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.     

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.     

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.     

A truncated laminin chain homologous to the B2 chain: structure, spatial expression, and chromosomal assignment

We describe the identification of a novel laminin chain. Overlapping clones were isolated from a human fibrosarcoma HT1080 cell cDNA library spanning a total of 5,200 bp. A second set of clones contained an alternative 3' end sequence giving a total of 4,316 bp. The longer sequence contained an open reading frame for a 1,193-residue-long polypeptide. The alternative sequence was shortened at the carboxyl-terminal end coding for a 1,111-residue-long polypeptide. The amino acid sequence contained 21 amino acids of a putative signal peptide and 1,172 residues or alternatively 1,090 residues of a sequence with five distinct domains homologous to domains I-V in laminin chains. Comparison of the amino acid sequences showed that the novel laminin chain is homologous to the laminin B2 chain. However, the structure of the novel laminin chain isolated here differs significantly from that of the B2 chain in that it has no domain VI and domains V, IV, and III are shorter, resulting in a truncated laminin chain. The alternative sequence had a shortened domain I/II. In accordance with the current nomenclature, the chain characterized here is termed B2t. Calculation of possible chain interactions of laminin chains with the B2t chain domain I/II indicated that the B2t chain can replace the B2 chain in some laminin molecules. The gene for the laminin B2t chain (LAMB2T) was localized to chromosome 1q25-q31 in close proximity to the laminin B2 chain gene. Northern analysis showed that the B2t chain is expressed in several human fetal tissues but differently from the laminin B1 and B2 chains. By in situ hybridization expression of the B2t chain was localized to specific epithelial cells in skin, lung, and kidney as opposed to a general epithelial and endothelial cell expression of the laminin B2 chain in the same tissues.     

Primary structure of the Drosophila laminin B2 chain and comparison with human, mouse, and Drosophila laminin B1 and B2 chains

Laminin, a major component of basement membranes, is a large glycoprotein consisting of three disulfide-bonded subunits, A, B1, and B2. We have isolated and sequenced a Drosophila laminin B2 chain cDNA clone that spans 5737 nucleotides. The deduced amino acid sequence predicts that the mature and nonglycosylated polypeptide has a chain length of 1606 residues (Mr = 178,665). This B2 chain contains 100 half-cystine residues, most of which are located in two cysteine-rich domains, and 11 N-X-S or N-X-T sequences which are potential sites of N-linked glycosylation. The predicted secondary structure reveals the presence of six structurally distinct domains, of which two are mainly alpha-helical, two are cysteine-rich with homologous repeats, and two are globular regions. The Drosophila B2 chain is 40.3 and 41.1% identical to the human and mouse B2 chains, respectively, and 29.6, 30.0, and 29.4% identical to the Drosophila, human, and mouse B1 chains, respectively.     

Human laminin B1 chain. A multidomain protein with gene (LAMB1) locus in the q22 region of chromosome 7

We report the isolation and characterization of six overlapping cDNA clones that provide the first and complete amino acid sequence of the human laminin B1 chain. The cDNA clones cover 5613 nucleotides with 5358 nucleotides in an open reading frame encoding 1786 amino acids, including a 21-residue signal peptide-like sequence. Sequence analysis demonstrated the presence of two types of internal homology repeats that were found in clusters within the polypeptide chain. The type A repeats contain about 50 amino acids of which 8 are cysteine. These repeats are present in two clusters toward the NH2-terminal end of the chain and are separated from each other by about 220 amino acids. The two clusters contain five and eight consecutive repeats each. There are two copies of consecutive type B repeats of about 40 amino acids close to the COOH-terminal end. Computer analysis of the amino acid sequence of the B1 chain revealed the presence of structurally distinct domains that contain cysteine-rich repeats, globular regions, and helical structures. Using somatic cell hybrid methodology and in situ hybridization to metaphase chromosomes it was established that the human laminin B1 gene (LAMB1) is located in the q22 region of chromosome 7.     

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.     

Drosophila substrate adhesion molecule: sequence of laminin B1 chain reveals domains of homology with mouse

Laminin, a substrate adhesion molecule in vertebrates, is a large glycoprotein complex in basement membranes that promotes cell adhesion, cell migration, and neurite outgrowth. Here we report on the cloning of the genes encoding the three subunits of Drosophila laminin. Sequence analysis of cDNA clones encoding the Drosophila B1 chain reveals a multidomain structure similar to that of its mouse homolog. The Drosophila sequence has only 25% amino acid identity with the mouse sequence in domains I, II, and IV. However, in one of the putative collagen-binding regions (domain VI) and the two cysteine-rich domains of EGF-like repeats (domains III and V), the amino acid identity between these two evolutionarily distant species jumps to 55%. Moreover, the number, length, and unique amino acid sequences of each of the 13 EGF-like repeats are highly conserved between Drosophila and mouse, suggesting that each may serve a unique function in protein-protein interactions.     

