Insulin binds to type V collagen with retention of mitogenic activity

The abilities of eight extracellular matrix proteins, fibronectin, vitronectin, laminin, and collagen types I, II, III, IV, and V to bind insulin were examined by binding studies with insulin conjugated with peroxidase. At a physiological pH and ionic strength, type V collagen bound to insulin most strongly. The other types of collagen, laminin, and vitronectin also bound insulin with affinity lower than that of type V collagen. The insulin-binding site of type V collagen was in a 30-kDa CNBr fragment of the alpha 1 (V) chain. Analysis of the amino acid sequence showed that this 30-kDa fragment was identical to the heparin-binding fragment of type V collagen. The insulin-binding sites of laminin and vitronectin were located in the A chain and in the heparin-binding domain, respectively. Insulin bound to type V collagen stimulated the synthesis of DNA by mouse mammary tumor MTD cells, indicating that bound insulin retained mitogenic activity.     

Inhibition of cell adhesion by type V collagen.

Human umbilical vein endothelial cells grew well in dishes coated with collagen types I, II, III, or IV. However, the same cells tended to detach themselves from dishes coated with type V collagen, and cell proliferation in these dishes was inhibited. Such anti-adhesive activity was partially retained by heat-denatured type V collagen or by its alpha 1 chain, but not by its alpha 2 chain. Several other cell types did not adhere to the type V collagen substratum even in the presence of 10% serum. The cell types strongly inhibited from adhering by type V collagen included Swiss mouse 3T3 cells and their MSV-transformants, BALB/c 3T3 cells and their methylcholanthrene-transformants, NIH 3T3 cells and their ras-transformants, BHK cells, CHO-9 cells, CHO-K1 cells, and mouse melanoma B16-F10 cells. Using Swiss mouse 3T3, we studied the effects of type V collagen on cell adhesion to fibronectin in serum-free medium. When the culture dishes were coated with a mixture of fibronectin with various concentrations of type V collagen, the adhesion of the cells was inhibited depending on the concentration of type V collagen. The inhibition of cell adhesion by type V collagen was competitively overcome by increased concentrations of fibronectin. The activity that interferes with the effects of fibronectin was retained mainly by the alpha 1 chain of heat-denatured type V collagen.     

Fibronectin binding site in type I collagen regulates fibronectin fibril formation

Mov13 fibroblasts, which do not express endogenous alpha 1(I) collagen chains due to a retroviral insertion, were used to study the role of type I collagen in the process of fibronectin fibrillogenesis. While Mov13 cells produced a sparse matrix containing short fibronectin fibrils, transfection with a wild type pro alpha 1(I) collagen gene resulted in the production of an extensive matrix containing fibronectin fibrils of normal length. To study the amino acids involved in the fibronectin-collagen interaction, mutations were introduced into the known fibronectin binding region of the pro alpha 1(I) collagen gene. Substitution of Gln and Ala at positions 774 and 777 of the alpha 1(I) chain for Pro resulted in the formation of short fibronectin fibrils similar to what was observed in untransfected Mov13 cells. Type I collagen carrying these substitutions bound weakly to fibronectin- sepharose and could be eluted off with 1 M urea. The effect of this mutation on fibronectin fibrillogenesis could be rescued by adding either type I collagen or a peptide fragment (CB.7) which contained the wild type fibronectin binding region of the alpha 1(I) chain to the cell culture. These results suggest that fibronectin fibrillogenesis in tissue culture is dependent on type I collagen synthesis, and define an important role for the fibronectin binding site in this process.     

Cloning, expression, and characterization of a cDNA encoding snake venom metalloprotease

A cDNA clone, MT-c, encoding metalloprotease was isolated from snake (Agkistrodon halys brevicadus) venom gland cDNA library. Deduced amino acid sequence indicated that MT-c is composed of a signal sequence, amino-terminal propeptide, a central metalloprotease domain, and a Lys-Gly-Asp (KGD) disintegrin domain. The partial cDNA encoding metalloprotease and disintegrin domain was subcloned and expressed in E. coli. The expressed MT-c protein was purified and successfully refolded into functional form retaining the enzyme activity. Analyses of the purified recombinant protease activity revealed that the enzyme hydrolyzes extracellular matrix proteins including type I gelatin, type IV and type V collagen, while type I, II, III collagens and fibronectin were insensitive to the proteolytic digestion. The recombinant enzyme was also able to degrade fibrinogen by specifically cleaving A alpha chain of the protein.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Production of a biologically active epidermal growth factor fusion protein with high collagen affinity

