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PubMed2000

Type V collagen modulates alloantigen-induced pathology and immunology in the lung.

Perivascular and peribronchiolar tissues are targets of the immune response during lung allograft rejection. Collagen type V (col[V]) is located within these tissues. Col(V) may be major histocompatibility complex (MHC)-like, and MHC-derived peptides have been used to induce immunologic tolerance and prevent rejection in allografts other than the lung. The current study tests the hypothesis that col(V) could be used to downregulate immune responses to lung alloantigen in vivo. We developed a murine model in which instillations of allogeneic bronchoalveolar lavage (BAL) cells (C57BL/6, I-a(b), H-2(b)) into lungs of BALB/c mice (I-a(d), H-2(d)) induce histology similar to grades 1 and 2 acute lung allograft rejection, apoptosis of airway epithelium and vascular endothelium, and upregulate tumor necrosis factor (TNF)-alpha production locally. The current study reports that instillations of col(V) into lungs before allogeneic BAL cells prevent development of rejection pathology and apoptosis, downregulate alloantigen-induced T-lymphocyte proliferation, and abrogate local TNF-alpha production. In addition, instillation of col(V)-pulsed autologous BAL cells into lungs of mice primed with allogeneic BAL cells perpetuates rejection pathology. Collectively, these data show that col(V) is a novel antigen involved in the rejection process, and suggest that col(V) could be used to modulate the rejection response to lung allografts.

Animals↗

PubMed1988

Changes in type V collagen in the kidneys of DOCA-NaCl treated rats.

Type V collagen was extracted and quantitated in the kidneys of unilaterally nephrectomized rats exposed to desoxycorticosteron acetate (DOCA-NaCl) treatment. A highly elevated content of collagen type V was found in these kidneys compared with controls. A higher content of collagen type V was present both in the pepsin digest of the total kidney and in the isolated glomerular basement membranes. It was previously reported that one of the factors involved in the growth of the remaining kidney in unilaterally nephrectomized DOCA-NaCl treated rats is the increase of collagen type IV, the effect being more prominent in rats nephrectomized at a younger age. The present study documents that the increase of type V collagen follows the same pattern.

Animals↗

PubMed1989

Type V collagen synthesis and deposition by chicken embryo corneal fibroblasts in vitro.

Chick embryo corneal fibroblasts were grown in culture to study the processes whereby fibroblasts regulate the deposition and organization of the collagenous, secondary stroma. The effects of an existing type I collagen substratum, cell density, and serum concentration on type V collagen synthesis were investigated. Type V collagen represented approximately 20% of the total fibrillar collagen synthesized, regardless of whether the cells were subcultured, grown on untreated or collagen-coated plastic, grown under confluent or subconfluent conditions, or grown in the presence of low (0.1%) or high (10.0%) serum concentrations. The synthesis of type V collagen remained constant at 20% of the total collagen when cells were grown in 1.0% serum, even though total collagen synthesis increased nearly twofold when compared to total synthesis in 0.1% or 10.0% serum. Immunocytochemistry with anti-collagen, type-specific monoclonal antibodies revealed a homogeneous population of cells synthesizing types I and V collagen. The fibrils deposited by cells grown in a three-dimensional collagen matrix contained a helical epitope on the type V molecule that was inaccessible unless the fibrillar structure was disrupted, mimicking the situation in situ. The production in vitro of heterotypic fibrils, with a constant I/V ratio and molecular packing mimicking the natural stroma, offers opportunities for studying in more detail this important process, which is essential for optical transparency.

Animals↗

PubMed1990

Primary structure of the heparin-binding site of type V collagen.

The abilities of collagens, type I, II, III, IV, and V, to bind heparin were examined by heparin-affinity chromatography and binding studies with [35S]heparin. At a physiological pH and ionic strength, only type V collagen bound to heparin. Collagens type I and II showed higher affinities than types III and IV for heparin, but did not bind to a heparin column at a physiological ionic strength. The heparin binding site of type V collagen was located in a 30 kDa CNBr fragment of the alpha 1(V) chain, and the amino acid sequence of this fragment was determined. The 30 kDa fragment contained a cluster of basic amino acid residues, and enzymatic cleavage within this basic domain greatly reduced the heparin-binding activities of the resulting peptides. Thus this basic region is probably the heparin-binding site of type V collagen.