Structure of the human laminin B2 chain gene reveals extensive divergence from the laminin B1 chain gene

The exon-intron structure of the human laminin B2 chain gene was elucidated from genomic lambda phage clones spanning 2 kilobase pairs (kb) of the 5'-flanking region, 58 kb of the structural gene and 10 kb of the 3'-flanking region. The entire gene was shown to contain 28 exons. The promoter region has no TATA or CAAT boxes whereas it contains five GC boxes and three AP-2-like binding sites. Comparison with the promoter region of the mouse gene revealed six highly conserved sequences of 14 to 42 base pairs in length. Sequencing of the last exon of the gene showed that the 3'-untranslated region of the mRNA can be up to 2797 nucleotides with five AATAAA potential polyadenylation signals. The similarity of the human 3'-untranslated sequence with that of mouse was shown to be 68.8%. The exon-intron structure of the laminin B2 chain gene demonstrated extensive divergence from the human laminin B1 chain gene, which has 34 exons. Only three intron locations are conserved in these two genes. The overall exon profile of the laminin B2 chain gene correlates only marginally with the pattern of structural domains and internal cysteine-rich repeats in the laminin B2 polypeptide chain.     

Localization of a major nidogen-binding site to domain III of laminin B2 chain

The large pepsin fragments P1 and P1X, which comprise most of the rod-like domains III of the three short arms of laminin from the mouse Engelbreth-Holm-Swarm tumor, possess full binding activity for nidogen in radioligand assays. Partial reduction (70-80%) of disulfide bonds in P1 did not reduce binding activity and allowed the separation of domain III segments originating from the A, B1 and B2 chains of laminin as demonstrated by sequence analysis. Only the B2 chain segment consisting of seven cysteine-rich repeats with similarity to epidermal growth factor showed substantial nidogen-binding activity. Further degradation of this component to an active 28-kDa fragment was achieved by a second pepsin digestion of partially reduced P1. This indicates that a major binding structure for nidogen is located within three or four cysteine-rich repeats occupying sequence positions 755 to about 920 in the B2 chain. The data also show that fragments P1 and P1X differ by the absence or presence of a large portion, domain IIIb, of the laminin A chain but are indistinguishable in nidogen binding.     

Identification of cDNA clones encoding different domains of the basement membrane heparan sulfate proteoglycan

We have used antibodies to the basement membrane proteoglycan to screen lambda gt11 expression vector libraries and have isolated two cDNA clones, termed BPG 5 and BPG 7, which encode different portions of the core protein of the heparan sulfate basement membrane proteoglycan. These clones hybridize to a single mRNA species of approximately 12 kilobases. Amino acid sequences obtained on peptides derived from protease digests of the core protein were found in the deduced sequence, confirming the identity of these clones. BPG 5 spanned 1986 base pairs and has an open reading frame of 662 amino acids. The amino acid sequence deduced from BPG 5 contains two cysteine-rich domains and two internally homologous domains lacking cysteine. The cysteine-rich domains show homology to the cysteine-rich domains of the laminin chains. A globule-rod structure, similar to that of the short arms of the laminin chains, is proposed for this region of the proteoglycan. The other clone, BPG 7, is 2193 base pairs long and has an open reading frame of 731 amino acids. The deduced sequence contains eight internal repeats with 2 cysteine residues in each repeat. These repeats show homology to the neural-cell adhesion molecule N-CAM and the plasma alpha 1B-glycoprotein. Looping structures similar to these proteins and to other proteins of the immunoglobulin gene superfamily are proposed for this region of the proteoglycan. The sequence DSGEY was found four times in this domain and could be heparan sulfate attachment sites.     

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.     

Amino acid sequence and domain structure of entactin. Homology with epidermal growth factor precursor and low density lipoprotein receptor

Entactin (nidogen), a 150-kD sulfated glycoprotein, is a major component of basement membranes and forms a highly stable noncovalent complex with laminin. The complete amino acid sequence of mouse entactin has been derived from sequencing of cDNA clones. The 5.9-kb cDNA contains a 3,735-bp open reading frame followed by a 3'- untranslated region of 2.2 kb. The open reading frame encodes a 1,245- residue polypeptide with an unglycosylated Mr of 136,500, a 28-residue signal peptide, two Asn-linked glycosylation sites, and two potential Ca2+-binding sites. Analysis of the deduced amino acid sequence predicts that the molecule consists of two globular domains of 70 and 36 kD separated by a cysteine-rich domain of 28 kD. The COOH-terminal globular domain shows homology to the EGF precursor and the low density lipoprotein receptor. Entactin contains six EGF-type cysteine-rich repeat units and one copy of a cysteine-repeat motif found in thyroglobulin. The Arg-Gly-Asp cell recognition sequence is present in one of the EGF-type repeats, and a synthetic peptide from the putative cell-binding site of entactin was found to promote the attachment of mouse mammary tumor cells.     

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.     

