UNIPROT:P00790 - FACTA Search

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Query: UNIPROT:P00790 (PGA)
2,475 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To investigate the nature of the 140 kDa glycoprotein in the trabecular meshwork, polypeptides were extracted with either urea/sodium dodecyl sulfate (SDS)/beta-2-mercaptoethanol (BME) or guanidine hydrochloride followed by pepsin digestion. After electrophoresis and immunoblotting with anti-type-VI-collagen antibodies, a single fraction of molecular weight 140 kDa was identified in the urea/SDS/BME extracts. Pepsin solubilization revealed two immunoreactive fractions (molecular weights 75 and 85 kDa) that comigrated with purified, pepsin-solubilized type VI collagen. By using the polymerase chain reaction (PCR) and primers specific for the alpha 2(VI) chain of type VI collagen, a single PCR product was obtained, which corresponded to the expected size of 137 base pairs, from the total RNA extracted from the trabecular meshwork ex vivo. Southern hybridization with the antisense oligonucleotide probe of the alpha 2(VI) chain confirmed that the amplified sequence was specific. The results show that the trabecular meshwork contains a significant amount of type VI collagen and that trabecular cells express the mRNA coding for the alpha 2(VI) chain of this glycoprotein. The presence of type VI collagen in the trabecular meshwork is implicated in cell-extracellular matrix interactions at this site, and its abnormal accumulation in glaucomatous and aging eyes probably signifies a defect in the function of the trabecular cells in these eyes.
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PMID:Identification of type VI collagen in the trabecular meshwork and expression of its mRNA by trabecular cells. 815 10

Matrix vesicles (MV), microstructures which rapidly accumulate Ca2+ and induce mineral formation in vitro, are linked to type II and X collagens and proteoglycans in the hypertrophic cartilage. However, the roles of these matrix proteins on MV function are not known. This led us to investigate the influence of type II and X collagen binding on Ca2+ uptake by MV. MV isolated from chicken growth plate cartilage were treated with pure bacterial collagenase and 1 M NaCl in synthetic cartilage lymph to selectively and completely remove associated type II and X collagens. Uptake of 45Ca2+ by these collagen-depleted vesicles was markedly reduced. Further treatment with detergent, which disrupted the membrane, restored Ca2+ uptake, indicating that the vesicle membrane structure and the nucleational core inside the vesicle lumen were still intact after the collagenase and 1 M NaCl treatments. Readdition of either native type II or X collagen to the collagenase, 1 M NaCl-treated MV stimulated their Ca2+ uptake to levels similar to those of untreated vesicles. Pepsin-treated type II and X collagens were less effective in stimulating Ca2+ uptake, indicating that non-triple helical domains of these collagens were involved. The pepsin treatment of these collagens also decreased their binding to annexin V (anchorin CII), one of three annexins found in MV, suggesting that annexin V is involved in mediating the binding of type II and X collagens to the MV surface. Furthermore, treatment of collagenase, 1 M NaCl-treated MV with chymotrypsin, which damaged annexin V as well as many other MV proteins, prevented the stimulation of Ca2+ uptake by these collagens. Thus, the interaction between type II and X collagens with MV activates the influx of Ca2+ into MV and may play an important role in calcification of the vesicles.
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PMID:Stimulation of calcification of growth plate cartilage matrix vesicles by binding to type II and X collagens. 815 77

