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.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Structure and expression of fibulin-2, a novel extracellular matrix protein with multiple EGF-like repeats and consensus motifs for calcium binding

A new protein, fibulin-2, was predicted from sequence analysis of cDNA clones obtained from a mouse fibroblast library. This protein consists of a 1195-residue polypeptide preceded by a 26-residue signal peptide. The COOH-terminal region of 787 amino acids contained three anaphylatoxin-related segments (domain I), 11 EGF-like repeats (domain II), 10 of which had a consensus motif for calcium-binding, and a 115- residue globular domain III. Except for two additional EGF-like repeats, this COOH-terminal region showed 43% sequence identity with the previously described fibulin-1 (BM-90). The NH2-terminal 408 residues, unique to fibulin-2, showed no sequence homology to other known proteins and presumably form two additional domains that differ in their cysteine content. Recombinant fibulin-2 was produced and secreted by human cell clones as a disulfide-bonded trimer. Rotary shadowing visualized the protein as three 40-45 nm long rods which are connected at one end in a globe-like structure. No significant immunological cross-reaction could be detected between fibulin-1 and fibulin-2. Production of the fibulin-2 was demonstrated by Northern blots and radioimmunoassay in fibroblasts but not in several tumor cell lines. Together with the observation that the serum level of fibulin-2 is 1,000-fold lower than that of fibulin-1, the data indicate that these two isoforms are not always coordinately expressed. This is also suggested by Northern blots of tissue mRNAs and by immunofluorescence localizations using mouse tissues. The latter studies also demonstrated an extracellular localization for fibulin-2 in basement membranes and other connective tissue compartments.     

Complete primary structure and genomic organization of the mouse Col14a1 gene.

The entire mouse cDNA sequence for type XIV collagen was determined using overlapping PCR products. The 6456 nucleotide (nt) cDNA sequence contains a 5391-nt open reading frame encoding 1797 amino acid residues. The amino terminus has a 28-residue signal peptide that is followed by the mature polypeptide of 1769 amino acid residues with a calculated molecular mass of 193.2 kDa. The mouse alpha1(XIV) collagen chain is predicted to contain all the structural domains described for the polypeptide in chicken and human. These include fibronectin type III repeats, von Willebrand factor A domains, thrombospondin-N-terminal-like domains and two triple-helical domains similar to those of other collagen family members. The amino acid residue sequence of human alpha1(XIV) collagen showed an overall identity of 74% to the chicken sequence and 88% to the human sequence. The entire mouse genomic structure has been determined and is made up of 48 exons. Alternatively spliced forms of mouse type XIV, collagen were not identified corresponding to the findings for the human form.     

Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans.

Synaptotagmin (p65) is an abundant synaptic vesicle protein that contains two copies of a sequence that is homologous to the regulatory region of protein kinase C. Full length cDNAs encoding human and Drosophila synaptotagmins were characterized to study its structural and functional conservation in evolution. The deduced amino acid sequences for human and rat synaptotagmins show 97% identity, whereas Drosophila and rat synaptotagmins are only 57% identical but exhibit a selective conservation of the two internal repeats that are homologous to the regulatory region of protein kinase C (78% invariant residues in all three species). The two internal repeats of synaptotagmin are only slightly more homologous to each other than to protein kinase C, and the differences between the repeats are conserved in evolution, suggesting that they might not be functionally equivalent. The cytoplasmic domains of human and Drosophila synaptotagmins produced as recombinant proteins in Escherichia coli specifically bound phosphatidylserine similar to rat synaptotagmin. They also hemagglutinated trypsinized erythrocytes at nanomolar concentrations. Hemagglutination was inhibited both by negatively charged phospholipids and by a recombinant fragment from rat synaptotagmin that contained only a single copy of the two internal repeats. Together these results demonstrate that synaptotagmin is highly conserved in evolution compatible with a function in the trafficking of synaptic vesicles at the active zone. The similarity of the phospholipid binding properties of the cytoplasmic domains of rat, human, and Drosophila synaptotagmins and the selective conservation of the sequences that are homologous to protein kinase C suggest that these are instrumental in phospholipid binding. The human gene for synaptotagmin was mapped by Southern blot analysis of DNA from somatic cell hybrids to chromosome 12 region cen-q21, and the Drosophila gene by in situ hybridization to 23B.     

