Intercellular accumulation of type V collagen fibrils in accordance with cell aggregation

We reported previously that human fibroblasts form clumps when cultured on a dish coated with reconstituted type V collagen fibrils. Essentially all the type V collagen fibrils, initially coated on the dish, were recovered in the cell clumps that had eventually formed during the culture. We interpreted that type V collagen fibrils adhere to cells more strongly than to the dish and are detached by cell movements. In this study, type V collagen was suspended with fibroblasts to examine the fate of the type V collagen fibrils and to determine whether the fibrils affect the behaviour of the cells directly adherent to the dish. The added type V collagen accumulated in the intercellular space concomitantly with the local aggregation of fibroblasts. scanning electron microscope examination indicated that type V collagen fibrils were found in the vicinity of cells in cultures without ascorbic acid where essentially no collagen secretion takes place. These results indicate that type V collagen forms fibrils and the fibrils are accumulated in the intercellular spaces. The accumulated type V collagen fibrils work as a cementing material for cell clump formation. This phenomenon is discussed in relation to the possible involvement of type V collagen fibrils in tissue organization.     

Collagen fibrils of an invertebrate (Sepia officinalis) are heterotypic: immunocytochemical demonstration

Collagen fibrils from the dermis of Sepia officinalis were processed for immunoelectron microscopy to reveal reactions to antibodies against mammalian types I, III, and V, teleost type I and cephalopod type I-like collagens, by single and double immunogold localization. The fibrils were observed: (a) in suspensions of prepared fibrils, (b) in ultrathin sections of embedded fibril preparations, and (c) in ultrathin sections of dermal tissue. Some samples were subjected to acetic acid or urea dissociation. It was found that collagen fibrils from Sepia dermis are heterotypic in that they are composed of type I-like and type V collagens. Type I-like collagen epitopes were present mainly at the periphery of the fibrils; type V collagen epitopes were present throughout the fibrils. This is the first demonstration that collagen fibrils from an invertebrate are heterotypic, suggesting that heterotypy may be an intrinsic characteristic of the fibrils of fibrillar collagens, independent of evolutionary or taxonomic status.     

Collagen types I, III, and V in human embryonic and fetal skin

The dermis of human skin develops embryonically from lateral plate mesoderm and is established in an adult-like pattern by the end of the first trimester of gestation. In this study the structure, biochemistry, and immunocytochemistry of collagenous matrix in embryonic and fetal dermis during the period of 5 to 26 weeks of gestation was investigated. The dermis at five weeks contains fine, individual collagen fibrils draped over the surfaces of mesenchymal cells. With increasing age, collagen matrix increases in abundance in the extracellular space. The size of fibril diameters increases, and greater numbers of fibrils associate into fiber bundles. By 15 weeks, papillary and reticular regions are recognized. Larger-diameter fibrils, larger fibers, denser accumulations of collagen, and fewer cells distinguish the deeper reticular region from the finer, more cellular papillary region located beneath the epidermis. The distribution of collagen types I, III, and V were studied at the light microscope level by immunoperoxidase staining and at the ultrastructural level by transmission (TEM) and scanning electron microscopy (SEM) with immunogold labeling. By immunoperoxidase, types I and III were found to be evenly distributed, regardless of fetal age, throughout the dermal and subdermal connective tissue with an intensification of staining at the dermal-epidermal junction (DEJ). Staining for types III and V collagen was concentrated around blood vessels. Type V collagen was also localized in basal and periderm cells of the epidermis. By immuno-SEM, types I and III were found associated with collagen fibrils, and type V was localized to dermal cell surfaces and to a more limited extent with fibrils. The results of biochemical analyses for relative amounts of types I, III, and V collagen in fetal skin extracts were consistent with immunoperoxidase data. Type I collagen was 70-75%, type III collagen was 18-21%, and type V was 6-8% of the total of these collagens at all gestational ages tested, compared to 85-90% type I, 8-11% type III, and 2-4% type V in adult skin. The enrichment of both types III and V collagen in fetal skin may reflect in part the proportion of vessel- and nerve-associated collagen versus dermal fibrillar collagen. The accumulation of dermal fibrillar collagen with increasing age would enhance the estimated proportion of type I collagen, even though the ratios of type III to I in dermal collagen fibrils may be similar at all ages.     