Pepsin-solubilized collagen VI in triple-helical and heat-denatured, unfolded form was shown to promote Mg(2+)- and Mn(2+)-dependent attachment and spreading of various cell lines. On the triple-helical substrate no inhibition of cell adhesion was observed with several synthetic RGD peptides except in the case of A375 melanoma cells. In contrast, adhesion to the unfolded substrate was highly sensitive to RGD inhibition. Nine synthetic peptides were designed according to 10 RGD sequences present in the triple-helical sequence of human collagen alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains. Only one peptide, corresponding to the C-terminal end of alpha 3(VI) chain, showed substantial inhibitory activity, whereas several peptides were active in direct adhesion assays when used as albumin conjugates. Inhibition tests with antibodies to integrin subunits, affinity chromatography, and ligand binding with purified integrins (alpha 1 beta 1, alpha 2 beta 1, alpha V beta 3, and alpha IIb beta 3) were used to identify collagen VI receptors. Binding to the triple-helical substrate is mediated by alpha 1 beta 1 and alpha 2 beta 1 integrins. Binding of both integrins to collagen VI was weaker than that to collagens I and/or IV. Recognition of the denatured substrate is mediated by beta 1 and beta 3 integrins. Activity was shown for alpha 5 beta 1 and alpha V beta 3 and weakly for alpha IIb beta 3 but not all alpha subunits possibly involved were identified. Distinct sets of receptors were also involved in A375 cell binding to triple-helical (beta 1-mediated) and denatured (beta 3-mediated) collagen VI, even though in this case both interactions could be efficiently inhibited by RGD peptides.
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PMID:Integrin and Arg-Gly-Asp dependence of cell adhesion to the native and unfolded triple helix of collagen type VI. 838 21

An autosomal dominant mutation in the COL2A1 gene was identified in a child with the Kniest form of spondyloepiphyseal dysplasia. A C to T transition at nucleotide 35 of exon 12 changed the codon GCG for alanine 102 of the triple helical domain of alpha 1(II) chains of type-II collagen to GTG for valine. The transition also introduced a GT dinucleotide into exon 12. Analysis of cDNA prepared from Kniest cartilage showed that in vivo the transition resulted in an alternatively spliced mRNA that lacked the 213' nucleotides from exon 12. The cartilage cDNA contained approximately equal amounts of normal cDNA and shortened mutant cDNA. The deletion of 21 nucleotides from the mutant cDNA maintained the translational reading frame but resulted in the loss of alanine 102 to lysine 108, which interrupted the repetitive glycine-X-Y triplet sequence required for formation of the triple helix. Type-II collagen molecules containing one or more mutant chains were expected, therefore, to contain interrupted triple helices with a short amino-terminal helical domain A and a large carboxy-terminal helical domain B. Kniest cartilage contained a reduced amount of pepsin-solubilized type-II collagen that consisted of overmodified alpha 1(II) chains. Peptide mapping showed that the overmodifications extended to the carboxy terminus of the alpha 1(II) chains. Pepsin digestion also yielded shortened alpha 1(II) chains corresponding to helical domain B. Kniest chondrocytes cultured in alginate beads produced type-II collagen that was not stably incorporated into the pericellular matrix. This study highlights the importance of dominant negative mutations of COL2A1 in producing Kniest dysplasia.
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PMID:Alternative splicing of exon 12 of the COL2A1 gene interrupts the triple helix of type-II collagen in the Kniest form of spondyloepiphyseal dysplasia. 889 63

The tissue engineering area henceforth calls more and more for bioabsorbable substrata made of biopolymers (collagen, laminin...) or polymers (PLA, PLGA, PGA...) to realize the three-dimensional culture of tissue equivalents. The poly (beta-hydroxybutyrate-beta-hydroxyvalerate), a biopolymer considered as being biodegradable and biocompatible, has been recently introduced for orthopaedic biomaterials and regeneration purposes. In our study, a PHB/9% HV polymer was transformed into 3D foams, then applied to the culture 3D of ovine chondrocytes (fibrous rings & growth plates) and osteoblasts (periostum). Sponges made of bovine type I collagen were used as references. Orthopaedic cells were isolated, prepared and sown by simple injection to the geometrical center of the substrata, then incubated from 0 to 35 days by changing the culture medium all 4 days. Maximal densities were reached after 21 days: 18-24.10(6) cells/g for the chondrocytes, 8-10.10(6) cells/g for the osteoblasts. The cellular proliferation was more marked, with highest cell densities, for the collagen sponges. Laser confocal microscopy shows that the cellular diffusion take place throughout the entire volume of the porous artificial substrata. Future studies will allow to apply the porous bioabsorbable substrata to high-density cell cultures, to the tissue engineering and regeneration, for example for orthopaedic tissues: cartilage, fibrocartilage and bone.
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PMID:[Bio-absorbable synthetic polyesters and tissue regeneration. A study of three-dimensional proliferation of ovine chondrocytes and osteoblasts]. 903 39

