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.     

Aldehyde content and cross-linking of type III collagen.

Three phosphorylated dinucleosides designated HS1, HS2, and HS3, isolated from the water-mould $\text{Achlya}$, were shown to significantly inhibit ribonucleotide reductase activity from $\text{Achlya}$. All three compounds decreased CDP reduction in fungal extracts by 50% at concentrations of 0.1mM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least 80% but showed only 10% inhibition with enzyme from $\text{E.}\text{coli}$. ADP reductase activity from $\text{Achlya}$ was inhibited 50% by both HS1 and HS3 at 0.1mM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of $\text{Achlya}$ correlated with thymidine uptake into DNA and with the synthesis of HS compounds.     

Dermal collagen fibrils are hybrids of type I and type III collagen molecules

It has been suggested that dermal collagen fibrils with 67-nm periodicity consist of hybrids of type I and type III collagens. This is based on the assumption that all these banded fibrils are coated with type III collagen regardless of their diameter. However, conclusive evidence for this form of hybridization is lacking. In order to clarify this problem dermal collagen fibrils were disrupted into microfibrils using 8 M urea. Single and double indirect immunoelectron microscopy showed type III collagen at the periphery of intact collagen fibrils but no labeling with type I collagen antibodies, suggesting that the epitopes for this collagen were masked. Disrupted collagen fibrils revealed type I collagen throughout the fibril except for the periphery which was coated with type III collagen. Almost no type III collagen was noted in the interior of the collagen fibrils. Since type III collagen is present only at the periphery it suggests that this collagen has a different role than type I collagen and may have a regulatory function in fibrillogenesis.     

Immunolocalization of type VIII collagen in vascular tissue

Type VIII collagen was first detected in the culture medium of aortic endothelial cells. Subsequently its synthesis by a number of other cell lines was demonstrated. Its presence in vascular tissue is reported here. It is a component of sheep aorta and carotid artery but could not be demonstrated in the jugular vein. It is principally localized in the subendothelial region but this can only be demonstrated after pretreatment of the tissue with proteases. Thus type VIII collagen is a constituent of blood vessels.     

A simplified method for quantitation of the relative amounts of type I and type III collagen in small tissue samples

A method is described for the quantitation of the relative amounts of types I and III collagens in rabbit lung tissue. This involved (i) repeated homogenization in the presence of 2% sodium dodecyl sulfate and the production of an acetone dried powder, (ii) reaction with cyanogen bromide, (iii) polyacrylamide gel electrophoresis, and (iv) densitometric scanning of proteins stained by Coomassie blue R-250. Several features of this procedure were shown to offer advantages over methods previously employed. First, the sodium dodecyl sulfate solution was shown to remove the bulk of noncollagen proteins leaving an insoluble residue which could then be reacted with cyanogen bromide without further purification. Second, cyanogen bromide was shown to solubilize essentially all of the collagen in the residue leaving an insoluble pellet with an amino acid analysis similar to elastin. Finally, to facilitate accurate quantitation, types I and III collagen standards were included with each gel so that a standard curve of protein versus staining density could be constructed. This method is assessed to be simpler and more accurate than those employed previously for the quantitation of collagens and can be applied to small tissue samples (<100 mg) such as would be obtained by lung biopsy.     

A new platelet receptor specific to type III collagen. Type III collagen-binding protein

Platelet interaction with type III collagen is mediated by several platelet receptors that recognize specific sequences in collagen. We previously described an octapeptide KP*GEP*GPK within the alpha(1)III-CB4 fragment that binds to platelets and specifically inhibits platelet aggregation induced by type III collagen. In this study, we demonstrated that the octapeptide prevented platelet contact and spreading on type III collagen and subendothelium under static and flow conditions. Platelets adhered to the immobilized octapeptide, and anti-bodies directed against other platelet collagen receptors (glycoprotein (GP) Ia/IIa, GP IV, p65, p47) did not impair this adhesion. The platelet octapeptide receptor was identified by ligand blotting as a protein doublet with molecular masses of 68 and 72 kDa and does not correspond to any other already known platelet collagen receptors (GP Ia, GP IV GP VI, and p65). Our results indicate that a specific type III collagen receptor, expressed on the platelet surface, is involved in the first stages of platelet type III collagen interaction.     