Structure of type I and type III heterotypic collagen fibrils: an X-ray diffraction study

The molecular packing arrangement within collagen fibrils has a significant effect on the tensile properties of tissues. To date, most studies have focused on homotypic fibrils composed of type I collagen. This study investigates the packing of type I/III collagen molecules in heterotypic fibrils of colonic submucosa using a combination of X-ray diffraction data, molecular model building, and simulated X-ray diffraction fibre diagrams. A model comprising a 70-nm-diameter D- (approximately 65 nm) axial periodic structure containing type I and type III collagen chains was constructed from amino acid scattering factors organised in a liquid-like lateral packing arrangement simulated using a classical Lennard-Jones potential. The models that gave the most accurate correspondence with diffraction data revealed that the structure of the fibril involves liquid-like lateral packing combined with a constant helical inclination angle for molecules throughout the fibril. Combinations of type I:type III scattering factors in a ratio of 4:1 gave a reasonable correspondence with the meridional diffraction series. The attenuation of the meridional intensities may be explained by a blurring of the electron density profile of the D period caused by nonspecific or random interactions between collagen types I and III in the heterotypic fibril.     

Copolymerization of pNcollagen III and collagen I. pNcollagen III decreases the rate of incorporation of collagen I into fibrils, the amount of collagen I incorporated, and the diameter of the fibrils formed

Previous observations suggested that pNcollagen III, the partially processed form of type III procollagen, coats fibrils of collagen I and thereby helps regulate the diameter of fibrils formed by collagen I. The previous observations, however, did not exclude the possibility that pNcollagen III was deposited on preformed collagen I fibrils after the fibrils were assembled. Here, mixtures of pNcollagen III and collagen I were generated simultaneously by enzymatic cleavage of precursor forms of the proteins. The results demonstrated that pNcollagen III forms true copolymers with collagen I. The presence of pNcollagen III both inhibited the rate at which collagen I assembled into fibrils and decreased the amount of collagen I incorporated into fibrils at steady-state equilibrium. In addition, the results demonstrated that copolymerization of pNcollagen III with collagen I generated fibrils that were thinner than fibrils generated under the same conditions from collagen I alone. Increasing the initial molar ratio of pNcollagen III to collagen I in the solution-phase increased the amount of pNcollagen III copolymerizing with collagen I and progressively decreased the diameter of the fibrils. Therefore, the copolymers were heterogeneous in that the stoichiometry of the two monomers in the fibrils varied. The results are consistent with a model in which pNcollagen III can regulate the diameter of collagen I fibrils by coating the surface of the fibrils and thereby allow tip growth but not lateral growth of the fibrils.     

Separation of type III collagen from type I collagen and pepsin by differential denaturation and renaturation.

Types I and III collagens were solubilized from fetal human skin by limited digestion with pepsin and precipitated by dialysis against 0.02 M Na₂HPO₄. Heat denaturation of the collagens in 2 M guanidine-HCl, pH 7.5, resulted in the precipitation of the contaminant pepsin which could be removed by centrifugation. Renaturation of the denatured collagens by dialysis against deionized water at 22° for 2 hours selectively precipitated the type III collagen fibrils. Type I collagen remained in solution. The simplicity and high recovery (77%) make this a suitable approach for the rapid estimation of type III collagen in small tissue samples.     

Upregulation of HSP47 and collagen type III in the dermal fibrotic disease, keloid

Keloid is a dermal fibrotic disease characterized by excessive accumulation of mainly type I collagen in extracellular matrix of the dermis. We have studied the expression levels of collagen types I and III, and its molecular chaperone HSP47 in keloid lesions and surrounding unaffected skin using Northern and Western blotting and immunohistochemical analyses. Collagen types I and III mRNA levels were found to be upregulated 20-fold in keloid tissues, contradicting previous reports of nearly normal type III collagen levels in this disease. HSP47 expression in keloid lesions was also highly upregulated; eightfold at mRNA level and more than 16-fold at the protein level. Strong upregulation of these three proteins in keloid was confirmed by immunohistochemical staining. These results suggest that accumulation of both type I and type III collagen is important for the development of keloid lesions, and that HSP47 plays a role in the rapid and extensive synthesis of collagen in keloid tissues.     

Cultured epidermis influences the fibril organization of purified type I collagen gels

Purified type I collagen gel used as culture substrate was composed of unstriated fibrils. Before culture, gel fragments were coated with culture medium with or without fetal calf serum (FCS+ coated or FCS- coated gels). Each gel fragment was apposed to a fragment of frog skin at the medium/air interface in Trowell culture chamber. After 7 days at 20 degrees C, the coated gels were covered with newly formed epidermis containing fibronectin localized around the keratinocytes, whose morphology was considerably modified. Fibroblast-shaped keratinocytes were localized in the anterior zone of the newly formed epidermis on FCS+ gels. The long axis of the cells was parallel to the gel surface, where numerous unstriated fibrils were located. Polyhedral keratinocytes were located in the posterior zone on FCS+ gels or the anterior and posterior zones on FCS- gels with the long axis perpendicular to the gel surface. Numerous cross-striated fibrils were found under the cultured keratinocytes in the vicinity of the basal filipodia. This model is useful for the study of collagen gel reorganization by keratinocytes.     