Structure of the chicken neuron-glia cell adhesion molecule, Ng-CAM: origin of the polypeptides and relation to the Ig superfamily

The neuron-glia cell adhesion molecule (Ng-CAM) mediates both neuron-neuron and neuron-glia adhesion; it is detected on SDS-PAGE as a predominant 135-kD glycoprotein, with minor components of 80, 190, and 210 kD. We have isolated cDNA clones encoding the entire sequence of chicken Ng-CAM. The predicted extracellular region includes six immunoglobulin-like domains followed by five fibronectin-type III repeats, structural features that are characteristic of several neural CAMs of the N-CAM superfamily. The amino acid sequence of chicken Ng-CAM is most similar to that of mouse L1 but the overall identity is only 40% and Ng-CAM contains a short fibronectin-like segment with an RGD sequence that has no counterpart in L1. These findings suggest that Ng-CAM and L1 may not be equivalent molecules in chicken and mouse. The amino-terminal sequences of the 210-, 190-, and 135-kD components of Ng-CAM are all the same as the predicted amino terminus of the molecule, whereas the 80-kD component begins within the third fibronectin repeat. The cDNA sequence is continuous across the junction between the 135- and 80-kD components, and a single 170-kD Ng-CAM polypeptide was isolated from tunicamycin-treated cells. In addition, all cDNA probes hybridized on Northern blots to a 6-kb RNA, and most hybridized to single bands on Southern blots. These results indicate that the Ng-CAM components are derived from a single polypeptide encoded by a single gene, and that the 135- and 80-kD components are generated from the 210/190-kD species by proteolytic cleavage. The 135-kD component contains most of the extracellular region including all of the immunoglobulin-like domains. It has no transmembrane segment, but it is tightly associated with the membrane. The 80-kD component contains two and a half type III repeats plus the RGD-containing segment, as well as the single transmembrane and cytoplasmic domains. These structural features of Ng-CAM provide a framework for understanding its multiple functions in neuron-neuron interactions, neurite fasciculation, and neuron-glia interactions.     

Amino acid sequence of mouse nidogen, a multidomain basement membrane protein with binding activity for laminin, collagen IV and cells.

The whole amino acid sequence of nidogen was deduced from cDNA clones isolated from expression libraries and confirmed to approximately 50% by Edman degradation of peptides. The protein consists of some 1217 amino acid residues and a 28-residue signal peptide. The data support a previously proposed dumb-bell model of nidogen by demonstrating a large N-terminal globular domain (641 residues), five EGF-like repeats constituting the rod-like domain (248 residues) and a smaller C-terminal globule (328 residues). Two more EGF-like repeats interrupt the N-terminal and terminate the C-terminal sequences. Weak sequence homologies (25%) were detected between some regions of nidogen, the LDL receptor, thyroglobulin and the EGF precursor. Nidogen contains two consensus sequences for tyrosine sulfation and for asparagine beta-hydroxylation, two N-linked carbohydrate acceptor sites and, within one of the EGF-like repeats an Arg-Gly-Asp sequence. The latter was shown to be functional in cell attachment to nidogen. Binding sites for laminin and collagen IV are present on the C-terminal globule but not yet precisely localized.     

Two different genes encode fibronectin binding proteins in Staphylococcus aureus. The complete nucleotide sequence and characterization of the second gene.

A gene encoding a fibronectin binding protein (FnBP) has recently been isolated and sequenced from Staphylococcus aureus strain 8325-4. In the same bacterial strain, 682 bp downstream to the stop codon of this gene (fnbA), a second gene termed fnbB has not been discovered, encoding another FnBP (FnBPB). The two genes show in large parts striking sequence homologies. The complete amino acid sequence encoded by fnbB has been deduced and compared to that deduced from fnbA. In FnBPB a stretch of 66 amino acids downstream to the signal peptide has 75% identity with the corresponding region in FnBPA. At the C-terminal site another 394 amino acid stretch is almost identical in both gene products. This stretch contains the 38 amino acid long D repeats, the wall spanning Wr repeats and the hydrophobic membrane spanning domain. In FnBPA each of the three D repeats has been identified as a fibronectin binding structure. These structures are highly conserved in FnBPB and most likely represent the major Fn-binding domain of this protein. However, a subclone of gene fnbB lacking the coding region for the D repeats also clearly expresses fibronectin binding activity. This additional binding site is so far unique for FnBPB and interacts like the D domains with the N-terminal 24-31-kDa fragment of fibronectin. The purified recombinant FnBP fragment (not containing the D repeats) completely inhibits the binding of fibronectin to whole cells of S. aureus.     

Sequence of contactin, a 130-kD glycoprotein concentrated in areas of interneuronal contact, defines a new member of the immunoglobulin supergene family in the nervous system

The primary amino acid sequence of contactin, a neuronal cell surface glycoprotein of 130 kD that is isolated in association with components of the cytoskeleton (Ranscht, B., D. J. Moss, and C. Thomas. 1984. J. Cell Biol. 99:1803-1813), was deduced from the nucleotide sequence of cDNA clones and is reported here. The cDNA sequence contains an open reading frame for a 1,071-amino acid transmembrane protein with 962 extracellular and 89 cytoplasmic amino acids. In its extracellular portion, the polypeptide features six type 1 and two type 2 repeats. The six amino-terminal type 1 repeats (I-VI) each consist of 81-99 amino acids and contain two cysteine residues that are in the right context to form globular domains as described for molecules with immunoglobulin structure. Within the proposed globular region, contactin shares 31% identical amino acids with the neural cell adhesion molecule NCAM. The two type 2 repeats (I-II) are each composed of 100 amino acids and lack cysteine residues. They are 20-31% identical to fibronectin type III repeats. Both the structural similarity of contactin to molecules of the immunoglobulin supergene family, in particular the amino acid sequence resemblance to NCAM, and its relationship to fibronectin indicate that contactin could be involved in some aspect of cellular adhesion. This suggestion is further strengthened by its localization in neuropil containing axon fascicles and synapses.     