Covalent binding of acetaldehyde to type III collagen

Incubation of neutral salt soluble type III pN-collagen with [14C]acetaldehyde in vitro resulted in the formation of spontaneously stable acetaldehyde-protein adducts. This reaction occurred primarily at lysine residues and it was not affected by 0.2-2 mM concentrations of ascorbate but addition of sodiumcyanoborohydride increased the stable adducts by 3-5-fold. When confluent cultures of human skin fibroblasts were incubated with physiologically relevant concentrations of acetaldehyde, it became covalently bound to type III procollagen secreted into the medium. We propose that acetaldehyde binding to collagen fibrils occurs in vivo following chronic alcohol consumption.     

The tissue form of chicken type VI collagen

We have purified intact type VI collagen from chicken gizzard. The protein was found to consist of a 130 kDa, a 140 kDa and a 180-200 kDa subunit. The 130 kDa and 140 kDa subunits were obtained in equimolar amounts and identified as the alpha 2 (VI) and the alpha 1 (VI) chains, respectively. The third subunit was usually obtained in the form of 3-4 closely related polypeptides, which may represent different processing or modification products of the alpha 3 (VI) chain.     

Cleavage of native type III collagen in the collagenase susceptible region by thermolysin.

Viscometric assays were used to demonstrate the activity of thermolysin (EC 3.4.24.4) on native type III collagen in solution. Analysis of the reaction products by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and electron microscopic visualisation of segment long spacing aggregates demonstrated localised cleavage of the collagen in the collagenase susceptible region.     

Defining requirements for collagenase cleavage in collagen type III using a bacterial collagen system

Degradation of fibrillar collagens is important in many physiological and pathological events. These collagens are resistant to most proteases due to the tightly packed triple-helical structure, but are readily cleaved at a specific site by collagenases, selected members of the matrix metalloproteinases (MMPs). To investigate the structural requirements for collagenolysis, varying numbers of GXY triplets from human type III collagen around the collagenase cleavage site were inserted between two triple helix domains of the Scl2 bacterial collagen protein. The original bacterial CL domain was not cleaved by MMP-1 (collagenase 1) or MMP-13 (collagenase 3). The minimum type III sequence necessary for cleavage by the two collagenases was 5 GXY triplets, including 4 residues before and 11 residues after the cleavage site (P4-P11'). Cleavage of these chimeric substrates was not achieved by the catalytic domain of MMP-1 or MMP-13, nor by full-length MMP-3. Kinetic analysis of the chimeras indicated that the rate of cleavage by MMP-1 of the chimera containing six triplets (P7-P11') of collagen III was similar to that of native collagen III. The collagenase-susceptible chimeras were cleaved very slowly by trypsin, a property also seen for native collagen III, supporting a local structural relaxation of the triple helix near the collagenase cleavage site. The recombinant bacterial-human collagen system characterized here is a good model to investigate the specificity and mechanism of action of collagenases.     

Human skin collagen. Presence of type I and type III at all levels of the dermis.

Human skin was sliced with a dermatome, and the ratio of type I to type III collagens at various depths was assayed by comparing the quantities of peptides of each derived from cyanogen bromide digestion of the cut skin. Although immunofluorescent studies have suggested type III collagen is located predominantly beneath the epidermis and around appendages, biochemical determination demonstrates the same ratio of type I to type III collagen at all levels of the dermis even in the absence of cutaneous appendages.     

Identification of cyanogen bromide peptides involved in intermolecular cross-linking of bovine type III collagen.

