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PubMed1984

Purification of a type V collagen degrading metalloproteinase from rabbit alveolar macrophages.

A proteinase capable of degrading the helical region of native human type V collagen was identified in serum-free culture medium from "in vivo-activated" rabbit alveolar macrophages. This enzyme was purified to homogeneity using a combination of gel-filtration, ion-exchange and affinity chromatography. Analysis of the purified material by gel electrophoresis revealed a single broad band with relative molecular mass of 82,000 daltons Enzyme activity was eluted from the gel in a region corresponding to the stained band. The protein band was also found to stain positively using the periodic acid-Schiff technique indicating that it was glycosylated. Amino acid analysis revealed a composition rich in acidic residues. The enzyme cleaved type V collagen into three large molecular weight doublets consistent with two cleavage sites within the helix but was inactive against native type I collagen. However, when type I or type V collagen was heat-denatured, the enzyme degraded the alpha chains to small molecular weight peptides.

Amino Acids↗
PubMed1986

Para osteo arthropathy--ectopic ossification of healing tendon about the rodent ankle joint: histologic and type V collagen changes.

Ectopic bone formation, or para osteo arthropathy (POA), is commonly seen in humans following lesions to the central nervous system. The condition also occurs after severe trauma, burns, or tetanus, and, rarely, after poliomyelitis or cauda equina lesions. The lack of a suitable laboratory animal model has hampered study of its etiology and treatment. The lesion occurs just above the rodent ankle joint 7 weeks after repair of the severed achilles tendon. Tendon repair using a single silk suture resulted in ectopic calcification in 22 of 23 animals. Ectopic ossification, with extensive mineralization, occurred in 11 animals, 5 of which showed bone marrow. Ectopic mineralization was moderate in 8 others, minimal in 4, and absent in one. Collagen typing thrice during healing indicated that type V collagen increased by one-third at 10 days, doubled at 20 days, and returned to near normal amounts at 50 days. Since type V collagen is a major component of the nonfibrous collagen of blood vessels, increased amounts of type V collagen are consistent with the presence of many new blood vessels in the granulation tissue at 10 and 20 days. Vascularity is less dense at 50 days when ectopic mineralization and new bone formation are observed. These findings suggest that POA and the degree of maturation of healing tendon may be linked to reduced amounts of type V collagen. It is concluded that the sutured tendon of the rodent ankle joint offers an animal model by which to study ectopic bone formation, or para osteo arthropathy.

Achilles Tendon↗
PubMed2004

Reconstituted type V collagen fibrils as cementing materials in the formation of cell clumps in culture.

Previous studies have reported that type V collagen is an anti-adhesive substrate for cultured cells in that the cells detach from culture dishes coated with type V collagen molecules or polypeptides derived from them. We have noticed that human fetal lung fibroblasts (TIG-1) initially show no reduction in adherence to and spreading on a dish coated with reconstituted type V collagen fibrils but eventually detach from the dish and form cell clumps. To determine the way in which reconstituted type V collagen fibrils are involved in cell clump formation, we have followed the fate of the fluorescence of type V collagen fibrils pre-labeled with fluorescein isothiocyanate. Essentially, all the fluorescence disappeared from the dish surface as the cells detached and was condensed in the cell clumps. The cells that were recovered from clumps and dissociated into separate cells by trypsin treatment proliferated normally after they were seeded on a bare culture dish. This result and those from gel electrophoresis, fluorescence microscopy, and a cell proliferation assay indicate that the cell detachment from the dish is not caused by cell necrosis or apoptosis but by cellular motility together with the unique features of type V collagen fibrils. Not only the adherence of type V collagen fibrils to TIG-1 cells is much stronger than that to the culture dish, but the fibrils are retained on the cellular surface. The strong adherence of type V collagen fibrils to cells plays a role in cementing TIG-1 cells together.

Cell Adhesion↗
PubMed2000

The influence of corneal stromal matrix proteins on the migration of human corneal fibroblasts.