Amino Acid Sequence↗

PubMed1994

Structural characteristics of cross-linking sites in type V collagen of bone. Chain specificities and heterotypic links to type I collagen.

To understand the role of type V collagen and its spatial interrelationship with type I collagen in bone matrix, the molecule's covalent intermolecular cross-links were structurally characterized. Type V collagen containing alpha 1(V), alpha 2(V) and alpha 1(XI) chains was isolated from bovine bone and reacted with NaB3H4 to label the cross-linking residues. Radiolabeled native molecules and isolated alpha chains were treated with sodium metaperiodate to cleave the divalent cross-linking bonds. Sequence analysis of the periodate-released peptides matched two of them to alpha 1(V) and alpha 1(XI) aminopropeptide domains. A third peptide was derived from the alpha 1(I) carboxytelopeptide domain of type I collagen. This latter peptide, therefore, came from a site of heterotypic cross-linking between types I and V collagens and accounted for about 15% of the total cross-linked peptides. Sequence analysis of isolated cross-linked tryptic peptides defined the helical sites of attachment of the periodate-released telopeptides and revealed that the putative aminoproteinase-cleavage sites in the alpha 1(V) and alpha 1(XI) chains are located in the molecule interior to the cross-linking residue. These data imply that type V collagen molecules in the extracellular matrix are primarily cross-linked to each other in a head-to-tail linear polymer that is linked laterally to type I collagen molecules in copolymeric fibrils.

Amino Acid Sequence↗

PubMed1989

Binding of heparan sulfate to type V collagen. A mechanism of cell-substrate adhesion.

The functions and molecular interactions of type V collagen in the pericellular matrix are unclear. Our studies show that type V collagen adsorbed on a surface binds heparin/heparan sulfate with apparent higher affinity than do collagen types I, II, III, IV, or VI, fibronectin, or laminin. Therefore, heparin-like molecules may mediate interactions between cells and type V collagen. Hence, type V collagen may act as an anchor for proteoglycans in the extracellular matrix and function as a substrate for glycosaminoglycan-mediated cell attachment. This model is supported by studies showing that Chinese hamster ovary (CHO) cell mutants which are deficient in glycosaminoglycan synthesis attach poorly to type V collagen substrates compared to wild-type cells, whereas attachment of CHO cell mutants to fibronectin substrates is not affected. Also, exogenous heparin reduces attachment of CHO, endothelial, and smooth muscle cells to type V collagen but does not affect cell attachment to fibronectin. The inhibitory activity of the exogenous heparin/heparan sulfate depends on the size and sulfate content of the polysaccharide chains. At tested concentrations, chondroitin sulfate does not affect the attachment of CHO cells or the binding of biotin-conjugated heparan sulfate to wells coated with type V collagen. These data suggest that a certain degree of structural specificity is involved in glycosaminoglycan binding to type V collagen.

Amino Acid Sequence↗

PubMed1996

Separation of the subtypes of type V collagen molecules, [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), by chain composition-dependent affinity for heparin: single alpha 1(V) chain shows intermediate heparin affinity between those of the type V collagen subtypes composed of [alpha 1(V)]2 alpha 2(V) and of alpha 1(V) alpha 2(V) alpha 3(V).

The heparin affinities of heat-treated type V collagen alpha-chains and the triple-helical molecules were evaluated in terms of the NaCl concentration required for prevention of binding to a heparin-Sepharose column. After heat treatment, alpha 1(V) chain required approximately two-fold higher NaCl concentration to pass through the column than the other two chains, alpha 2(V) and alpha 3(V). Thus, the heparin affinity of alpha 1(V) may be approximately two-fold higher than those of the other alpha (V)-chains. The type V collagen molecules in triple-helical conformation were separated into two fractions at 170 mM NaCl in 20 mM phosphate buffer, pH 7.2, containing 2 M urea; bound and non-bound. The ratio of the three alpha-chains, alpha 1(V): alpha 2(V): alpha 3(V) was 2 : 1 : 0 and 1 : 1 : 1 in the bound and flow-through fractions, respectively, on analysis by SDS-PAGE. The differential affinity of the two fractions could be accounted for by the number of alpha 1(V) chains in the triple-helical molecule, if these fractions contained triple-helical subtypes with the chain compositions of [alpha 1(V)]2 alpha 2(V) and alpha 1(V) alpha 2(V) alpha 3(V), respectively. From the comparison of the NaCl concentration required for prevention of the binding, [alpha 1(V)]2, alpha 2(V) had about two-fold higher affinity than alpha 1(V) alpha 2(V) alpha 3(V), and the separated alpha 1(V) chain showed an intermediate affinity. A possible explanation for difference in heparin affinity among the subtypes of molecules and the separated alpha-chains is that the heparin affinity of type V collagen molecule is governed by the number of alpha 1(V) chains contained in the molecule and that steric restraint in a triple-helical conformation weakens the binding of alpha 1(V) chain to heparin.

