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PubMed2000

Type V collagen is increased during rabbit medial collateral ligament healing.

To understand the reparative process of medial collateral ligament (MCL), fibrillar collagen and their relative ratios in healing MCL with anterior cruciate ligament (ACL) reconstruction were analyzed. Skeletally mature New Zealand white rabbits were subjected to a mop-end tear of MCL without repair with ACL reconstruction. Rabbits were killed 6 and 52 weeks after injury. Ligamentous tissues from the injury site and sham controls were soaked in 0.5 M acetic acid for 24 h, minced, and treated with pepsin to solubilize collagen. Pepsin solubilized about 80% of the total collagen as determined by hydroxyproline analysis of the pepsin residues. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the solubilized collagen revealed presence of fibrillar collagen types I, III, and V. Densitometric scanning of the protein bands corresponding to types I, III, and V collagen indicated that in sham controls types III and V collagen represented about 8% and 12%, respectively, of the type I collagen whereas the healed MCL ligaments at 6 weeks showed significant increase in type III and V collagen to about 19% and 24%, respectively. By 52 weeks type III collagen in the healed MCL had returned to that of sham controls while type V collagen remained elevated at approximately 18%. These data suggest that presence of type V collagen in high concentration in healing ligaments may have an influence on collagen fibril diameters seen in healed ligament and should be included in the analysis when evaluating ligament healing.

Animals↗
PubMed1995

Improved chromatographic purification of human and bovine type V collagen sub-molecular species and their subunit chains from conventional crude preparations. Application to cell-substratum adhesion assay for human umbilical vein endothelial cell.

Two human type V collagen sub-molecular species, designated [alpha 1(V)]2 alpha 2(V) and alpha 1(V)alpha 2(V)alpha 3(V), were purified chromatographically from a commercially available preparation, in which cystine-rich collagenous contaminants were contained, with a column packed with Fractogel EMD SO3-. From bovine crude preparations, the [alpha 1(V)]2 alpha 2(V) form free from the collagenous contaminants was purified. Type V collagen subunit chains were isolated from each type V collagen molecule by anion-exchange HPLC with a Bakerbond PEI Scout column. The highly purified human type V collagen molecules and their subunit chains were used to examine the inhibitory effect on human umbilical vein endothelial cell proliferation. It was confirmed that the alpha 1(V) chain has inhibitory activity and it was found that the inhibitory effect of the [alpha 1(V)]2 alpha 2(V) form is stronger than that of the alpha 1(V)alpha 2(V)alpha 3(V) form and that the alpha 3(V) chain has no inhibitory activity.

Animals↗
PubMed1996

Reduction of type V collagen using a dominant-negative strategy alters the regulation of fibrillogenesis and results in the loss of corneal-specific fibril morphology.

A number of factors have been implicated in the regulation of tissue-specific collagen fibril diameter. Previous data suggest that assembly of heterotypic fibrils composed of two different fibrillar collagens represents a general mechanism regulating fibril diameter. Specifically, we hypothesize that type V collagen is required for the assembly of the small diameter fibrils observed in the cornea. To test this, we used a dominant-negative retroviral strategy to decrease the levels of type V collagen secreted by chicken corneal fibroblasts. The chicken alpha 1(V) collagen gene was cloned, and retroviral vectors that expressed a polycistronic mRNA encoding a truncated alpha 1(V) minigene and the reporter gene LacZ were constructed. The efficiency of viral infection was 30-40%, as determined by assaying beta-galactosidase activity. To assess the expression from the recombinant provirus, Northern analysis was performed and indicated that infected fibroblasts expressed high steady-state levels of retroviral mRNA. Infected cells synthesized the truncated alpha 1(V) protein, and this was detectable only intracellularly, in a distribution that colocalized with lysosomes. To assess endogenous alpha 1(V) protein levels, infected cell cultures were assayed, and these consistently demonstrated reductions relative to control virus-infected or uninfected cultures. Analyses of corneal fibril morphology demonstrated that the reduction in type V collagen resulted in the assembly of large-diameter fibrils with a broad size distribution, characteristics similar to fibrils produced in connective tissues with low type V concentrations. Immunoelectron microscopy demonstrated the amino-terminal domain of type V collagen was associated with the small-diameter fibrils, but not the large fibrils. These data indicate that type V collagen levels regulate corneal fibril diameter and that the reduction of type V collagen is sufficient to alter fibril assembly so that abnormally large-diameter fibrils are deposited into the matrix.