Collagen is generally incapable of capturing polypeptides such as growth factors in a specific manner. In this study, we established a collagen-binding growth factor (FNCBD-EGF) consisting of epidermal growth factor (EGF) and the fibronectin collagen-binding domain. A typical yield of FNCBD-EGF was approximately 200 microg/ml culture in an Escherichia coli expression system. This fusion protein bound to gelatin and fibrillar collagen sponges, and the bound protein was not effectively eluted even with 2 M NaCl. In addition, FNCBD-EGF bound to type I, II, III, or IV collagen-coated plates, and the specificity of binding was confirmed by competitive inhibition using fibronectin. FNCBD-EGF substantially stimulated cell growth after binding to collagen-coated culture plates, whereas EGF had no effect, indicating that this fusion protein acted as a collagen-associated growth factor. In an animal model of impaired wound healing, FNCBD-EGF, but not EGF, was retained with collagen sponges at wound sites 4 d after implantation, and repair of epidermis was observed underneath the sponges. These results suggested that our fusion protein with high collagen affinity would be useful for wound healing.     

The cell adhesion domain of type XVII collagen promotes integrin-mediated cell spreading by a novel mechanism

Type XVII collagen (BP180) is a keratinocyte transmembrane protein that exists as the full-length protein in hemidesmosomes and as a 120-kDa shed ectodomain in the extracellular matrix. The largest collagenous domain of type XVII collagen, COL15, has been described previously as a cell adhesion domain (Tasanen, K., Eble, J. A., Aumailley, M., Schumann, H., Baetge, J, Tu, H., Bruckner, P., and Bruckner-Tuderman, L. (2000) J. Biol. Chem. 275, 3093-3099). In the present work, the integrin binding of triple helical, human recombinant COL15 was tested. Solid phase binding assays using recombinant integrin alpha(1)I, alpha(2)I, and alpha(10)I domains and cell spreading assays with alpha(1)beta(1)- and alpha(2)beta(1)-expressing Chinese hamster ovary cells showed that, unlike other collagens, COL15 was not recognized by the collagen receptors. Denaturation of the COL15 domain increased the spreading of human HaCaT keratinocytes, which could migrate on the denatured COL15 domain as effectively as on fibronectin. Spreading of HaCaT cells on the COL15 domain was mediated by alpha(5)beta(1) and alpha(V)beta(1) integrins, and it could be blocked by RGD peptides. The collagen alpha-chains in the COL15 domain do not contain RGD motifs but, instead, contain 12 closely related KGD motifs, four in each of the three alpha-chains. Twenty-two overlapping, synthetic peptides corresponding to the entire COL15 domain were tested; three peptides, all containing the KGD motif, inhibited the spreading of HaCaT cells on denatured COL15 domain. Furthermore, this effect was lost by mutation from D to E (KGE instead of KGD). We suggest that the COL15 domain of type XVII collagen represents a specific collagenous structure, unable to interact with the cellular receptors for other collagens. After being shed from the cell surface, it may support keratinocyte spreading and migration.     

The type XIII collagen ectodomain is a 150-nm rod and capable of binding to fibronectin, nidogen-2, perlecan, and heparin

Type XIII collagen consists of a short N-terminal intracellular domain, a transmembrane domain, and a collagenous ectodomain, and it is found at many sites of cell adhesion. We report on the characterization of recombinant type XIII collagen. The shed ectodomain was purified from insect cell culture medium and shown to form 240-kDa trimers with a T(m) of 42 degrees C. Correct chain association into a triple-helical conformation was confirmed by limited pepsin digestion and CD spectroscopy. Rotary shadowing electron microscopy of the ectodomain revealed it to be a 150-nm rod with two flexible hinges separating 31-, 52-, and 68-nm portions. The rods represent the collagenous domains 1-3, and the hinges coincide with the non-collagenous domains 2 and 3. By using surface plasmon resonance analysis, the ectodomain showed interaction with immobilized fibronectin, nidogen-2, and perlecan with K(D) values in the nanomolar range. The binding sites of type XIII collagen for fibronectin were localized to the collagenous domains, whereas the binding activities for nidogen-2 and perlecan resided in the pepsin-sensitive portions of the ectodomain. Furthermore, the ectodomain bound significantly to heparin, which also inhibited shedding of the ectodomain in insect cell cultures. The results reveal that type XIII collagen is notably distinct in its structure compared with other cell-surface proteins, and the in vitro binding with fibronectin, heparin, and two basement membrane components is indicative of multiple cell-matrix interactions in which this ubiquitously expressed protein participates.     