Animals↗

PubMed1992

Biological activity of a proteoglycan form of macrophage colony-stimulating factor and its binding to type V collagen.

Two different types of macrophage colony-stimulating factors (M-CSF) were found, one with an apparent molecular mass of 85 kDa and the other greater than 200 kDa. The high molecular mass M-CSF was identified as a proteoglycan carrying chondroitin sulfate glycosaminoglycan and was designated as the proteoglycan form of M-CSF (PG-M-CSF). In this study, we compared the biological activity of the 85-kDa M-CSF and PG-M-CSF and examined the binding properties of these two M-CSF to certain extracellular matrix proteins, i.e. types I-V collagen and fibronectin, using a modified enzyme-linked immunosorbent assay. PG-M-CSF was capable of supporting the formation of murine macrophage colonies, and pretreatment of PG-M-CSF with chondroitinase AC, which degrades chondroitin sulfate, did not alter its colony-stimulating activity. The specific activity of PG-M-CSF was similar to that of the 85-kDa M-CSF. The 85-kDa M-CSF had no apparent affinity for the extracellular matrix proteins examined, whereas PG-M-CSF had an appreciable binding capacity to type V collagen, but did not bind to types I, II, III, and IV collagen or to fibronectin. Pretreatment of PG-M-CSF with chondroitinase AC completely abolished the binding of the species to type V collagen. Addition of exogenous chondroitin sulfate inhibited the binding of PG-M-CSF to type V collagen in a dose-dependent manner. These data indicated that the interaction between PG-M-CSF and type V collagen was mediated by the chondroitin sulfate chain of PG-M-CSF. PG-M-CSF bound to type V collagen could stimulate the proliferation of bone marrow macrophages, indicating that the matrix protein-bound PG-M-CSF retained its biological activity. This interaction between PG-M-CSF and type V collagen implies that the role of PG-M-CSF may be distinct from that of 85-kDa M-CSF.

Animals↗

PubMed1992

Disassembly of F-actin filaments in human endothelial cells cultured on type V collagen.

We examined the inhibitory activity of type V collagen on cell attachment and cell growth and the role of stress fibers and beta 1 integrin in cultured human endothelial cells. Human endothelial cells cultured on type V collagen attached temporarily to the substrate and formed stress fibers. However, the cells failed to proliferate and gradually detached from the substrate. After 24 h, the cells on type V collagen lacked discernible stress fibers (F-actin filaments) and exhibited dots in small aggregates of F-actin. In addition, the cells expressed little or no proteins as focal adhesions, including vinculin and beta 1 integrin. In contrast, the cells on fibronectin and type I collagen formed complete F-actin filaments, exhibited sufficient vinculin and beta 1 integrin, and grew logarithmically from 2 days. On the other hand, human smooth muscle cells formed complete F-actin filaments, revealed typical focal adhesions, and started to proliferate rapidly after 24 h on type V collagen as well as on fibronectin and type I collagen. Thus, the disassembly of F-actin filaments was observed as a specific phenomenon in human endothelial cells cultured on type V collagen. Moreover, the F-actin filaments disappeared from endothelial cells treated with cytochalasin D after 24 h and the cells detached from fibronectin and type I collagen with time, a result consistent with the observations on type V collagen. Accordingly, the disassembly of F-actin filaments in focal adhesions may result in the detachment of endothelial cells from type V collagen.

Actins↗

PubMed1987

Localization of the alpha 3 (V) chain of type V collagen in human skin.