Human basement membrane heparan sulfate proteoglycan core protein: a 467-kD protein containing multiple domains resembling elements of the low density lipoprotein receptor, laminin, neural cell adhesion molecules, and epidermal growth factor

The primary structure of the large human basement membrane heparan sulfate proteoglycan (HSPG) core protein was determined from cDNA clones. The cDNA sequence codes for a 467-kD protein with a 21-residue signal peptide. Analysis of the amino acid sequence showed that the protein consists of five domains. The amino-terminal domain I contains three putative heparan sulfate attachment sites; domain II has four LDL receptor-like repeats; domain III contains repeats similar to those in the short arms of laminin; domain IV has lg-like repeats resembling those in neural cell adhesion molecules; and domain V contains sequences resembling repeats in the G domain of the laminin A chain and repeats in the EGF. The domain structure of the human basement membrane HSPG core protein suggests that this mosaic protein has evolved through shuffling of at least four different functional elements previously identified in other proteins and through duplication of these elements to form the functional domains. Comparison of the human amino acid sequence with a partial amino acid sequence from the corresponding mouse protein (Noonan, D. M., E. A. Horigan, S. R. Ledbetter, G. Vogeli, M. Sasaki, Y. Yamada, and J. R. Hassell. 1988. J. Biol. Chem. 263:16379-16387) shows a major difference between the species in domain IV, which contains the Ig repeats: seven additional repeats are found in the human protein inserted in the middle of the second repeat in the mouse sequence. This suggests either alternative splicing or a very recent duplication event in evolution. The multidomain structure of the basement membrane HSPG implies a versatile role for this protein. The heparan sulfate chains presumably participate in the selective permeability of basement membranes and, additionally, the core protein may be involved in a number of biological functions such as cell binding, LDL-metabolism, basement membrane assembly, calcium binding, and growth- and neurite-promoting activities.     

Analysis of the assembly of laminin and the laminin-entactin complex with laminin chain specific monoclonal and polyclonal antibodies

Antibodies specific for the A, B1, and B2 chains of laminin have been obtained and characterized. Lam V, a rat X mouse monoclonal antibody, was obtained by immunizing Lewis rats with the extracellular matrix derived from the mouse endodermal line M1536-B3. The antibody was shown to recognize a conformation-sensitive epitope present on the A chain of laminin. The antibody exhibited high avidity for native laminin and uncomplexed newly synthesized laminin A chains. cDNA clones in the vector lambda-gt11 containing sequences for the B1 and B2 chains of laminin were shown to synthesize beta-galactosidase fusion proteins in the host cells induced with IPTG. The fusion protein F3 contained amino acid residues 822-1765 of the B1 chain of mouse laminin, and the fusion protein E4 contained 219 amino acids at the carboxyl terminus of the B2 chain of rat laminin. These two fusion proteins were used to obtain rabbit polyclonal antibodies which were characterized for their specificity and ability to immunoprecipitate laminin and the B chains of laminin. The chain-specific antibodies were used to analyze the assembly and processing of laminin in the mouse endodermal cell line M1536-B3. The results indicated that the covalent assembly of the A and B chains of laminin was initiated as early as 3 min after labeling cells. At this time point uncomplexed A chain of laminin could be observed even though there was an excess of B1 and B2 chains. As early as 4 min after labeling monomeric, dimeric, and oligomeric forms of the B chains of laminin were observed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular cloning of the rat integrin alpha 1-subunit: a receptor for laminin and collagen

Integrin heterodimers mediate a variety of adhesive interactions, including neuronal attachment to and process outgrowth on laminin. We report here the cloning and primary sequence of an M-200 kD integrin alpha subunit that associates with the integrin beta 1 subunit to form a receptor for both laminin and collagen. Similarities in ligand-binding specificity, relative molecular mass and NH2-terminal sequence make this a strong candidate for the rat homologue of the alpha subunit of the human integrin VLA-1. The full-length rat alpha 1 cDNAs encode a protein containing a purative signal sequence and a mature polypeptide of 1,152 amino acids, with extracellular, transmembrane and cytoplasmic domains. Several structural features are conserved with other integrin alpha chains, including (a) a sequence motif repeated seven times in the NH2-terminal half; (b) potential Ca2+/Mg2+ binding sites in repeats 5, 6, and 7, and (c) alignment of at least 14 of 23 cysteine residues. This rat alpha 1 sequence also contains a 206-amino acid I domain, inserted between repeats 2 and 3, that is homologous to I domains found in the same position in the alpha subunits of several integrins (VLA-2, Mac-1, LFA-1, p150). The rat alpha 1 and human VLA-2 apha subunits share greater than 50% sequence identity in the seven repeats and I domain, suggesting that these sequence identities may underlie some of their similar ligand-binding specificities. However, the rat integrin alpha 1 subunit has several unique features, including a 38-residue insert between two Ca2+/Mg2+ binding domains, and a divergent 15-residue cytoplasmic sequence, that may potentially account for unique functions of this integrin.     

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.