Motivated by the alterations seen in the corneal matrix composition after photorefractive keratectomy and the migration of corneal keratocytes seen following this procedure, the locomotor response of corneal stromal fibroblasts to various extracellular matrix proteins was determined. In addition, the involvement of integrin mediated attachment to the matrix proteins was investigated. Quantitative invasion assays were performed using collagen gels, supplemented with either fibronectin, tenascin, collagen type V, collagen type VI, chondroitin sulfate or keratan sulfate. The ultrastructure of the gels was visualized by scanning electron microscopy and related to the migration results. The extent of alpha(1)beta(1), alpha(2)beta(1), alpha(3)beta(1)and alpha(5)beta(1)integrin mediated attachment to the matrix proteins was evaluated using blocking antibodies. Fibronectin increased corneal fibroblast migration significantly, and served as an excellent substrate for cellular attachment, mediated by the alpha(5)beta(1)integrin. Addition of tenascin to the fibronectin-containing gels disrupted these effects, while attachment to this matrix also involved the integrins alpha(2)beta(1)and alpha(3)beta(1). Chondroitin sulfate and collagen types V and VI primarily altered the structure of the collagen matrix, resulting in an inhibition of migration by the collagens and an increase by chondroitin sulfate. They all served as poor substrates for attachment. Thus, the migratory activity of corneal fibroblasts in vitro is influenced by the composition of the surrounding extracellular matrix, either by integrin mediated cell-matrix interactions or through matrix-matrix interactions. This study provides evidence that the provisional matrix deposited in a corneal stromal wound may facilitate the entry of migrating corneal fibroblasts.

Antibodies, Blocking↗
PubMed1983

Type V collagen. Quantitation in normal lungs and in lungs of rats with bleomycin-induced pulmonary fibrosis.

Type V collagen was first isolated in 1976; there is still controversy as to how many molecular species of type V collagen exist. Although its structural and functional roles remain unclear, reports of changes in the relative amount of type V collagen from that present in normal tissue have been reported in such diverse pathologic conditions as atherosclerotic aortas, prolapsed mitral valves, and fibrotic lungs. Methods for quantitating type V collagen relative to other collagens have consisted of solubilizing the collagen with pepsin and then analyzing the ratios of the intact chains by gel electrophoresis or by column chromatography. In tissues in which only a small percentage of the total collagen can be solubilized by pepsin, such analyses may not accurately reflect changes in the total collagen present. In this report, a method for quantitating type V collagen relative to types I and III collagens based on CNBr peptide mapping is presented. CNBr solubilizes virtually all the collagen present in any tissue. The method is applied to a model of bleomycin-induced pulmonary fibrosis in rats. It was found that type I collagen increased relative to types III and V collagens, which seemed to remain at values comparable to those observed in lungs from control (normal) rats, both in terms of newly synthesized collagen (collagen synthesized by lung minces during 4 h in culture) and total unlabeled lung collagen (collagen synthesized during the life of the animal).

Amino Acids↗
PubMed1989

Immunoelectron microscopical evidence that type V collagen is a fibrillar collagen: importance for an aggregating capability of the preparation for reconstituting banding fibrils.

Type V collagen has already been shown, in many immunohistochemical studies, to be widely distributed in connective tissues. Its supramolecular structure, however, has been unclear. We demonstrate that the major aggregates formed from type V collagen solution in vitro are fine fibrils with a D-periodic banding pattern. Further, by using the immunogold labeling method, we find that these fibrils react strongly with anti-type V collagen antibody. Electronmicroscopic examination showed three kinds of aggregate: fine fibrils with periodic banding pattern, fine fibrils without banding pattern, and non-fibrillar materials. Both striated and nonstriated fibrils, when incubated with rat polyclonal anti-human type V collagen IgG followed by incubation with 15 nm-gold conjugated goat anti-rat IgG, were labeled with colloidal gold. We conclude that type V can be classified as a fibrillar collagen. Also, from the present findings together with previous studies, we believe type V collagen may exist in vivo in various connective tissues as fine fibrils with a 67 nm-periodic banding pattern, by itself, or with type I or type III fibrillar collagen, being located between, and connecting the basal lamina and interstitial collagen fibers.

Collagen↗
PubMed1998

Expression of type I and type V collagen mRNAs in the elasmoid scales of a teleost fish as revealed by in situ hybridization.

The ability of scale-forming cells to produce both type I and type V collagens was investigated by in situ hybridization at the light and electron microscope levels. Biochemical analyses reported that type I collagen, the predominant component, was associated with the minor type V collagen in the collagenous matrix of the teleost scales where, thin and thick collagen fibrils formed distinct layers. Thin collagen fibrils of the external layer were produced by the episquamal scleroblasts scattered on the outer scale surface, while thick collagen fibrils forming the compact basal plate were produced by the hyposquamal scleroblasts lining the inner surface of the scale. We demonstrated that episquamal and hyposquamal scleroblasts contained mRNAs for alpha1(I) and alpha1(V) collagens. Quantification by image analysis of the relative amount of alpha1(I) and alpha1(V) mRNAs in episquamal and hyposquamal scleroblasts suggests that the gene expression of type V collagen was proportionally higher in episquamal scleroblasts. These results support our hypothesis that the diameter of the thin fibrils of the external layer is regulated by the significant amount of type V collagen that interacts with type I collagen.