The aim of these studies was to see if a combination of osteopromotive membranes of expanded polytetrafluoroethylene (e-PTFE; GORE-TEX) and BMP, known to separately promote bone healing, would be stimulatory if combined. Bilateral transosseous 'critical size' defects at the mandibular angles in adult male rats were treated with either (i) various BMP preparations only; ii) lateral and medial coverage with e-PTFE membranes only; iii) a combination of various BMPs and membranes; or iv) left untreated. Two semi-purified bovine BMPs (bBMP), comprising collagen as carrier material, and recombinant human (rh) BMP-2 (1, 2 or 8 micrograms), delivered in either biodegradable beads of polylactide/polyglycolide acid (PLA/PGA) or purified collagen, were tested. After 12 or 24 days, block biopsies were taken for histological analysis. The different materials were well tolerated by the hosts and elicited large amounts of newly formed bone widely spread in the surrounding soft tissue. The combinations of bBMP and membranes did not improve the rate of bone healing compared to membranes only. In contrast, the combination of rhBMP-2 in PLA/PGA with membranes was more efficacious than membrane placement only and resulted in bone bridging at 24 days, with a bone contour resembling the original anatomy. The combination of membrane placement with rhBMP-2 may be of value in the clinic for bone regenerative purposes. The results are also of general importance for choosing carrier materials for delivery of other growth-stimulatory substances in combination with membranes.
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PMID:Bone regeneration by a combination of osteopromotive membranes with different BMP preparations: a review. 908 67

This study was undertaken to investigate whether the choice of carrier/delivery system might be crucial for rhBMP-2 induced osteogenesis beneath osteopromotive membranes. Standardized 5-mm transosseous rat mandibular defects were implanted with recombinant human BMP-2 (rhBMP-2) with or without membrane placement. Two doses of rhBMP-2 (1 microg and 8 microg per defect) were delivered with either collagen sponge or bioabsorbable poly(D,L-lactide-coglycolide) (PLA/PGA) beads plus allogenic blood as carriers. Membrane-covered defects (no BMP) served as controls. Virtually all defects treated with rhBMP-2 without membrane placement already were bridged by new bone after 12 days, independent of rhBMP-2 dose or carrier material, and lateral bone growth was extensive outside the original defect. Membrane placement significantly decreased the stimulatory activity of the BMP, as seen after 12 days, even though osteogenesis was more advanced with rhBMP-2 and membrane compared to membrane alone. After 24 days, defects treated with membrane and rhBMP-2 in the PLA/PGA carrier were totally bridged with regenerated bone, whereas defects covered with membrane without BMP implantation displayed an average bone bridging of only 53%. In an overall analysis of the bone regeneration, the PLA/PGA carrier material was found to be superior to the collagen carrier in the presence of membranes, which was, in turn, more efficient than membrane placement alone (no rhBMP-2). There was much less lateral bone growth when BMP implantation was combined with membrane placement. It was concluded that bone formation beneath osteopromotive membranes may be significantly enhanced by rhBMP-2 and that the delivery system can affect the amount of bone formation obtained. For eventual clinical use, membrane placement has the advantage of keeping the growth-stimulatory implant in place as well as obtaining the desired anatomical contour of the bone formed.
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PMID:Importance of delivery systems for growth-stimulatory factors in combination with osteopromotive membranes. An experimental study using rhBMP-2 in rat mandibular defects. 913 67

