1,721,159 research outputs found
Biosynthesis of chick type VI collagen. II: Processing and secretion in fibroblasts and smooth muscle cells.
No full textThe biosynthesis of type VI collagen was studied in "matrix-free" chick embryo smooth muscle cells and fibroblasts. Omission of ascorbate from the culture affected to a great extent the secretion in fibroblasts but had a very minor effect on smooth muscle cells. Quantitative analysis of the secretion process in continuous time course and in pulse-chase experiments confirmed that fibroblasts and smooth muscle cells secreted type VI collagen with the same chain composition but with different kinetics: after 4 h of chase more than 60% of the labeled type VI collagen was present in the culture medium of fibroblasts, whereas at the same time interval less than 25% was secreted by smooth muscle cells. The different kinetics depends on intrinsic properties of the cells, since it was detected also in adherent cells. However, even in fibroblasts, secretion of type VI collagen was much slower than secretion of fibronectin, of which more than 50% was already in the cell medium after 1 h of chase. Treatment of the cells with inhibitors of hydroxylation and glycosylation caused a shift in mobility that revealed a size heterogeneity in the Mr = 260,000 subunit. No evidence of processing was observed in chick cells for any of the subunits that were synthesized and secreted uncleaved. In addition, after several days of chase the Mr of the subunits of type VI collagen isolated from the matrix remained unchanged, thus excluding that in the chick even a partial or incomplete processing takes place
The carboxyl terminus of the chicken alpha3 chain of collagen VI is a unique mosaic structure with glycoprotein Ib-like, fibronectin type III, and Kunitz modules.
No full textThe primary amino acid sequence of the carboxyl-terminal portion of the alpha 3 chain of chick type VI collagen as deduced from the nucleotide sequence is reported. This carboxyl-terminal segment is not present in the alpha 1 and alpha 2 chains of chick type VI collagen and is specific for a mosaic region with extensive similarities to several other proteins. This unique segment, beginning with a stretch (73 residues) very rich in serine and threonine, is preceded by sequences analogous to the platelet glycoprotein Ib. This region is followed by one segment that closely resembles the type III domains of fibronectin. At the end of the sequence, there is a 58-residue motif very similar to sequences characteristic of the Kunitz-type proteinase inhibitors. The present findings and our recent observation that the alpha 3(VI) chain contains 11 repeats similar to type A repeats of von Willebrand factor raise interesting questions about the peculiar mosaic structure and the multiple functions that this unique collagen might play in growth and remodeling of connective tissues
The superfamily of proteins with von Willebrand Factor type A-like domains: one theme common to components of extracellular matrix, hemostasis, cellular adhesion, and defense mechanisms.
No full textType A modules: interacting domains found in several non-fibrillar collagens and in other extracellular matrix proteins.
No full textA 200-amino acid long motif first recognized in von Willebrand Factor (type A module) has been found in components of the extracellular matrix, hemostasis, cellular adhesion, and immune defense mechanisms. At present the extracellular matrix is the predominant site of expression of type A modules since at least four non-fibrillar collagens and two non-collagenous proteins contain a variable number of modules ranging from one to twelve. The modules conform to a consensus motif made of short conserved subregions separated by stretches of variable length. The proteins that incorporate type A modules participate in numerous biological events such as cell adhesion, migration, homing, pattern formation, and signal transduction after interaction with a large array of ligands
Isolation of cDNA clones corresponding to the Mr=150,000 subunit of chick type VI collagen.
No full textType VI collagen is a disulfide-bonded protein with an unusual structure in that the molecule contains three short triple-helical domains and very extended non-collagenous regions. The molecule is a heterotrimer composed in the chick of two polypeptides of similar apparent size in SDS-PAGE (Mr = 140- and 150,000) but different structure, and a third component that is much larger (Mr = 260,000) than the other two chains. We report here on the isolation of several overlapping cDNA clones from a chicken aorta mRNA expression library in the plasmid vector pEX1. Antibodies affinity purified onto the fusion proteins recognized the chick type VI collagen Mr = 150,000 subunit. Northern blots using the cDNA inserts from the above clones revealed a single RNA species of about 4,600 nucleotides sufficient to code for a protein with the size of the Mr = 150,000 subunit
Stable expression of chicken type VI collagen alpha1, alpha2, and alpha3 cDNAs in murine NIH/3T3 cells.