Animals↗
PubMed1991

Distribution of collagens type V and VI in the normal human alveolar mucosa: an immunoelectronmicroscopic study using ultrathin frozen sections.

The ultrastructural localization of collagens type V and VI in normal human gingival mucosa was investigated by immunoelectron microscopy. Twenty biopsies were fixed in dimethylsuberimidate and shock-frozen in slush nitrogen. Collagen type V was mainly located to meshworks of uniform nonstriated microfibrils of 12 to 20 nm width, which preferentially appeared in larger spaces between cross-striated major collagen fibrils. Occasionally single microfibrils of collagen type V fanned out from the ends of major collagen fibrils, which may indicate a role as a core fibril. Collagen type V was not found in the subepithelial basement membrane and the immediately adjacent stroma. Collagen type VI was detected in a loose reticular network of unbanded microfilaments that were morphologically distinguishable by knoblike protrusions every 100-110 nm. These microfilaments were found in the vicinity, but not as an intrinsic component, of the subepithelial basement membrane. Single filaments of collagen type VI filaments appeared to form bridges between neighboring cross-striated major collagen fibrils, suggesting an interconnecting role for this collagen type. The method presented appears to be excellently suited to study the normal and pathological supramolecular organization of the oral extracellular matrix.

Adolescent↗
PubMed1991

Monoclonal antibodies to type V collagen for immunohistological examination of new tissue deposition associated with biomaterial implants.

We developed a panel of highly specific monoclonal antibodies (MAb) against dog Type V collagen. Each antibody showed differential reactivities towards Type V collagen from other species. All the antibodies were highly reactive in conventional ELISA, as well as with electroblots of collagen after polyacrylamide gel electrophoresis using non-denaturing conditions. The MAb were shown to be suitable for the immunohistological detection of Type V collagen in tissue sections, although this normally required pre-treatment of sections with 50 mM acetic acid. In particular, the antibodies were shown to be useful for examining samples of a collagen-based biomaterial, a vascular prosthesis, after explant from evaluation in an animal model. This showed that Type V collagen was most prominent in regions of new tissue formation within the neointima, close to the inner surface of the prosthesis. The broad spectrum of differential reactivities allows the antibodies to be used for a wide range of experimental models. These MAb therefore provide a novel approach for the evaluation of biomaterial performance, particularly for collagen-based implants.

Animals↗
PubMed1993

Biochemical remodeling of collagen in the heart of spontaneously hypertensive rats--prominent increase in type V collagen.

To determine how collagen is remodeled in the heart during the development of cardiac hypertrophy, the collagen concentration and the proportions of types I, III, and V collagen were analyzed in the hearts of 10-, 20-, 32-, and 40-week-old spontaneously hypertensive (SHR) and Wistar-Kyoto (WKY) rats. The collagen concentration was calculated after the hydroxyproline content was measured and the proportions of types I, III, and V collagen were determined by non-interrupted SDS-polyacrylamide gel electrophoresis (SDS-PAGE). There was no significant difference between the collagen concentrations in 10-week-old SHR and WKY. At 20 weeks, the collagen concentration in the hearts of the SHR had decreased significantly (p < 0.01) when compared to that in the WKY, at 40 weeks, this concentration in SHR had increased significantly (p < 0.05) when compared to that in the WKY. Type V collagen in WKY increased with age and type I collagen in the 20- and 40-week-old WKY decreased significantly (p < 0.05) when compared to that in the 10-week-old WKY. However, the proportion of type I collagen in the 20-week-old WKY did not differ from that of the 40-week-old WKY. There was no significant difference between the proportions of the various types of collagen in 10-week-old SHR and those in age- and sex-matched WKY. However, the proportion of type V collagen in the 20- and 40-week-old SHR was significantly (p < 0.01) higher than that in age- and sex-matched WKY.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Immunofluorescent localization of type-V collagen as a fibrillar component of the interstitial connective tissue of human oral mucosa, artery and liver.

Polyclonal antibodies against native human type-V collagen were produced in rabbits and goats. Following purification, crossreaction of the antibodies with highly immunogenic peptides of basement membranes or the interstitial matrix was excluded on the basis of sensitive radioimmunoassays. These antibodies, when applied to cryostat sections of human oral mucosa, liver and arterial walls, never stained basement membranes as did antibodies against type-IV collagen or laminin. On the contrary, we observed delicate arborizing fibers in the interstitial compartment with extensions contacting structures such as subepidermal basement membranes. Arterioles contained a unilamellar sheath of longitudinally oriented fibers limited to the intimal layer. Larger arteries exhibited a multilamellar fibrous fluorescence over the whole intima, whereas the media showed a much weaker staining. The data identified type-V collagen as an interstitial fibrillar collagen rather than a basement membrane collagen, with a tissue pattern completely different from that of collagens types I, III, VI or fibronectin. A reinterpretation of the role of type-V collagen in connective tissue function is warranted.