Animals↗
PubMed1998

Type V collagen is an epithelial cell cycle inhibitor that is induced by and mimics the effects of transforming growth factor beta1.

The regulation of epithelial cell cycle progression by extracellular matrix proteins was investigated in mink lung epithelial cells (Mv1Lu cells) and primary human keratinocytes. Exogenous type V collagen was able to mimic all of the inhibitory effects of type 1 transforming growth factor beta (TGF-beta1). No significant inhibitory effect was observed with collagen types I, III, and IV; laminin; or fibronectin. The type V collagen used was not contaminated with TGF-betas. TGF-beta1 increased the rate of type V collagen protein secretion in Mv1Lu cells, which occurred coincident with DNA synthesis inhibition. Both TGF-beta1 and type V collagen inhibited retinoblastoma protein phosphorylation and the expression of cyclin-dependent kinase (cdk) 4 and cdk2, but not p27Kip expression. Mv1Lu cells constitutively expressing the SV40 T antigen or cdk4 were resistant to the inhibitory effects of both TGF-beta1 and type V collagen. Our results demonstrate that type V collagen is a novel and specific epithelial cell cycle inhibitor and suggest that it may act as an autocrine mediator of the inhibitory effects of TGF-beta1.

Animals↗
PubMed1980

Collagen-platelet interaction. Type V(A-B) collagen induced platelet aggregation.

One of the more recently discovered collagens, type V (or A-B) collagen, in its native fibillar form mediates human platelet aggregation and the release of serotonin. In agreement with a recent report, it has no detectable effect on human platelets in the soluble or amorphous form. The possibility that the observed results might be due to contaminating interstitial collagens was eliminated by taking advantage of unusual solubility properties of type V collagen. Type V collagen dissolved in 0.1M acetic acid formed native-type fibrils when dialyzed against PBS and amorphous fibrils when dislyzed against 0.05M Tris/0.13M NaCl, pH 7.4, at 4 degrees C. Interstitial collagens remained in solution under both of these conditions. In addition, type V collagen treated with sufficient, purified synovial collagenase to digest all contaminating interstitial collagen retained its platelet-aggregating properties. The purity of type V collagen was confirmed by SDS-PAGE of CNBr digests. These data indicate that the quaternary structure of type V collagen is important in its recognition by platelet membranes.

Amino Acids↗
PubMed1994

Diversity in the processing events at the N-terminus of type-V collagen.

The processing of human collagen type-V chains was studied using anti-peptide polyclonal antibodies raised against peptide sequences at the N-terminal non-triple-helical region of pro-alpha 1(V) and pro-alpha 2(V) chains. The anti-peptide polyclonal antibody raised against positions 48-57 of the N-terminal alpha 2(V) sequence recognized the mature form of the human alpha 2(V) chain extracted without any proteolytic treatment from several tissues in the presence of a mixture of protease inhibitors. It also recognized the pro-alpha 2(V) and pN-alpha 2(V) collagen chains secreted in the cell-culture media of the rhabdomyosarcoma A204 cell line. The pN-alpha 2(V) collagen chain from this cell line migrated during electrophoresis with the alpha 2(V) chain obtained from tissues. This demonstrates that the alpha 2(V) chain in tissues is incompletely processed and is present as the pN-alpha 2(V) collagen chain which lacks the C-propeptide. In comparison, an anti-peptide polyclonal antibody raised against residues at positions 284-299 of the N-terminal alpha 1(V) human sequence failed to recognize the mature form of the alpha 1(V) chain while it reacted with the pN-alpha 1(V) collagen chain form. These results suggest that the alpha 1(V) chain undergoes a processing event in the N-terminal region that involves the removal of at least the first 284 residues. Amino acid sequence analysis was performed on cyanogen-bromide-generated or trypsin-generated peptides of the two electrophoretic bands obtained for the tissue form of collagen V. The slower-migrating band corresponding to the intact alpha 1(V) chain gave, as expected, only sequences corresponding to the alpha 1(V) chain. However, the band previously considered to be the intact alpha 2(V) chain also gave sequences for the alpha 1(V) chain in addition to the alpha 2(V) chain. This result indicates the presence in tissue extracts of a further processed form of alpha 1(V) chain which migrates with the intact alpha 2(V) chain. On further analysis, we observed that the two bands of the tissue form of collagen V occurred in a 1:1 ratio whereas, after the pepsin digestion to remove non-collagenous regions, two bands were observed with an alpha 1(V)/alpha 2(V) chain ratio of 3:1. These results indicate that the alpha 1(V) chain exists in an additional stoichiometry, different from [alpha 1(V)]2 alpha 2(V).(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Distribution and synthesis of type V collagen in the rat mammary gland.