No full textAs a component of an extensive network of microfibrils interwoven with large collagen fibers and in close contact with cell surfaces, type VI collagen plays an important role in cell-matrix interactions. To investigate the behaviour of chicken type VI collagen chains in heterologous host cells as a means to understanding the pattern of assembly of this collagen, we transfected murine NIH/3T3 cells with cDNAs encoding chicken alpha 1(VI), alpha 2(VI) and alpha 3(VI) chains. Cell lines that constitutively expressed the individual chains were analyzed by metabolic labeling and immunoprecipitation with specific antibodies. No self-association was observed for either alpha 1(VI) or alpha 2(VI) chains which were secreted as monomeric polypeptides. Furthermore, neither the chicken alpha 1(VI) nor alpha 2(VI) chains associated with the endogenous murine chains to form chimeric chicken/murine heterotrimers. In contrast, chimeric chicken/murine heterotrimers were detected in cell lines transfected with chicken alpha 3(VI) cDNA. These chimeric forms appeared to be properly aligned since their triple helices were stable to pepsin digestion. In addition, the chimeric heterotrimers coassembled and gave rise to disulfide-linked type VI collagen molecules
Secretion and matrix assembly of recombinant type VI collagen.
No full textA monomer of type VI collagen is composed of three different chains of 140 (alpha 1), 130 (alpha 2), and 250-350 kDa (alpha 3). Monomers assemble into dimers (6 chains) and tetramers (12 chains) that are stabilized by disulfide bonds and, once associated one to another, give rise to a microfilamentous network in close apposition with cell surfaces and banded collagen fibers. We have derived murine NIH/3T3 cell lines that were transfected with the cDNAs for the three chains and that constitutively expressed chicken type VI collagen. Cotransfection was efficient because, in three out of six isolated cell lines, all chicken chains were expressed. Southern blotting demonstrated that several copies of each cDNA were integrated approximately in equal number. Expression of the three polypeptide chains was consistent with the levels of the respective mRNAs. The three chicken chains assembled by disulfide bonding to form correctly folded triple helical aggregated composites with sizes corresponding to type VI collagen monomers, dimers, and tetramers. These functional recombinant assemblies were secreted and became incorporated into the extracellular matrix, where they formed an extensive fibrillar network
Efficient expression of chicken alpha1(VI) collagen chain in transiently transfected mammalian cells.
No full textType VI collagen is a component of the extracellular matrix made of three subunits, alpha 1(VI) and alpha 2(VI) (Mr = 140,000), and alpha 3(VI) (Mr = greater than 300,000). Triple helical monomers assemble intracellularly into disulfide-linked dimers and tetramers, with the tetramers being the "building blocks" that give rise to higher order extracellular structures by head-to-head association, the microfilaments. To study the pattern of assembly and the structure-function relationships of type VI collagen, we transfected mammalian cells with a full-length cDNA coding for chicken alpha 1(VI) under the control of SV40 early and late promoters and assayed the expression, secretion, and assembly of the protein by immunoperoxidase and immunoprecipitation of metabolically labeled cells. First, conditions were determined that allowed efficient transfection both in African monkey kidney COS-1 and CV-1 cells and in mouse fibroblasts. In our hands the late promoter was most efficient in CV-1 cells; whereas the early promoter was efficient in L cells at three days post-transfection. Chicken alpha 1(VI) could be isolated from cell extracts as well as from cell medium. Both the intracellular and the secreted forms of alpha 1(VI) are present as a monomer polypeptide and as disulfide-linked dimers and trimers that migrate in SDS gels with apparent Mr of about 130,000, 240,000 and 360,000, respectively. In L cells, endogenous mouse type VI collagen also was isolated by immunoprecipitation with specific antibodies. However, heterologous molecules made of the chicken alpha 1(VI) chain and the mouse alpha 2(VI) and alpha 3(VI) chains were not detected in the present experiments. Digestion with pepsin of the non-reduced chicken alpha 1(VI) polypeptides immunoprecipitated from the cell medium resulted in the disappearance of the bands, suggesting improper or non-stable assembly of alpha 1(VI) homotrimers. These data support predictions from sequence analysis that type VI collagen heterotrimeric molecules are more stable than other assembly alternatives
Multiple forms of chicken alpha3(VI) collagen chain generated by alternative splicing in type A repeated domains.