Antibody Specificity↗
PubMed2006

Collagen type V enhances matrix contraction by human periodontal ligament fibroblasts seeded in three-dimensional collagen gels.

Extracellular matrix components play an important role in modulating cellular activity. To study such capacities of the matrix, fibroblasts are frequently cultured in a three-dimensional gel and contraction is assessed as a measure of cellular activity. Since a connective tissue contains several types of collagen, we investigated the effect of gels composed of collagen I alone or in combination with 10% collagen III and/or 5% collagen V on contraction by human periodontal ligament fibroblasts. Gels containing collagen V contracted much faster than those without this type of collagen. Blocking of the integrin beta1-subunit with an activity-blocking antibody delayed (gels with collagen V) or almost completely blocked (gels without collagen V) contraction. Use of an antibody directed against integrin alpha2beta1 resulted in delay of gel contraction for gels both with and without collagen V. Anti-integrin alpha v beta3 or RGD peptides partially blocked contraction of gels containing collagen V, but had no effect on gels consisting of collagen I alone. The beta1-containing integrins are involved in the basal contraction by fibroblasts that bind to collagens I and III. The enhanced contraction, stimulated by collagen V, appears to be mediated by integrin alpha v beta3. We conclude that collagen V may play an important modulating role in connective tissue contraction. Such a modulation may occur during the initial stages of wound healing and/or tissue regeneration.

Adult↗
PubMed1993

Type V collagen: molecular structure and fibrillar organization of the chicken alpha 1(V) NH2-terminal domain, a putative regulator of corneal fibrillogenesis.

Previous work from our laboratories has demonstrated that: (a) the striated collagen fibrils of the corneal stroma are heterotypic structures composed of type V collagen molecules coassembled along with those of type I collagen, (b) the high content of type V collagen within the corneal collagen fibrils is one factor responsible for the small, uniform fibrillar diameter (25 nm) characteristic of this tissue, (c) the completely processed form of type V collagen found within tissues retains a large noncollagenous region, termed the NH2-terminal domain, at the amino end of its alpha 1 chain, and (d) the NH2-terminal domain may contain at least some of the information for the observed regulation of fibril diameters. In the present investigation we have employed polyclonal antibodies against the retained NH2-terminal domain of the alpha 1(V) chain for immunohistochemical studies of embryonic avian corneas and for immunoscreening a chicken cDNA library. When combined with cDNA sequencing and molecular rotary shadowing, these approaches provide information on the molecular structure of the retained NH2-terminal domain as well as how this domain might function in the regulation of fibrillar structure. In immunofluorescence and immunoelectron microscopy analyses, the antibodies against the NH2-terminal domain react with type V molecules present within mature heterotypic fibrils of the corneal stroma. Thus, epitopes within at least a portion of this domain are exposed on the fibril surface. This is in marked contrast to mAbs which we have previously characterized as being directed against epitopes located in the major triple helical domain of the type V molecule. The helical epitopes recognized by these antibodies are antigenically masked on type V molecules that have been assembled into fibrils. Sequencing of the isolated cDNA clones has provided the conceptual amino acid sequence of the entire amino end of the alpha 1(V) procollagen chain. The sequence shows the location of what appear to be potential propeptidase cleavage sites. One of these, if preferentially used during processing of the type V procollagen molecule, can provide an explanation for the retention of the NH2-terminal domain in the completely processed molecule. The sequencing data also suggest that the NH2-terminal domain consists of several regions, providing a structure which fits well with that of the completely processed type V molecule as visualized by rotary shadowing.

Amino Acid Sequence↗
PubMed1986

The staining of type V collagen obtained from FBJ virus-induced osteosarcoma with concanavalin A.

Type V collagen from FBJ virus-induced osteosarcoma of mice has a high content of saccharide as has been noted for type V collagens from different sources. In the present study, this collagen was found to contain significant amounts of mannose and hexosamine. Three alpha chains of this collagen were electrophoretically separated and cleaved by cyanogen bromide. The cyanogen bromide peptides, following their separation by SDS-polyacrylamide gel electrophoresis, were transferred onto nitrocellulose paper and stained with concanavalin A. Several bands derived only from alpha 3(V) stained positively, but this was inhibited by the presence of alpha-methylmannoside. Thus, at least one of these three chains appears to have an asparagine-linked oligosaccharide.