The interactions of type VI collagen have been investigated, using solid phase binding assays, with two components of the fibrillin-containing microfibrils, the elastin-binding protein, MAGP-1 and its structural relative MAGP-2. Both native and pepsin-treated forms of type VI collagen specifically bound to MAGP-1 but not to MAGP-2. Pepsin type VI collagen was shown to block the binding of MAGP-1 to native type VI collagen indicating that the major MAGP-1-binding site was in the triple-helical region of the molecule. MAGP-1 was found not to bind to collagens I, III, and V. Affinity blotting of pepsin-treated type VI collagen showed that MAGP-1 binding was specific for the collagenous domain of the alpha3(VI) chain. Decorin and biglycan were found not to inhibit the interaction of pepsin-treated type VI collagen with MAGP-1, indicating that its binding site on the collagen is not close to that for the proteoglycans. Reduction and alkylation of disulfide bonds in MAGP-1 did not destroy its type VI collagen-binding properties, indicating that the binding site was likely to be in the cysteine-free, N-terminal domain of MAGP-1. Interestingly, the interaction of MAGP-1 with type VI collagen was inhibited by tropoelastin, suggesting that the binding sites for tropoelastin and type VI collagen may be in the same domain of MAGP-1. A peptide, corresponding to amino acids 29-38 of MAGP-1, was found to inhibit the interactions of MAGP-1 with type VI collagen and tropoelastin. The results suggest that the peptide may contain the binding sequences for both type VI collagen and tropoelastin, and thus that these two proteins may share the same binding site on MAGP-1. The interactions of MAGP-1 with type VI collagen and tropoelastin were both determined to be of moderately high affinity, with Kd values of 5.6 x 10(-7) M and 2.6 x 10(-7) M, respectively. The findings indicate that MAGP-1 may mediate a molecular interaction between type VI collagen microfibrils and fibrillin-containing microfibrils, structures which are often found in close proximity to each other in a wide range of extracellular matrices.
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PMID:Microfibril-associated glycoprotein-1 (MAGP-1) binds to the pepsin-resistant domain of the alpha3(VI) chain of type VI collagen. 927 43

The biochemical consequences of a type II procollagen mutation that contained a Gly574Ser amino acid substitution were analyzed in a transgenic mouse strain. The mutation correlated with one previously characterized in a patient with the lethal human chondrodysplasia, hypochondrogenesis (Horton et al., 1992), and resulted in a similar shortlimbed phenotype. There were fewer collagen fibrils present in the transgenic cartilage and reduced immunofluorescence of cartilage matrix using a type II collagen antibody. Pepsin-extracted collagen from transgenic mouse embryo cartilage was analyzed electrophoretically and indicated less type II as well as type XI collagen compared to their wild-type littermates. A pulse-chase experiment was performed to evaluate the biosynthesis and fate of type II collagen. Chondrocytes isolated from transgenic tissue synthesized fewer stable molecules, resulting in decreased secretion of the procollagen chains. By amino acid sequence analysis of the type II collagen peptides from cartilage of transgenic mouse embryos, serine was not detected at residue 574, the site mutated in the transgene. Based on sequence data, we believe that the molecules incorporated into collagen fibrils of the extracellular matrix, while fewer in number, were composed of normal alpha 1(II) chains.
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PMID:Type II collagen pro-alpha-chains containing a Gly574Ser mutation are not incorporated into the cartilage matrix of transgenic mice. 931 59

The collagen isotypes present at early (6 week) and late (5 month) stages of growing deer antler were isolated and identified. Pepsin-digested collagens were separated by differential salt fractionation, SDS-PAGE and Western blotting and subsequently identified by immunostaining. Cyanogen bromide digestion of antler tissue was used to establish a collagen type-specific pattern of peptides, and these were also identified by immunoblotting. Collagen type I was found to be the major collagen in both early- and late-stage antler. Collagen type II was present in the young antler in small amounts but was not confined to the soft "cartilaginous" tip of the antler. Collagen type XI was found in the pepsin digest of the young antler, but collagen type IX was not present at either stage of antler growth. Collagen type X was found in the young antler in all fractions studied. Microscopic study showed that the deer antler did not possess a discrete growth plate as found in endochondral bone growth. Unequivocal immunolocalization of the different collagen types in the antler were unsuccessful. These results show that, despite the presence in the antler of many cartilage collagens, growth does not occur through a simple endochondral process.
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PMID:Deer antler does not represent a typical endochondral growth system: immunoidentification of collagen type X but little collagen type II in growing antler tissue. 944 Feb 22

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