Cell attachment properties of collagen type VI and Arg-Gly-Asp dependent binding to its alpha 2(VI) and alpha 3(VI) chains

Twelve of sixteen different cell types including fibroblasts and tumor cells were able to attach and spread on substrates of pepsin-solubilized or intact collagen VI, and on its triple helical domain. Attachment and spreading were independent of soluble mediator proteins (fibronectin, laminin) and collagen VI was distinct from collagens I, IV and V in the cells with which it interacted. Many of the same cells bound and spread on substrates prepared from unfolded alpha 2(VI) and alpha 3(VI) chains but not on the alpha 1(VI) chain. The interactions with the chains were inhibited by low concentrations (10-100 microM) of synthetic RGDS and RGDT but not RGES peptides while the binding of cells to pepsin-solubilized collagen VI was more than 20-fold less sensitive to these peptides. The data indicate that cells have the ability to bind to collagen VI in a specific manner suggesting a similar function for collagen VI in situ.     

Fibronectin binding to complement subcomponent C1q. Localization of their respective binding sites.

The interaction of purified human plasma fibronectin with the C1q subcomponent of complement was investigated by using a solid-phase radiobinding assay. 125I-fibronectin binding to native C1q, purified collagen domain (C1q-c) or globular domain (C1q-g) was compared. When the purified domains were insolubilized by binding to plastic, the C1q-c exhibited 59% of the binding demonstrated with intact C1q, whereas the C1q-g exhibited 35% of the binding. N-Terminal sequencing of the globular domain showed that a sequence of seven collagen-like amino acids was retained on each chain of the C1q-g fragment. 125I-fibronectin binding to C1q could be inhibited equally well by fluid-phase C1q and C1q-c, but not by fluid-phase C1q-g, implying that the collagen-like region retained on the C1q-g is masked in the fluid phase. In addition, studies were performed to determine which subunit(s) of C1q bind(s) fibronectin. The percentages of fibronectin bound by the A, B, and C chain of C1q were found to be 38, 21 and 41% respectively. Inhibition studies with purified 200-180 kDa, 50 kDa or 29 kDa fragments of fibronectin show that the binding site on fibronectin for C1q is the 50 kDa gelatin-binding domain.     

Collagen synthesis by bovine aortic endothelial cells in culture.

Endothelial cells isolated from bovine aorta synthesize and secrete type III procollagen in culture. The procollagen, which represents the major collagenous protein in culture medium, was specifically precipitated by antibodies to bovine type III procollagen and was purified by diethyl-aminoethylcellulose chromatography. Unequivocal identification of the pepsin-treated collagen was made by direct comparison with type III collagen isolated by pepsin digestion of bovine skin, utilizing peptide cleavage patterns generated by vertebrate collagenase, CNBr, and mast cell protease. The type III collagen was hydroxylated to a high degree, having a hydroxyproline/proline ratio of 1.5:1.0. Pulse-chase studies indicated that the procollagen was not processed to procollagen intermediates or to collagen. Pepsin treatment of cell layers, followed by salt fractionation at acidic and neutral pH, produced several components which were sensitive to bacterial collagenase and which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with alpha A, alpha B, and type IV collagen chains purified from human placenta by similar techniques. Bovine aortic endothelial cells also secreted fibronectin and a bacterial collagenase-insensitive glycoprotein which, after reduction, had a molecular weight of 135,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (using procollagen molecular weight standards) and which was not precipitable by antibodies to cold-insoluble globulin or to alpha 2-macroglobulin. Collagen biosynthesis by these cells provides an interesting model system for studying the polarity of protein secretion and the attachment of cells to an extracellular matrix. The presence of type III collagen in the subendothelium and the specific interaction of this protein with fibronectin and platelets suggest the involvement of this collagen in thrombus formation following endothelial cell injury.     

Evidence that the collagen in the culture medium of Chinese hamster lung cells contains components related at the primary structural level to the alpha1(V) collagen chain

