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.     

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.     

Human laminin B2 chain. Comparison of the complete amino acid sequence with the B1 chain reveals variability in sequence homology between different structural domains

The complete amino acid sequence of the human laminin B2 chain has been determined by sequencing of cDNA clones. The six overlapping clones studied cover approximately 7.5 kilobases of which 5312 nucleotides were sequenced from the 5' end. The open reading frame codes for a 33-residue signal peptide and a 1576-residue B2 chain proper, which is 189 residues less than in the highly homologous B1 chain (Pikkarainen, T., Eddy, R., Fukushima, Y., Byers, M., Shows, T., Pihlajaniemi, T., Saraste, M., and Tryggvason, K. (1987) J. Biol. Chem. 262, 10454-10462). Computer analysis revealed that the B2 chain consists of distinct domains that contain helical structures, cysteine-rich repeats, and globular regions, as does the B1 chain. However, domain alpha and domain beta of the B1 chain have no counterpart in B2, and the number of cysteine-rich repeats is 12, or 1 less than in the B1 chain. The degree of homology between the two chains is highest in the cysteine repeat-containing domains III and V where 40% of the residues match. However, results demonstrate that the B1 and B2 chains of laminin are highly homologous proteins that are probably the products of related genes.     

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.     

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.     

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.     

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 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.     

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.     

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)     

Identification of redundant angiogenic sites in laminin alpha1 and gamma1 chains

The degradation of the extracellular matrix is one of the first steps involved in angiogenesis, the formation of new vessels from preexisting ones. Laminin, a large extracellular matrix protein, has many biological activities, including the promotion of angiogenesis. Screening of the laminin-1 chains identified 20 angiogenic peptides, of which, A13 and C16, from the alpha1 and gamma1 chains, respectively, were the most active. We recently identified the receptors for C16 as the integrins alpha5beta1 and alphavbeta3. Here, we show unexpectedly that A13 is a redundant active site to C16 present in the N-terminal globular domain of the alpha1 chain. The peptides are located in homologous sites present in the last globular domains of their respective chains, and their amino acids are 66% conserved, as compared to the inactive homologous site in the beta1 chain, B19 to B20, which is only 18%-23% conserved. Cell attachment studies demonstrated that both A13 and C16 reciprocally inhibited their adhesion activity, whereas the corresponding laminin beta1 chain peptides were inactive. Chorioallantoic membrane assays showed that the in vivo angiogenic activity of A13 is blocked by a C16 antagonist, C16S, which also binds to the same integrin receptors. A13 affinity chromatography and immunoprecipitation analysis showed that the alphavbeta3 and alpha5beta1 integrin receptors bind to this sequence. We have therefore identified redundant activity on two laminin chains. These highly conserved functional sites are likely important mediators of the biological responses of laminins because either one or both of these chains (active sites) are present in almost all laminin isoforms identified to date.     

Laminins and other strange proteins.

Laminins are large multidomain proteins of the extracellular matrix (ECM) with important functions in the development and maintenance of cellular organization and supramolecular structure, in particular in basement membranes. Each molecule is composed of three polypeptide chains, A (300-400 kDa) and B1 and B2 (180-200 kDa), which together form the characteristic cross-shaped laminin structure with three short arms and one long arm. Many different domains have been identified in laminin by sequence analysis, structural investigations, and functional studies. Each short arm is formed by homologous N-terminal portions of one of the three chains. Structurally, each short arm contains two or three globular domains which are connected by rows of manyfold-repeated Cys-rich "EGF-like" domains. In all three chains this region is followed by a long heptad repeat region similar to those found in many alpha-helical coiled-coil proteins. These parts of the three laminin chains constitute a triple-stranded coiled-coil domain, which forms the extended rodlike structure of the long arm. This is the only domain in the protein which is made up of more than one chain and consequently serves the function of chain assembly. The two B chains are terminated by the coiled-coil domain, but the A chain contains an additional C-terminal segment which accounts for five globular domains located at the tip of the long arm. Several important functions of laminin have been assigned to individual domains in either the short arms or terminal regions of the long arm.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential expression of the laminin A and B chains in chimeric kidneys

The expression of laminin in embryonic kidneys growing in ovo is followed with mouse-specific, affinity-purified antibodies against the laminin A and B chains. In mouse kidneys growing on the chicken chorioallantoic membrane, the epithelium and nephrogenic mesenchyme are derived from mouse and the vasculature from chicken chorioallantoic vessels. Hence, with the mouse-specific antibodies, it is possible to analyze the deposition of laminin chains by the nephrogenic tissue, because laminin derived from the chicken vasculature remains unstained. In these chimeras, only the laminin B chain, but not the A chain, is expressed in the undifferentiated nephrogenic mesenchyme. The basement membrane around the ureter bud is labeled by the antibodies against both laminin A and B chains. In the mesenchyme, the laminin A chain appears when the mesenchyme converts into tubules. The results suggest that the laminin A and B chains are synthesized differentially in the embryonic nephrogenic tissue.     

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.     

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.     

Sequencing of laminin B chain cDNAs reveals C-terminal regions of coiled-coil alpha-helix.

cDNAs for laminin B chains have been isolated from a parietal endoderm cDNA library in pUC8 and pUC9. Identification is based on: ability to direct the synthesis in Escherichia coli of polypeptides carrying laminin antigen determinants, in vitro translation of hybrid selected mRNA, and hybridization to high mol. wt. RNA differentially expressed in cells synthesizing large amounts of laminin. The plasmid pPE9 hybrid selects mRNA for the B2 (mol. wt. 185 000) chain and provides 217 residues of C-terminal amino acid sequence. The plasmids pPE386 and 49 both hybrid select mRNAs for the B1a (mol. wt. 205 000) and B1b (mol. wt. 200 000) chains. These two cDNAs are identical over much of their sequence, but pPE386 includes 133 nucleotides of 3' non-coding sequence and a poly(A) tail. Together they provide 495 residues of C-terminal amino acid sequence. Analysis of the predicted sequences reveals a striking heptad repeat, with a high probability that residues a and d are hydrophobic. Such a repeat is typical of the coiled-coil alpha-helices found in proteins such as myosin, tropomyosin and desmin (2-stranded) and fibrinogen (3-stranded).