Extracellular matrix production by embryonic epithelium cultured on type IV collagen Deposition of a primary corneal stroma-like structure containing large irregular type I fibrils without type II collagen

The corneal stroma of the chick embryo is deposited in two steps. The primary stroma is laid down by the corneal epithelium and it contains type I, type II and type IX collagens. Its formation is subsequent to the presumptive epithelial cells' migration onto the lens capsule (which is rich in type IV collagen). The secondary, ultimate stroma is synthesized by fibroblasts whcih, on day 5 of development, invade the swollen primary stroma. It is composed of a matrix of thin (25 nm), regular fibrils containing type I and type V collagens.We found that a chick corneal epithelium isolated from either a 6-day or a 14-day embryo was able to produce, in vitro, stroma-containing type I collagen fibrils. However, the amount of collagen deposited and its organization were highly dependent on the substratum used. Plastic or purified bovine type I collagen substrata led to the release of very few fibrils. Purified human type IV collagen induced the production of an abundant matrix made of large irregular collagen fibrils.When compared to native corneal stroma, there were two aspects in which this matrix differed: (1) it contained only type I collagen, as shown by indirect immunofluorescence, and (2) there were numerous large, irregular fibrils of about 100 to 130 nm in diameter.In conclusion, it is suggested that purified type IV collagen substitutes, in part, for the basement membrane and allows the production of a corneal stroma-like matrix by an embryonic corneal epithelium in culture. This production is possible even with a 14-day epithelium which, in vivo, is no more involved in the synthesis of the stroma collagens. Moreover, the regulatory effect of type II collagen, previously suggested by in vivo observations, may be confirmed in this in vitro system by the appearance of large fibrils in the newly deposited stroma that are made only by type I collagen.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell-layer associated pepsin-resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils.     

Type III collagen can be present on banded collagen fibrils regardless of fibril diameter

Monoclonal antibodies that recognize an epitope within the triple helix of type III collagen have been used to examine the distribution of that collagen type in human skin, cornea, amnion, aorta, and tendon. Ultrastructural examination of those tissues indicates antibody binding to collagen fibrils in skin, amnion, aorta, and tendon regardless of the diameter of the fibril. The antibody distribution is unchanged with donor age, site of biopsy, or region of tissue examined. In contrast, antibody applied to adult human cornea localizes to isolated fibrils, which appear randomly throughout the matrix. These studies indicate that type III collagen remains associated with collagen fibrils after removal of the amino and carboxyl propeptides, and suggests that fibrils of skin, tendon, and amnion (and presumably many other tissues that contain both types I and III collagens) are copolymers of at least types I and III collagens.     

Reduced type I collagen utilization: a pathogenic mechanism in COL5A1 haplo-insufficient Ehlers-Danlos syndrome

To examine mechanisms by which reduced type V collagen causes weakened connective tissues in the Ehlers-Danlos syndrome (EDS), we examined matrix deposition and collagen fibril morphology in long-term dermal fibroblast cultures. EDS cells with COL5A1 haplo-insufficiency deposited less than one-half of hydroxyproline as collagen compared to control fibroblasts, though total collagen synthesis rates are near-normal because type V collagen represents a small fraction of collagen synthesized. Cells from patients with osteogenesis imperfecta (OI) and haplo-insufficiency for proalpha1(I) chains of type I collagen also incorporated about one-half the collagen as controls, but this amount was proportional to their reduced rates of total collagen synthesis. Collagen fibril diameter was inversely proportional to type V/type I collagen ratios (EDS > control > OI). However, a reduction of type V collagen, in the EDS derived cells, was associated with the assembly of significantly fewer fibrils compared to control and OI cells. These data indicate that in cell culture, the quantity of collagen fibrils deposited in matrix is highly sensitive to reduction in type V collagen, far out of proportion to type V collagen's contribution to collagen mass.     

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.The present study was supported in part by Grant-in-Aid for Developmental Scientific Research (07558249), by The Japan Society for the Promotion of Science, Research for the Future Program (JSPS-RFTF96I00201), by the Program for Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR), by Grant-in-Aid for the Creation of Innovations through Business-Academic-Public Sector Cooperation to T.H., and by Grant-in-Aid for Scientific Research (B) to Y.I.     

