains for the self-aggregation of laminin, while fragments E8 and P1' do not. Furthermore, the individual domain-specific interactions that contribute to assembly are calcium dependent and of low affinity.     

Primary structure of the mouse laminin B2 chain and comparison with laminin B1

One of the major components of basement membranes is the glycoprotein laminin, made up of three disulfide-bonded subunits, the A, B1, and B2 chains. We have isolated and sequenced overlapping mouse laminin B2 chain cDNA clones covering 7562 base pairs. The deduced amino acid sequence predicts that the mature B2 chain consists of 1572 residues, has an unglycosylated molecular weight of 173,541, and possesses 14 potential N-linked glycosylation sites. Analysis of the predicted secondary structure shows the presence of six domains, two rich in alpha-helical structure, two composed of homologous cysteine-rich repeat units, and two globular regions. The organization of the molecule is very similar to that of the mouse laminin B1 chain, and significant sequence homology between the B1 and B2 chains was found in their two cysteine-rich domains and in their amino-terminal globular domains.     

Evidence for coiled-coil alpha-helical regions in the long arm of laminin.

Three new laminin fragments, E8, E9 and 25K with mol. wt. 50 000-280 000, were prepared from a limited elastase digest of laminin and from tissue extracts. They were similar with respect to their rod-like structure, a high alpha-helix content, the assembly from two chain segments and immunological cross-reactivity. Two of the fragments (E8 and E9) possess in addition globular domains which lack alpha-helices. Chemical, immunological and physical data together with sequence analysis strongly indicate that the alpha-helical segments are assembled in coiled-coil structures which are located in the rod of the long arm of laminin. These data give new insights into the overall structure of the protein.     

Laminins in basement membrane assembly

The heterotrimeric laminins are a defining component of all basement membranes and self-assemble into a cell-associated network. The three short arms of the cross-shaped laminin molecule form the network nodes, with a strict requirement for one α, one β and one γ arm. The globular domain at the end of the long arm binds to cellular receptors, including integrins, α-dystroglycan, heparan sulfates and sulfated glycolipids. Collateral anchorage of the laminin network is provided by the proteoglycans perlecan and agrin. A second network is then formed by type IV collagen, which interacts with the laminin network through the heparan sulfate chains of perlecan and agrin and additional linkage by nidogen. This maturation of basement membranes becomes essential at later stages of embryo development.     

Localization of three distinct heparin-binding domains of laminin by monoclonal antibodies

Monoclonal antibodies were utilized to localize novel heparin-binding domains of laminin. A solid-phase radioligand binding assay was designed such that [3H] heparin bound to laminin in a time- and concentration-dependent manner. Tritiated heparin binding to laminin was saturable and specific as determined by competition with unlabeled heparin, dextran sulfate, and dermatan sulfate. By Scatchard analysis, two distinct dissociation constants were calculated (Kd = 50 and 130 nM), suggesting the presence of at least two binding sites for heparin on laminin. Tritiated heparin bound to thrombin-resistant (600 kDa) and chymotrypsin-resistant (440 kDa) laminin fragments, both known to lack the terminal globular domain of the long arm. Sodium dodecyl sulfate-polyacrylamide gels of chymotrypsin- and thermolysin-digested laminin chromatographed on a heparin-Sepharose column showed multiple proteolytic fragments binding to the column. Monoclonal antibodies generated against laminin were tested for their ability to inhibit [3H]heparin binding to laminin. Four monoclonal antibodies significantly inhibited the binding of [3H]heparin to laminin in the range of 15-21% inhibition. Laminin-monoclonal antibody interactions examined by electron microscopy showed that one antibody reacted at the terminal globular domain of the long arm, domain Hep-1, while epitopes for two of these monoclonal antibodies were located on the lateral arms of laminin, domain Hep-2, and the fourth monoclonal antibody bound below the cross-region of laminin, domain Hep-3. When two monoclonal antibodies recognizing distinctly different regions of laminin were added concomitantly, the inhibition of [3H]heparin binding to laminin increased almost 2-fold. These results suggest that at least two novel heparin-binding domains of laminin may be located in domains distinct from the terminal globular domain of the long arm.     

Mapping of three major heparin-binding sites on laminin and identification of a novel heparin-binding site on the B1 chain

Laminin, a major basement membrane glycoprotein, interacts with many basement membrane- and cell surface-associated heparin-like macromolecules. In order to understand these interactions better, we have tried to map heparin-binding sites on laminin precisely. Electron microscopy revealed three major heparin-binding sites: 1) on the globule of the long arm; 2) on the outer globule of the short arms; and 3) on the inner globule of the short arms. Elution of heparin bound to a laminin affinity column with a linear salt gradient produced three peaks at 0.15, 0.17, and 0.20 M NaCl. When the laminin-heparin interaction was examined in the presence of increasing salt concentrations by the technique of rotary shadowing, the weakest binding was assigned to the inner globule of the short arms and the strongest to the globule of the long arm. One peptide termed AC15, with the sequence Arg-Ile-Gln-Asn-Leu-Leu-Lys-Ile-Thr-Asn-Leu-Arg-Ile-Lys-Phe-Val-Lys from the B1 chain, was identified as a heparin-binding sequence localized on the outer globule of the lateral short arm. Because the two stronger heparin-binding sites were mapped in domains participating in laminin self-association, the effect of heparin on this phenomenon was examined using turbidity and electron microscopy. At low heparin concentrations, laminin oligomer and polymer formation was slightly enhanced. At high heparin concentrations, a drastic inhibition of polymerization was observed, and laminin was detected to be mainly monomeric in rotary-shadowed samples. These results suggest that local variation in the concentration of heparin-like macromolecules might be a crucial factor in determining the association of matrix macromolecules and therefore the structure of basement membranes.     

Laminin, a multidomain protein. The A chain has a unique globular domain and homology with the basement membrane proteoglycan and the laminin B chains

Laminin (Mr = 800,000) is a glycoprotein consisting of three chains, A, B1, and B2, and has diverse biological activities. Previously we reported the complete primary structure of the B1 and B2 chains of mouse laminin deduced from cDNA sequence (Sasaki, M., Kohno, K., Kato, S., Martin, G. R., and Yamada, Y. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 935-939; Sasaki, M., and Yamada, Y. (1988) J. Biol. Chem. 262, 17111-17117). Here we describe the isolation, characterization, and sequence of cDNA clones spanning 9,520 bases which encode the entire A chain of mouse laminin. The nucleotide sequence of the clones contains an open reading frame of 3,084 amino acids including 24 amino acids of a signal peptide. The A chain contains some eight distinct domains including alpha-helices, cysteine-rich repeats and globules. There is considerable sequence and structural homology between the A chain and the B1 and B2 chains. However, the A chain has a unique globular structure containing homologous repeats at the carboxyl terminus and constituting one third of the molecular mass of the chain. Furthermore, the A chain contains three globules and three cysteine-rich domains at the amino terminus, whereas the B1 and B2 chains have only two each of such domains. The A chain shows homology to the basement membrane heparan sulfate proteoglycan core protein and the extracellular domain of the Drosophila neurogenic protein Notch. There is an RGD (Arg-Gly-Asp) sequence in one of the cysteine-rich domains of the A chain. This potential cell binding sequence could be active as another adhesion signal in addition to the previously identified cell binding sequence YIGSR (Tyr-Ile-Gly-Ser-Arg) of the B1 chain.