Serum from goats immunized with human type V collagen chains that were cut out of polyacrylamide gels contained an antibody that recognized only type V collagen in an enzyme-linked immunoabsorbent assay and did not label laminin, fibronectin, or types I and IV collagen. Western blot analysis of the antibody showed that its determinant was the alpha 3 (V) chain of type V collagen. Indirect immunofluorescent staining of intact human skin with the antibody produced staining of the dermal blood vessels but not of the dermal-epidermal junction (DEJ). In contrast, both the dermal blood vessels and the DEJ were labeled by the antibody if the skin substrate was first split through the lamina lucida region of the DEJ by incubation in 1 M NaCl solution. Indirect immunoelectron microscopy confirmed the staining pattern found by immunofluorescence and defined the ultrastructural localization of type V collagen in skin. Type V collagen is localized within the DEJ to the lamina lucida region and polar aspects of the basal cell keratinocyte plasma membrane.

Collagen↗

PubMed1991

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.

Amino Acid Sequence↗

PubMed1981

Type V collagen from the chick embryo: biochemical, physicochemical and ultrastructural characteristics.

Two collagen alpha chains have been isolated from whole 17 day chick embryos which are similar to the B chain or alpha 1(V) and the A chain or alpha 2(V) recently described in mammalian and avian tissues. A non-collagenous acidic protein was co-purified with the Type V collagen which was resistant to pepsin digestion. The molecular weight, circular dichroism spectrum, melting temperature and diffusion coefficient of the native Type V collagen and isolated alpha chains were similar to values obtained for other chick collagen types. The SLS crystallite of Type V collagen had a distinct pattern of banding as identified by electron microscopy. We consistently observed more alpha 2(V) than alpha 1(V) following both CM-cellulose and QAE-Sephadex ion exchange chromatography of denatured Type V collagen, but unusual solubility properties and recoveries of the alpha 1(V) chain may have diminished its relative amount. In addition we have found that the alpha 1(V) chains are chemically heterogeneous and one component electrophoreses as an alpha 2(V) chain on SDS gels.

Animals↗

PubMed1993

A single chain 19-kDa fragment from bovine thrombospondin binds to type V collagen and heparin.

Location of the type V collagen-binding domain within bovine thrombospondin (TSP) was investigated by using fragments of reduced and alkylated TSP. A fragment of relative molecular mass (19 kDa) was isolated, which inhibited binding of 125I-TSP to type V collagen in a solid-phase binding assay. A direct binding assay using the 125I-labeled fragment confirmed that the fragment actually bound to collagen. The fragment retained specificity for the native structure of type V collagen like the intact TSP molecule. Its binding to the collagen, however, was not inhibited by Ca2+ in contrast to intact TSP. Amino acid sequence analysis of the 19-kDa fragment suggested that this fragment corresponded to Val333-Lys412, a part of the stalklike region in the human TSP primary structure. It was found that the fragment also bound well to heparin in a specific and saturable manner and, furthermore, binding to type V collagen was inhibited by soluble heparin. Removal of the N-linked sugar chain from this fragment resulted in a 14-kDa fragment. The deglycosylated fragment retained the ability to bind to type V collagen as well as heparin. These results suggest that a type V collagen-binding site is present in the 80-residue portion of bovine TSP (Val333-Lys412) and is likely to be identical or lie very close to a heparin-binding site, which exists in the type I repeat structure.

Amino Acid Sequence↗

PubMed2001

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.

Amino Acid Sequence↗

PubMed1988

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.

Cell Adhesion↗

PubMed1984

Type V collagen in human amnion is a 12 nm fibrillar component of the pericellular interstitium.