The collagenous protein synthesized by cultured Chinese hamster lung (CHL) cells and present in the culture medium has been isolated after limited pepsin digestion and differential salt precipitation. Molecular size analysis of this material indicates that the CHL cell medium collagen contains chains which exhibit an apparent molecular mass of approximately 85,000 Da. When chromatographed on CM-cellulose under denaturing conditions, the reduced and alkylated CHL cell medium collagen chains elute slightly after the human alpha1(I) chain but well before the pepsin-derived alpha1(V) chain, which is the constituent chain present in the CHL cell cellular matrix collagen. Analysis of the peptides derived by CNBr cleavage of the CHL medium collagen chains by chromatography on CM-cellulose reveals, however, that these chains contain peptides which correspond both in size and in chemical properties to those derived from the alpha1(V) collagen chain, but clearly lack two peptides (alpha1(V)-CB4 and alpha1(V)-CB5) which are normally present in pepsin-derived alpha1(V) chains. Furthermore, analysis of the CHL cell culture medium collagenous material obtained without pepsin digestion indicates the presence of collagenous chains that exhibit after reduction a molecular mass of approximately 160,000 Da, which is smaller than the proposed size of the pro alpha1(V) collagen chain. These results demonstrate that the collagenous protein present in the culture medium of CHL cells is directly related at the primary structural level to the alpha1(V) collagen chain, and it is postulated that this material represents the large fragment derived from a collagenase cleavage of the [pro alpha1(V)]3 molecules present in the cell layer. Furthermore, these results and previous reports indicate that the only identifiable genetic type of procollagen chain synthesized by this cloned cell line in culture corresponds to the pro alpha1(V) chain.     

Schwann cell adhesion to a novel heparan sulfate binding site in the N-terminal domain of alpha 4 type V collagen is mediated by syndecan-3.

Previously we reported that type V collagen synthesized by Schwann cells inhibits the outgrowth of axons from rat embryo dorsal root ganglion neurons but promotes Schwann cell migration (Chernousov, M. A., Stahl, R. C., and Carey, D. J. (2001) J. Neurosci. 21, 6125-6135). Analysis of Schwann cell adhesion and spreading on dishes coated with various type V collagen domains revealed that Schwann cells adhered effectively only to the non-collagenous N-terminal domain (NTD) of the alpha4(V) collagen chain. Schwann cell adhesion to alpha4(V)-NTD induced actin cytoskeleton assembly, tyrosine phosphorylation, and activation of the Erk1/Erk2 protein kinases. Adhesion to alpha4(V)-NTD is cell type-specific because rat fibroblasts failed to adhere to dishes coated with this polypeptide. Schwann cell adhesion and spreading on alpha4(V)-NTD was strongly inhibited by soluble heparin (IC(50) approximately 30 ng/ml) but not by chondroitin sulfate. Analysis of the heparin binding activities of a series of recombinant alpha4(V)-NTD fragments and deletion mutants identified a highly basic region (not present in other type V collagen NTD) as the site responsible for high affinity heparin binding. Schwann cells adhered poorly to dishes coated with alpha4(V)-NTD that lacked the heparin binding site and failed to spread or assemble organized actin-cytoskeletal structures. Soluble alpha4(V)-NTD polypeptide that contained the heparin binding site inhibited spreading of Schwann cells on dishes coated with alpha4(V)-NTD. Affinity chromatography of Schwann cell detergent extracts on a column of immobilized alpha4(V)-NTD resulted in the isolation of syndecan-3, a transmembrane heparan sulfate proteoglycan. Together, these results suggest that Schwann cells bind to collagen type V via syndecan-3-dependent binding to a novel high affinity heparin binding site in the alpha4(V)-NTD.     

Participation of C1q and its receptor in adherence of human diploid fibroblast

C1q binds through its collagen-like domain to specific surface receptors of fibroblasts and to adhesive elements of extracellular matrix including fibronectin, collagens, proteoglycans, and laminin. To determine whether C1q participates in fibroblast adhesion, cells in serum-free medium were plated on surfaces coated with purified C1q at physiologic ionic strength and pH. Surfaces coated with fibronectin or collagen type I served as positive controls, and those coated with BSA were negative controls. Substratum-adsorbed C1q promoted fibroblast adhesion to a maximum of 73% of available cells within 90 min at 37 degrees C. Adhesion was C1q concentration dependent, saturable, specific, and dependent on the collagen-like domain of the molecule. De novo protein synthesis plays a role in adhesion: pretreatment of fibroblasts with cycloheximide reduced adherence about 50% of controls. Addition of exogenous fibronectin, collagen type I, or C1q as soluble mediators did not affect adhesion of the cycloheximide-treated cells to C1q substrate. Adhesion could be accounted for primarily, although not completely, by the C1q receptors. Antibodies raised against the Raji cell C1q receptors (alpha C1qR Ab) specifically inhibited fibroblast adhesion to C1q substrates about 60% of controls. The binding of fibroblasts to C1q substrates could be inhibited about 24% of controls with the GRGDTP cell recognition peptide. GRGDTP and alpha C1q Ab had an additive effect on adhesion that was inhibited 77 to 80% of controls. We conclude from these data that aggregated rather than monomeric C1q may be the natural ligand of the fibroblast C1q receptor, and the biologic function of the receptor in cells of the connective tissue may be cell adhesion.     