Animals↗
PubMed2003

T47-D cells and type V collagen: a model for the study of apoptotic gene expression by breast cancer cells.

We have previously reported that type V collagen is a poorly adhesive, anti-proliferative and motility-inhibitory substrate for the 8701-BC breast cancer cell line, which also triggers DNA fragmentation and impairs survival of the same cell line. In the present work we have extended to other breast cancer cell lines (T47-D, MDA-MB231, Hs578T) our investigation of type V collagen influence on the DNA status and cell survival, also examining whether adhesion and growth of cells on this collagen substrate could exert some effect on the expression level of selected apoptosis-related genes. We report here that, among the cell lines tested, only T47-D is responsive to the death-promoting influence of type V collagen. In addition, the latter induces changes in gene expression by up-regulating p53, Waf-1, Cas, Dap kinase and caspases 1, -5 and -14 and down-regulating Bcl-2. Our data validate the T47-D line as a suitable in vitro model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on mammary tumor cells.

Apoptosis↗
PubMed1983

Immunohistochemical localization of laminin, type IV and type V collagen in basal cell carcinoma.

Laminin, type IV and type V collagen were localized in twenty-five basal cell carcinomas (BCCs) by immunofluorescence. All basal cell carcinomas were separated from the connective tissue by a continuous basement membrane (BM) which always contained the three antigenic determinants. Nodular and cystic BCCs, but not pagetoid BCCs, contain linear and globular deposits of these BM components. The linear deposits probably represent BM debris that remains undigested and the globular deposits correspond to residual cytoplasmic fragments occurring during apoptosis. These results suggest that BM components are synthesized by epithelial cells and are secreted to polymerize at the dermo-epidermal junction.

Aged↗
PubMed2001

Domain structure of chondroitin sulfate E octasaccharides binding to type V collagen.

We demonstrated previously that chondroitin sulfate E (ChS-E) binds to type V collagen (Munakata, H., Takagaki, K., Majima, M., and Endo, M. (1999) Glycobiology 9, 1023--1027). In this study, we investigated the structure and binding of ChS-E oligosaccharides. Eleven oligosaccharides were isolated from ChS-E by gel filtration chromatography and anion-exchange high performance liquid chromatography after hydrolysis with testicular hyaluronidase. Separately, seven oligosaccharides were custom synthesized using the transglycosylation reaction of testicular hyaluronidase. Structural analysis was performed by enzymatic digestions in conjunction with high performance liquid chromatography and mass spectrometry. This library of 18 oligosaccharides was used as a source of model molecules to clarify the structural requirements for binding to type V collagen. Binding was analyzed by a biosensor based on surface plasmon resonance. The results indicated that to bind to type V collagen the oligosaccharides must have the following carbohydrate structures: 1) octasaccharide or larger in size; 2) a continuous sequence of three GlcAbeta1--3GalNAc(4S,6S) units; 3) a GlcAbeta1--3GalNAc(4S,6S) unit, GlcAbeta1--3GalNAc(4S) unit or GlcAbeta1--3GalNAc(6S) unit at the reducing terminal; 4) a GlcAbeta1--3GalNAc(4S,6S) unit at the nonreducing terminal. It is likely that these characteristic oligosaccharide sequences play key roles in cell adhesion and extracellular matrix assembly.

Binding Sites↗
PubMed1999

Interaction between collagens and glycosaminoglycans investigated using a surface plasmon resonance biosensor.

The interactions of glycosaminoglycans with collagens and other glycoproteins in extracellular matrix play important roles in cell adhesion and extracellular matrix assembly. In order to clarify the chemical bases for these interactions, glycosaminoglycan solutions were injected onto sensor surfaces on which collagens, fibronectin, laminin, and vitronectin were immobilized. Heparin bound to type V collagen, type IX collagen, fibronectin, laminin, and vitronectin; and chondroitin sulfate E bound to type II, type V, and type VII collagen. Heparin showed a higher affinity for type IX collagen than for type V collagen. On the other hand, chondroitin sulfate E showed the highest affinity for type V collagen. The binding of chondroitin sulfate E to type V collagen showed higher affinity than that of heparin to type V collagen. These data suggest that a novel characteristic sequence included in chondroitin sulfate E is involved in binding to type V collagen.

Animals↗
PubMed1994

Type V collagen and Bowman's membrane. Quantitation of mRNA in corneal epithelium and stroma.