Ehlers Danlos syndrome type VIIB. Incomplete cleavage of abnormal type I procollagen by N-proteinase in vitro results in the formation of copolymers of collagen and partially cleaved pNcollagen that are near circular in cross-section.

We have shown that a child with Ehlers Danlos syndrome (EDS) type VII has a G to A transition at the first nucleotide of intron 6 in one of her COL1A2 alleles. Half of the cDNA clones prepared from the proband's pro alpha 2(I) mRNA lacked exon 6. The type I procollagen secreted by the proband's dermal fibroblasts in culture was purified, and collagen fibrils were generated in vitro by cleavage of the procollagen with the procollagen N- and C-proteinases. Incubation of the procollagen with N-proteinase resulted in a 1:1 mixture of pCcollagen and uncleaved procollagen. Incubation of this mixture with C-proteinase generated collagen and abnormal pNcollagen (pNcollagen-ex6) that readily copolymerized into fibrils. By electron microscopy these fibrils resembled the hieroglyphic fibrils seen in the N-proteinase-deficient skin of dermatosparactic animals and humans and were distinct from the near circular cross-section fibrils seen in the tissues of individuals with EDS type VII. Further incubation of the hieroglyphic fibrils with N-proteinase resulted in partial cleavage of the pNcollagen-ex6 in which the abnormal pN alpha 2(I) chains remained intact. These fibrils were not hieroglyphic but were near circular in cross-section. Fibrils formed from collagen and pNcollagen-ex6 that had been partially cleaved with elevated amounts of N-proteinase prior to fibril formation were also near circular in cross-section. The results are consistent with a model of collagen fibril formation in which the intact N-propeptides are located exclusively at the surface of the hieroglyphic fibrils. Partial cleavage of the pNcollagen-ex6 by N-proteinase allows the N-propeptides to be incorporated within the body of the fibrils. The model provides an explanation for the morphology and molecular composition of collagen fibrils in the tissues of patients with EDS type VII.     

Type V collagen regulates the assembly of collagen fibrils in cultures of bovine vascular smooth muscle cells

Vascular smooth muscle cells (SMCs), the major cellular constituent of the medial layer of an artery, synthesize the majority of connective tissue proteins, including fibrillar collagen types I, III, and V/XI. Proper collagen synthesis and deposition, which are important for the integrity of the arterial wall, require the antioxidant vitamin C. Vitamin C serves as cofactor for the enzymes prolyl and lysyl hydroxylase, which are responsible for the proper hydroxylation of collagen. Here, the role of type V collagen in the assembly of collagen fibrils in the extracellular matrix (ECM) of cultured vascular SMCs was investigated. Treatment of SMCs with vitamin C resulted in a dramatic induction in the levels of the cell‐layer associated pepsin‐resistant type V collagen, whereas only a minor induction in the levels of types I and III collagen was detected. Of note, the deposition of type V collagen was accompanied by the formation of striated collagen fibrils in the ECM. Immunohistochemistry demonstrated that type V collagen, but not type I collagen, became masked as collagen fibrils matured. Furthermore, the relative ratio of type V to type I collagen decreased as the ECM matured as a function of days in culture, and this decrease was accompanied by an increase in the diameter of collagen fibrils. Together these results suggest that the masking of type V collagen is caused by its internalization on continuous deposition of type I collagen on the exterior of the fibril. Furthermore, they suggest that type V collagen acts as framework for the initial assembly of collagen molecules into heterotypic fibrils, regulating the diameter and architecture of these fibrils. J. Cell. Biochem. 80:146–155, 2000. © 2000 Wiley‐Liss, Inc.     

Type II achondrogenesis-hypochondrogenesis: identification of abnormal type II collagen.

We have extended the study of a mild case of type II achondrogenesis-hypochondrogenesis to include biochemical analyses of cartilage, bone, and the collagens produced by dermal fibroblasts. Type I collagen extracted from bone and types I and III collagen produced by dermal fibroblasts were normal, as was the hexosamine ratio of cartilage proteoglycans. Hyaline cartilage, however, contained approximately equal amounts of types I and II collagen and decreased amounts of type XI collagen. Unlike the normal SDS-PAGE mobility. Two-dimensional SDS-PAGE revealed extensive overmodification of all type II cyanogen bromide peptides in a pattern consistent with heterozygosity for an abnormal pro alpha 1(II) chain which impaired the assembly and/or folding of type II collagen. This interpretation implies that dominant mutations of the COL2A1 gene may cause type II achondrogenesis-hypochondrogenesis. More generally, emerging data implicating defects of type II collagen in the type II achondrogenesis-hypochondrogenesis-spondyloepiphyseal dysplasia congenita spectrum and in the Kniest-Stickler syndrome spectrum suggest that diverse mutations of this gene may be associated with widely differing phenotypic outcome.     