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.     

Identification and characterization of novel mouse and human ADAM33s with potential metalloprotease activity

The ADAM family of membrane-anchored proteins has a unique domain structure, with each containing a disintegrin and metalloprotease (ADAM) domain. We have isolated mouse and human cDNAs encoding a novel member of the ADAM family. The mouse and human predicted proteins consisted of 797 and 813 amino acids, respectively, and they shared 70% homology of the entire amino acid sequence. The mouse ADAM gene exists at a single gene locus. The human gene was ubiquitously expressed in tissues other than liver, was mapped to human chromosome 20p13, and was found to consist of 22 exons. Both proteins have domain organization identical to that of previously reported members of the ADAM family, and contain the typical zinc-binding consensus sequence (HEXGHXXGXXHD) in their metalloprotease domain and a pattern of cysteine localization (C(x)(3)C(x)(5)C(x)(5)CxC(x)(8)C) in their EGF-like domain that is typical of an EGF-like motif. The human protein shows homology with Xenopus ADAM13 (44%), human ADAM19 (40%), and human ADAM12 (39%). From the results of phylogenic analysis based on primary amino acid sequence and distribution of the mRNA, these novel ADAM genes were thus named ADAM33.     

Cloning and sequence of the cDNA encoding the β4 integrin subunit in rat peripheral nerve.

β4 and α6 integrin subunits dimerize to form an adhesion receptor that is necessary to nucleate hemidesmosomes and to anchor epithelial cells to their basal laminae. β4 is also expressed in Schwann cells (which do not contain hemidesmosomes) in peripheral nerve, where it may function in the formation or maintenance of myelin. The cDNA for β4 integrin has been cloned from epithelia-derived human and mouse tissues. We cloned cDNAs encoding β4 integrin from libraries derived from rat peripheral nerve, and determined the complete nucleotide sequence encoding the signal peptide and mature protein. Comparison of the deduced amino acid (aa) sequence revealed 95.1% and 87.5% identity with the mouse and human epithelia-derived sequences, respectively. The amino acid sequence of postulated signal transduction domains in β4 was 100% identical among rat, mouse, and human. Our cDNA clones included two of the four postulated alternatively spliced variants previously described in epithelial clones. Despite the potentially diverse functions of β4 integrin in Schwann cells and keratinocytes, the cDNAs for nerve-derived β4 integrin are highly similar to those cloned from epithelia.     

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 MUC3 gene encodes a transmembrane mucin and is alternatively spliced.

Epithelial mucins are a family of secreted and cell surface glycoproteins expressed by epithelial tissues and implicated in epithelial cell protection, adhesion modulation and signaling. The gene encoding human MUC3 (hMUC3), localised to chromosome 7q22, is most highly expressed in the small intestine. It has previously been reported to be a non-transmembrane mucin with minimal homology to its suggested orthologues from rat (rMuc3) and mouse (mMuc3). RT-PCR was performed to investigate the carboxyl terminus of the published sequence of hMUC3 from normal colon and small intestine tissues and also from a series of 10 colorectal cancer cell lines. Two distinct PCR products were identified. In contrast to the previously published hMUC3 sequence, which terminates shortly after a single cysteine-rich EGF-like domain, conceptual protein translation of the dominant and largest PCR product identified two extracellular cysteine-rich EGF-like domains separated by an N-glycosylation-rich domain and a potential coiled-coil region, followed by a putative transmembrane region and a 75 amino acid cytoplasmic tail. The smaller of the two PCR products was found to be an alternative splice variant of MUC3 including the first EGF-like domain but lacking part of the second EGF-like domain and the transmembrane region. Nine out of 10 colorectal cancer cell lines were found to express MUC3. Interestingly, one of the cell lines, LoVo, expressed predominantly the alternative splice form lacking a transmembrane domain. Structural homology of the new protein sequence of hMUC3 with rMuc3 and mMuc3 indicates it is closely related to the rodent proteins and is likely to be involved in ligand-binding and intracellular signaling. The new finding that MUC3 encodes a transmembrane molecule presents a new paradigm for the structure of this mucin and the manner in which it may function.     

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.