Immunoelectron microscopy was utilized to detect type V collagen in human amnion. Monospecific antibodies to type V collagen were detected with protein A-gold conjugates in tissue sections and epoxy-embedded sections of human amnion. Type V collagen was localized to the immediate vicinity of the basal lamina, but was distinct from laminin and type IV collagen, which localized only to the lamina lucida and lamina densa, respectively, of the basal lamina. At high magnification, 12 nm unbanded fibrils were seen to be labelled by anti-type V collagen antibody; these fibrils extended from the lamina densa of the basal lamina well into the interstitial matrix. In comparison, only the amorphous matrix of the lamina densa showed labelling with anti-type IV collagen antibodies. Anti-laminin antibodies labelled the lamina lucida. Quantitative analysis of grain distribution revealed the laminin labelling to be centered over the distal half of the lamina lucida (mean distance from the cell surface = 70 nm). In contrast, type IV collagen was centered over the lamina densa (mean = 115 nm). Both distributions were essentially Gaussian and distinct from the broad distribution of type V collagen. Type I collagen fibers with characteristic 67 nm periodicity were unlabelled with antibodies to type V collagen, although labelled type V fibrils were frequently enmeshed among the type I fibers. Antibodies to type I collagen labelled these fibers but not the type V fibrils. The results indicate that in human amnion, type V collagen is a 12 nm diameter, unbanded fibril which extends from the lamina densa of the basal lamina into the adjacent interstitial matrix. We hypothesize that type V collagen functions as a network of anchoring fibrils between the cell basal lamina and the extracellular matrix, especially type I collagen fibres. Type V collagen thus appears to be a unique interstitial collagen.

Amnion↗

PubMed1983

Biosynthesis and regulation of type V collagen in diploid human fibroblasts.

The biosynthesis of type V collagen and its regulation were studied using diploid human gingival fibroblasts. Cells were metabolically labeled with radioactive amino acids and labeled proteins were subjected to limited pepsin digestion, DEAE-cellulose chromatography at 15 degrees C, and polyacrylamide gel electrophoresis. Proteins eluted from DEAE-cellulose columns by 0.25 M NaCl contained a collagen species which was resistant to mammalian collagenase and had alpha chains with hydroxylysine/lysine ratios and CNBr peptide patterns similar to alpha 1(V) and alpha 2(V). Procollagen(V) fractions obtained by DEAE-cellulose chromatography and immunoprecipitates of type V collagen antibody contained polypeptides with Mr = 239,000, 219,000, 198,000, 174,000, 157,000, and 132,000. By comparing the CNBr peptide maps of these proteins with those of standard alpha 1(V) and alpha 2(V) chains, the first three polypeptides were shown to be related to alpha 1(V) and the others to alpha 2(V). It was concluded that the gingival fibroblasts synthesize type V collagen, that the pro alpha 1(V) and the pro alpha 2(V) chains have Mr = 239,000 and 174,000, respectively, and that the alpha 1(V) and alpha 2(V) chains laid in the form of fibrils have Mr = 198,000 and 132,000, respectively. A detectable amount of type V collagen was synthesized only at high cell density, and it was associated with the cell layer. The amount and proportion of type V synthesized were increased when the cells were labeled in the presence of serum, and the increase was accompanied by a decrease in type III. This effect was dependent on serum concentration. Serum obtained from platelet-poor plasma failed to elicit this effect, and it was restored by the addition of platelet-derived growth factor. Platelet-derived growth factor was effective in medium with and without platelet-poor serum. Thus, it appears that platelet-derived growth factor may be an important regulatory factor in the synthesis of types V and III collagens.

Amino Acids↗

PubMed1982

Increased content of Type V Collagen in desmoplasia of human breast carcinoma.

Type V (AB) collagen is present in increased amounts in desmoplasia of human breast carcinoma. Type V collagen occurs as linear deposits in the interstitium (desmoplastic stroma surrounding the tumor), as demonstrated by immunofluorescence and immunoperoxidase techniques utilizing antibodies to Type V collagen. In contrast, no demonstratable Type V occurs in the interstitium of fibroadenoma, fibrocystic disease, or normal breast tissue. As identified and quantitated by pepsin extraction, salt fractionation, and polyacrylamide gel electrophoresis, Type V constitutes 10% +/- 5% of all interstitial collagens (Types I, III, and V) in desmoplasia, compared with 1.5% +/- 0.5% in fibroadenoma and less than 0.1% in all types of fibrocystic disease, including sclerosing adenosis, and less than 0.1% in normal breast tissue. For infiltrating ductal carcinoma, Type V collagen is not secreted by the invasive carcinoma cells, nor is it present in an extracellular basement membrane location. By immunohistologic methods, Type V collagen can be seen exclusively in the desmoplastic stroma. The authors propose that desmoplasia of human breast carcinoma manifests a characteristic collagen profile and that the increased Type V may be produced by specialized cells such as myofibroblasts in the interstitium, which are recruited in response to invasive carcinoma.

Adenocarcinoma, Scirrhous↗