Bowman's membrane is an acellular matrix of the cornea which lies between the epithelial basal lamina and the corneal stroma. By immunoelectron microscopy, we have determined that types I and V collagen are components of the collagen fibrils in Bowman's membrane of the chick cornea. Although these same components are found in the fibrils of the stroma, the fibrils of Bowman's membrane are smaller in diameter and less uniform than those of the stroma. At early stages of development, the corneal epithelium synthesizes the types I and II collagen of the primary stroma. We therefore asked whether it might also be capable of synthesizing the type V collagen found in Bowman's membrane at later stages of development. Our results, using competitive polymerase chain reaction to quantitate mRNA from avian corneal cells, indicate that the amount of alpha 1(V) collagen mRNA present in epithelia, relative to alpha 2(I) collagen mRNA, is greater than that in stromal fibroblasts. We postulate that this enables the epithelium to synthesize a higher ratio of type V to type I collagen than the stroma and that this proportionally higher amount of type V might account for the ultrastructural appearance of the fibrils in Bowman's membrane.

Amino Acid Sequence↗
PubMed1999

Construction and characterization of mutations within the Klebsiella mrkD1P gene that affect binding to collagen type V.

The fimbria-associated MrkD1P protein mediates adherence of type 3 fimbriate strains of Klebsiella pneumoniae to collagen type V. Currently, three different MrkD adhesins have been described in Klebsiella species, and each possesses a distinctive binding pattern. Therefore, the binding abilities of mutants possessing defined mutations within the mrkD1P gene were examined in order to determine whether specific regions of the adhesin molecule were responsible for collagen binding. Both site-directed and chemically induced mutations were constructed within mrkD1P, and the ability of the gene products to be incorporated into fimbrial appendages or bind to collagen was determined. Binding to type V collagen was not associated solely with one particular region of the MrkD1P protein, and two classes of nonadhesive mutants were isolated. In one class of mutants, the MrkD adhesin was not assembled into the fimbrial shaft, whereas in the second class of mutants, the adhesin was associated with fimbriae but did not bind to collagen. Both hemagglutinating and collagen-binding activities were associated with the MrkD1P molecule, since P pili and type 3 fimbriae carrying adhesive MrkD proteins exhibited identical binding properties.

Adhesins, Bacterial↗
PubMed1994

The failure to reconstitute the second extracellular matrix-integrin-cytoskeleton system in human endothelial cells on type V collagen.

We examined the formation of F-actin filaments and the expression of beta 1 integrin in human vascular endothelial cells cultured on type V collagen. The cells attached to the exogenous substrate, formed complete F-actin filaments, and expressed vinculin and beta 1 integrin 0.5-1 h after inoculation. These phenomena are referred to as the first ECM-integrin-cytoskeleton system. After 3-6 h, disassembly of the F-actin filaments was observed to occur from the leading edges, and the cells developed focal adhesions only in their central regions. After 12-24 h, the cells on the type V collagen failed to form the second ECM-integrin-cytoskeleton system, and gradually detached from the substrate. In contrast, the cells on type I collagen developed both the first and second system, and acclimatized themselves to the environment. Thrombospondin (TSP), an anti-adhesive protein, was capable of inhibiting the spreading of the cells both after 1 h and 24 h. However, type V collagen treated with TSP inhibited the cell spreading after 1 h, but not after 24 h. The attachment and spreading of the cells on type V collagen were little affected by an anti-TSP antibody and the synthetic peptide GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), which significantly inhibited the attachment and spreading of the cells on TSP. Taken together, these results suggest that the cell detachment from type V collagen is not attributed to endogenously produced TSP (or member of TSP family) with anti-adhesive properties, but resulted from the failure to reconstitute the second ECM-integrin-cytoskeleton system in focal adhesion.

Actins↗
PubMed1983

Extraction, isolation and characterization of neutral salt soluble type V collagen from fetal calf skin.

Collagen was extracted with neutral salt solution and examined for the presence of type V collagen. A fraction which was insoluble in both 0.02 M Na2HPO4, pH 9.2 and phosphate-buffered saline (PBS) pH 7.2 at 4 degrees C contained both alpha 1(V)- and alpha 2(V)-chains demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, diethylaminoethyl cellulose ion exchange chromatography, amino acid analysis and segment long spacing (SLS) crystallites. SLS crystallites showed a globular N-terminal extension peptide attached to the type V collagen monomer. Ion exchange chromatography also demonstrated the presence of a third, minor component, which was identified as the alpha 3(V)-chain. In certain extractions, components corresponding to the partially processed procollagen chains of type V collagen were also observed.

Amino Acids↗