Type V collagen controls the initiation of collagen fibril assembly

Vertebrate collagen fibrils are heterotypically composed of a quantitatively major and minor fibril collagen. In non-cartilaginous tissues, type I collagen accounts for the majority of the collagen mass, and collagen type V, the functions of which are poorly understood, is a minor component. Type V collagen has been implicated in the regulation of fibril diameter, and we reported recently preliminary evidence that type V collagen is required for collagen fibril nucleation (Wenstrup, R. J., Florer, J. B., Cole, W. G., Willing, M. C., and Birk, D. E. (2004) J. Cell. Biochem. 92, 113-124). The purpose of this study was to define the roles of type V collagen in the regulation of collagen fibrillogenesis and matrix assembly. Mouse embryos completely deficient in pro-alpha1(V) chains were created by homologous recombination. The col5a1-/- animals die in early embryogenesis, at approximately embryonic day 10. The type V collagen-deficient mice demonstrate a virtual lack of collagen fibril formation. In contrast, the col5a1+/- animals are viable. The reduced type V collagen content is associated with a 50% reduction in fibril number and dermal collagen content. In addition, relatively normal, cylindrical fibrils are assembled with a second population of large, structurally abnormal collagen fibrils. The structural properties of the abnormal matrix are decreased relative to the wild type control animals. These data indicate a central role for the evolutionary, ancient type V collagen in the regulation of fibrillogenesis. The complete dependence of fibril formation on type V collagen is indicative of the critical role of the latter in early fibril initiation. In addition, this fibril collagen is important in the determination of fibril structure and matrix organization.     

In vitro and post-transplantation differentiation of human keratinocytes grown on the human type IV collagen film of a bilayered dermal substitute

Using human type IV and type I + III collagens and a new, nontoxic cross-linking procedure, we have developed a cell-free bilayered human dermal substitute for organotypic culture and transplantation of human skin keratinocytes. We have studied the formation of the basement membrane, and the differentiation of keratinocytes grown on the type IV collagen layer of this dermal substitute, in vitro and after grafting onto nude mice. These studies demonstrated the formation of essential constituents of the basement membrane in culture: hemidesmosomes and deposition of extracellular matrix on the top of the type IV collagen were observed as early as 6 days after plating of human keratinocytes. Although the keratinocytes formed a well-organized multilayered epithelium, they exhibited limited differentiation when grown submerged in liquid medium. However, the multilayered sheet obtained after 14 days in submerged culture was composed of a regular basal cell layer, several nucleated suprabasal cell layers containing granular cells, and several dense, anucleated cell layers. The grafting experiments have shown a good biocompatibility of the dermal substitute. It is repopulated by fibroblasts, newly synthesized collagen, vessels, and a few mononuclear cells. At Day 14 after grafting, the type IV collagen layer was still present and very dense, and the basement membrane appeared as in culture, with numerous well-structured hemidesmosomes and deposition of extracellular matrix resembling lamina densa. At Day 55 after transplantation, even if the epidermal graft did not exhibit all the characteristics of the normal epidermis in vivo, it was very close to it. At this stage, the basement membrane was complete, with structures clearly indicative of anchoring fibrils. This new dermal substitute offers many advantages. It is stable and easy to handle. Its production is standardized. The oxidation induced by periodic acid led to a nontoxic cross-linked matrix. This dermal substitute is the first one entirely composed of human collagens. The type I + III collagen underlayer is reorganized when grafted. It supports a type IV collagen top layer which offers an excellent substrate for keratinocytes, favors their anchorage, and favors the formation of the basement membrane in vitro. This dermal substitute could be useful for wound coverage or as an in vitro model for toxicological and pharmacological studies.     

Type I and type III collagen content of healing wounds in fetal and adult rats

Full-thickness, dermal wounds were surgically created on the dorsa of fetal rats on the 17th day of gestation. The granulation tissue which developed after 2 days (19 days of gestation) was harvested from six to nine animals and pooled and the collagen was extracted with 0.5 M acetic acid and acetic acid plus pepsin. The ratio of type III:type I collagen was estimated from densitometer scans of electrophoretically separated alpha-chains. Full-thickness (to fascia depth) wounds were also produced on the dorsa of adult rats and granulation tissue which had developed for different periods of time up to 30 days was excised. Relative proportions of type III and type I collagen were assessed in normal and granulation tissues taken from the adult rats. Both fetal and adult granulation tissues have elevated type III collagen content but normal fetal tissue has a much higher content of type III than does normal adult tissue.     

The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.