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.

Animals↗
PubMed1993

Platelets deficient in glycoprotein IIIb aggregate normally to collagens type I and III but not to collagen type V.

The aggregation of platelets induced by collagens is considered an important step in primary hemostasis. Glycoprotein (GP) IIIb (GPIIIb, GPIV, CD36) has been proposed as a blood platelet receptor for collagen. Platelets from three healthy blood donors were shown to be clearly deficient in GPIIIb. These platelets aggregated normally in response to type I and III collagens. In addition, platelet factor 4, beta-thromboglobulin, and adenosine triphosphate (ATP) secretion in response to type I and III collagens was normal. The findings indicate that GPIIIb is not the major, essential collagen receptor for type I and III collagens. This would explain why all individuals with GPIIIb-deficient platelets examined so far are healthy and, in particular, show no apparent evidence of hemostatic problems. However, in contrast to control platelets, no aggregation and impaired platelet factor 4, beta-thromboglobulin, and ATP secretion was observed in response to type V collagen. Therefore, it is postulated that for type V collagen-induced aggregation both GPIa/IIa and GPIIIb are essential.

Adult↗
PubMed1994

Localization of fibril/microfibril and basement membrane collagens in diabetic glomerulosclerosis in type 2 diabetes.

Collagen is one of the major components of the extracellular matrices of the kidney. Basement membrane collagen, type IV collagen, is the major component in normal glomeruli. Fibril and interstitial collagen such as type III collagen, type V collagen, and type VI collagen are minor components of glomerular extracellular matrices and are localized mainly in the interstitium. Diabetic glomerulosclerosis is characterized by the expansion of the glomerular mesangial matrix as well as by thickening of the glomerular basement membrane. In order to clarify the roles of these various types of collagen in the development of diabetic glomerulosclerosis, immunohistochemical studies were performed in kidney specimens from patients with Type 2 diabetes. Early glomerulosclerosis is characterized by expansion of mesangial matrix with basement membrane collagen. However, in later stages glomerulosclerosis is characterized by an increase in the minor collagen components, such as type V and type VI collagen or collagens not normally present, such as type III collagen. Mesangial cells are known to synthesize all these types of collagen. In diabetes, phenotypic change in mesangial cells might produce excess amounts of fibril and interstitial collagen such as type III, type V, and type VI collagen, thus, leading to glomerulosclerosis.

Aged↗
PubMed1993

Structural analysis of the extension peptides on matrix forms of type V collagen in fetal calf bone and skin.

The structure of the extension peptides retained on the tissue form of type V collagen molecules was determined. Type V collagen alpha chains containing extension peptides were extracted from fetal calf skin and bone by 4 M guanidine-HCl and 0.5 M acetic acid, respectively. Collagens present in both extracts were fractionated by sodium chloride precipitation. The collagen alpha(V) chains were then resolved by reverse-phase high performance liquid chromatography. The N-terminal extension peptides were characterized by direct sequence analysis after deblocking with pyroglutamate amino-peptidase and analysis of the products of digestion by bacterial collagenase, chymotrypsin, V8 protease and endoproteinase Lys-C. The results showed that the retained extension peptides on type V collagen molecules in the extracellular matrix of skin and bone were amino-propeptides and that the alpha 2(V) chain retains an intact amino-propeptide while the alpha 1(V) chain appears to be partially processed. The extended alpha 1(V) chain isolated from fetal calf bone gave an identical amino-terminal sequence to that of the alpha 1(V) chain isolated from fetal calf skin, suggesting that a specific enzyme may be involved in processing the alpha 1(V) amino-propeptide.

Amino Acid Sequence↗
PubMed1983

Type V collagen during granulation tissue development.

The collagen content, as determined by hydroxyproline assay, of experimental granulation tissue in rats was observed to increase rapidly 21 days, and less rapidly to 90 days of tissue development. Resistance of the collagen to pepsin digestion reached a maximum at 21 days, suggesting more extensive or more stable crosslinking at that time. Type V collagen and the expected collagen types I and III were present in pepsin extracts of the granulation tissue as determined by SDS-polyacrylamide gel electrophoresis. Over 3 months of tissue development the relative quantity of type V collagen, as evidenced by changes in the alpha B chain, varied in parallel with the changing vascularity of the tissue, suggesting an association with capillary endothelial cells and angiogenesis.