Morphine inhibits indolactam V-induced U937 cell adhesion and gelatinase secretion

We demonstrate that indolactam V, a non-phorbol protein kinase C activator, promotes U937 cell attachment to fibronectin, type IV collagen and laminin. In the absence of indolactam V, 2-4% of U937 cells attach to all test substrates, however, in the presence of 100 nM indolactam V, 25, 16 and 11% of U937 cells attach to fibronectin, type IV collagen and laminin, respectively. When added concomitantly, 90 microM H-7, a protein kinase C inhibitor, reduces indolactam V-induced U937 cell adhesion to fibronectin by 91%. Monoclonal antibodies directed against both the beta1 and alpha 5 integrin subunits inhibit indolactam V-induced U937 cell adhesion to fibronectin by 62 and 52%, respectively. Indolactam V also promotes homotypic aggregation in U937 cells, which is blocked with either anti-ICAM or anti-LFA-1 antibodies. In addition, indolactam V promotes U937 cell secretion of a 92 kDa gelatinase as demonstrated by zymography. In the presence of low levels of morphine (10 nM-1.0 microM), the U937 cell attachment to matrix proteins was not significantly affected. However, in the presence of 10 microM morphine, the indolactam V treated cells exhibit a 71-74% reduction in cell adhesion to the matrix proteins. Further, 10 microM morphine also blocks indolactam V-induced homotypic aggregation and gelatinase secretion. The inhibitory effect of morphine on cell-matrix adhesion and gelatinase secretion was not inhibited by the opiate receptor antagonist naloxone (1 microM). While 10 microM naloxone did partially counteract the effect of 10 microM morphine on U937 cell attachment, this effect was likely non-specific since 10 microM naloxone alone increased cell adhesion. Supporting this conclusion, PCR analysis revealed that U937 cells do not express the mu high affinity morphine receptor. Also, indolactam V did not induce mu receptor expression, suggesting that morphine acts on U937 cells in a non-specific fashion.     

Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v

We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.     

Structural and functional features of the alpha 3 chain indicate a bridging role for chicken collagen VI in connective tissues

Type VI collagen is a component of 100 nm long periodic filaments with a widespread distribution around collagen fibers and on the surface of cells. It is an unusual collagen constituted by three distinct chains, one of which (alpha 3) is much larger than the others and is encoded by a 9-kb mRNA. The amino acid sequence of the alpha 3(VI) deduced from the present cDNA clones specifies for a multidomain protein of at least 2648 residues made of a short collagenous sequence (336 residues), flanked at the N-terminus by nine 200 residue long repeating motifs and at the C-terminus by two similar motifs that share extensive identities with the collagen-binding type A repeats of von Willebrand factor. Type VI collagen and alpha 3(VI) fusion proteins bound to insolubilized type I collagen in a specific, time-dependent, and saturable manner. The alpha 3(VI) chain has three Arg-Gly-Asp sequences in the collagenous domain, and cell attachment was stimulated by the triple helix of type VI collagen and by alpha 3(VI) fusion proteins containing Arg-Gly-Asp sequences. This function was specifically inhibited by the Arg-Gly-Asp-Ser synthetic peptide. The type I collagen-binding and the cell-attachment properties of the alpha 3(VI) chain provide direct information for the role of type VI collagen in connective tissues.     

Isolation and purification of biopolymers using an affinity chromatography method. X. A biospecific approach to preparing collagen-like peptides containing segments binding fibronectin

A biospecific method for one-stage isolation of collagen peptides containing the fibronectin binding site is proposed. alpha 1CB7-peptide of the type I collagen cyanogen bromide cleavage was isolated by means of affinity chromatography on adsorbents containing an immobilized gelatin-binding domain (45 kDa) of fibronectin. The method allows one to obtain, in a short time, highly purified preparation of alpha 1CB7-peptide necessary for biochemical studies.     

Type V collagen selectively inhibits human endothelial cell proliferation

Type V collagen from human placenta remarkably inhibited human umbilical vein endothelial cell (HUVEC) proliferation in a dose-dependent manner when coated on the culture dishes. Other types of collagen (I, III, IV) and fibronectin enhanced HUVEC proliferation under the same conditions. The inhibitory activity of type V collagen was seen not only when it was coated on the dishes, but also when it was directly added into cell culture. The attachment effect of type V collagen did not differ from that of type I collagen. The inhibitory activity is a phenomenon selective for endothelial cells, since type V collagen did not affect the proliferation of human umbilical vein smooth muscle cells, aortic smooth muscle cells, or nasal mucosa fibroblasts.