No full textType VI collagen is a structurally unique component widely distributed in connective tissues. Its molecular structure consists of monomers that have the potential to assemble intracellularly into dimers and tetramers which, once secreted, can form microfilaments by end-to-end association. Individual monomers are composed of chains of Mr = approximately 140,000 (alpha 1 and alpha 2) and greater than 300,000 (alpha 3). Type VI collagen molecules contain a short triple helix with large globular domains at both ends. These domains are made for their greatest part of repetitive units similar to type A repeats of von Willebrand Factor. The alpha 3(VI) chain, contributing most of the mass of the NH2-terminal globule, appeared heterogenous both at the mRNA and protein level. Several alpha 3(VI)-specific clones that lack the sequences corresponding to repeats A8 and A6 were isolated from a chicken aorta cDNA library. Northern blot hybridization of poly (A+)-enriched RNA from chicken gizzard with cDNA fragments corresponding to several individual type A repeats showed that A8- and A6-specific probes did not hybridize to the lower Mr transcripts. Clones spanning approximately 20 kb of the 5'-end of the alpha 3(VI) gene were isolated from a chicken genomic library and subjected to analysis by restriction mapping, Southern blotting, and selective sequencing of the intron-exon boundaries. At the most 5'-end of the gene an additional type A repeat (A9), previously undetected in cDNA clones, was identified. Furthermore, it was determined that the presumed signal peptide and repeats A9 through A6 are encoded within individual exons. Reverse transcription and polymerase chain reaction of aorta RNA suggested that a mechanism of alternative mRNA splicing by a phenomenon of exon skipping generates alpha 3(VI) isoform variants that contain different numbers of type A repeats. Immunohistochemistry of frozen sections of chicken embryo tissues with repeat-specific mAbs showed that an antibody directed against a conditional exon has a more restricted tissue distribution compared to an antibody against a constitutive exon
Monoclonal antibodies against chick gp 115, a matrix glycoprotein with broad distribution.
No full textHybridoma cell lines were generated producing monoclonal antibodies to chick gp 115, a 115,000-dalton glycoprotein widely distributed in the connective tissue. The specificity of the antibodies was determined by indirect radioimmunobinding: the extent of binding was a function of i) antigen and ii) antibody concentration; iii) inhibition of binding of radiolabelled antibody by unlabelled antibody and iv) among many known extracellular collagenous or noncollagenous glycoproteins tested only gp 115 gave a strong positive binding reaction. The antibodies were used for indirect immunofluorescence and a strong staining reaction was detected in all blood vessels, around smooth muscle cells in several organs, and in the connective matrix of other tissues such as the liver, and the lung. Based on the competition of binding of [125I]-labeled purified antibody by unlabeled antibodies, two separate epitopes were identified on gp 115. Further analysis of the localization of the epitope was obtained by CNBr cleavage and partial digestion of gp 115 with Staphylococcus aureus V8 protease and alpha-chymotrypsin digestion. Following CNBr cleavage a major fragment of Mr = 35,000 was recognized by 4 monoclonal antibodies, and fragments of comparable Mr were detected following V8 protease and alpha-chymotrypsin digestion
- …