Animals↗
PubMed1989

A new lethal brittle bone syndrome with increased amount of type V collagen in a patient.

A new lethal brittle bone disease is described in three patients with slender long bones, thin ribs, hypomineralized calvaria, and normal facial appearance. In spite of several limb fractures this syndrome can be differentiated from the lethal forms of osteogenesis imperfecta and is better related to the thin-bone group of lethal dysplasias. Biochemical investigation of collagen from one of the patients by the use of gel electrophoresis and high-pressure liquid chromatography analyses failed to demonstrate any evident defect in the structure of type I collagen chains. Nevertheless collagen extractability from the dermis was altered owing to an increase in the proportion of acid-soluble material. Tritium-proline labeling of cultured fibroblasts confirmed the reduction in total collagen synthesis. This was attributed to a lower type I and type III amount whereas type V collagen level was markedly increased in the cell layer. RNA analysis of the three collagen types with the appropriate cDNA probes confirmed the protein data. Electron microscopic examination of bone and skin showed morphologically abnormal fibroblasts and osteoblasts with an abundant distended rough endoplasmic reticulum and an altered plasma membrane. Unexpected thin fibrils with a banding pattern and surrounding the type I fibrils were observed. They might represent type V collagen. We suggest that, in this patient, the moderate decrease in type I collagen amount is insufficient to account for the radiological findings and that type V collagen overproduction could play a role in the bone brittleness by interfering with the process of mineralization.

Bone Diseases, Developmental↗
PubMed1993

Human vascular endothelial cell attachment and growth inhibition by type V collagen.

PURPOSE: Human vascular prostheses develop a pseudointima that after time is devoid of a complete endothelial lining. The composition of this pseudointima consists of both cellular and noncellular components such as coagulation proteins and extracellular matrix proteins. Of these extracellular molecules, type V collagen has been reported to be localized to surfaces of vascular prostheses. We have hypothesized that type V collagen may be involved in the mechanisms of inhibition of endothelialization on vascular prostheses. In this study, interactions of human protein and cells with clinically used material and reference surfaces were analyzed in vitro. METHODS AND RESULTS: Human collagen types IV and V or human fibronectin was coated on disks punched from expanded polytetrafluoroethylene, bacteriologic polystyrene, and tissue culture-treated polystyrene. Fibronectin adsorbed equally to these surfaces, but differential adsorption of type V collagen occurred. The attachment and growth of human saphenous vein and umbilical vein endothelial cells and of human skin fibroblasts were also evaluated on protein-coated or uncoated surfaces. Type IV collagen and fibronectin promoted the attachment of these cells, but type V collagen reduced cellular adhesion. Growth of endothelial cells was significantly inhibited on surfaces coated with type V collagen even when additional growth substances such as serum, retinal-derived growth factor, and heparin were present in the medium. Human adult dermal fibroblast adhesion and cell growth were not affected by coating the surfaces with type V collagen. CONCLUSIONS: The components of the extracellular matrix of the pseudointima may directly influence endothelial cells by inhibiting cell proliferation, migration from within the graft or from anastomoses, or both. Type V collagen, a matrix protein found at luminal surfaces of vascular prostheses, may be one protein responsible for control of endothelial responses.

Cell Adhesion↗
PubMed1998

Mutations of the alpha2(V) chain of type V collagen impair matrix assembly and produce ehlers-danlos syndrome type I.

Ehlers-Danlos syndrome (EDS) is a heterogeneous connective tissue disorder that severely impairs the structure and function of the skin, joints, eyes and blood vessels. We have identified mutations of the COL5A2 gene, which encodes the alpha2(V) chain of type V collagen, in two unrelated patients with the severe type I form of EDS. The first proband was heterozygous for a 7 bp deletion that resulted in skipping of exon 27 while the second proband was heterozygous for a single nucleotide substitution that resulted in skipping of exon 28. Cultured dermal fibroblasts from both probands produced about equal amounts of the normal and mutant alpha2(V) mRNAs and protein chains. The dermis from the first proband contained a sparse collagen fibrillar network with great variability in collagen fibril sizes and shapes. The dermal collagens were also abnormally soluble. Bone cells from the first proband also produced about equal amounts of the normal and mutant alpha2(V) mRNAs. However, the collagen fibrillar architecture and collagen solubility of the bone matrix were normal. Our findings show that heterozygous mutations of the COL5A2 gene can produce the EDS type I phenotype. They also suggest that type V collagen plays a more important role in collagen fibrillogenesis of dermis than that of bone.

Adolescent↗