Cyanogn bromide peptides derived from bovine type III collagen and containing reducible cross-links were isolated and identified. Two peptides, alpha 1 (III)CB7 and alpha 1 (III)CB9B, from within the helical portion of the molecule were shown to contain the 'amino donor' residues cross-linked to non-helical 'aldehyde donor' residues in the formation of cross-links. This information, in conjunction with previously published data for the order of the cyanogen bromide peptides [Fietzek, Allman, Rauterberg & Wachter (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 84-86], suggests that in type III collagen intermolecular cross-links are located in the end-overlap regions, so as to stabilize a quarter-stagger arrangement of molecules within the fibre in a similar manner to that proposed for type I and type II collagens.     

Isolation of human type X collagen and immunolocalization in fetal human cartilage

Type X collagen was extracted with 1 M NaCl and 10 mM dithiothreitol at neutral pH from fetal human growth plate cartilage and purified to homogeneity by gel filtration and anion-exchange chromatography. The purified protein migrates in SDS/polyacrylamide gels with an apparent Mr of 66,000 under reducing conditions, and as a high-Mr oligomer under non-reducing conditions. Purified collagenase digests most of the molecule; pepsin digestion at 4 degrees C decreases the Mr of the monomer to 53,000. A rabbit antiserum was raised against purified human type X collagen; the IgG fraction was specific for this collagen by criteria of ELISA and immunoblotting after absorption with collagen types I, II, VI, IX and XI. Immunohistological studies localized type X collagen exclusively in the zone of hypertrophic and calcifying cartilage.     

Immunofluorescent localization of type III collagen and the N-terminal propeptide of type III procollagen in dentin matrix in osteogenesis imperfecta

Demineralized deciduous and permanent teeth from seven patients with six different types of osteogenesis imperfecta (OI) and from four unaffected controls were stained for type III collagen and for the N-terminal propeptide of type III procollagen using indirect immunofluorescence. Sillence types IA, IB and III OI were each represented by one patient. Two patients had type IVB and two had unclassifiable OI. After enzymatic treatment, the dentin matrix of one patient each with type IB OI, type IVB, and unclassifiable OI reacted with the specific antibodies against both type III collagen and the N-terminal propeptide. Positive staining was observed around the pathological canal-like structures and as delicate strands traversing the matrix. The similar patterns of immunofluorescence for both antigens in dentin in OI are suggestive of retention of the N-terminal propeptide in association with type III collagen identical to that in normal nonmineralized connective tissues. The abnormal presence of type III collagen in dentin in OI may be secondary to the aberrant structure of type I collagen. The failure of dentin matrix of all patients with OI to immunostain for type III collagen and the N-terminal propeptide may reflect heterogeneity or additional secondary changes in matrix macromolecule interactions.     

Co-ordinate increase in the expression of type I and type III collagen genes in progressive systemic sclerosis fibroblasts.

Progressive systemic sclerosis (PSS), is a connective tissue disease characterized by excessive accumulation of collagen in the skin and various internal organs which is due, at least in part, to increased collagen production by PSS fibroblasts. In order to examine the molecular mechanisms responsible for this abnormality, we compared the kinetics of collagen biosynthesis, the intracellular degradation of collagen and the expression of Types I and III procollagen genes between normal and PSS dermal fibroblasts in culture. Two age- and sex-matched normal and PSS dermal fibroblast cell lines were studied. The results showed that the PSS cultures produced higher amounts of collagen than did normal fibroblasts and displayed an abnormal kinetic pattern. Furthermore, the PSS cells showed a slight but statistically significant increase in the fraction of collagen degraded intracellularly when compared with normal cells (23% against 18% respectively). The levels of mRNA for procollagen Types I and III were determined by Northern and dot-blot hybridization with specific cloned cDNA probes for alpha 1(I), alpha 2(I) and alpha 1(III) and it was found that they were 2-3-fold higher for each of the three chains in the PSS cell lines compared with the controls. These findings indicate, therefore, that the overproduction of collagen characteristic of PSS fibroblasts can be largely accounted for by the increased levels of collagen mRNA.     

Differential expression of type I and type III collagen genes during tooth development

Collagen gene expression during mouse molar tooth development was studied by quantitative in situ hybridization techniques. Different expression patterns of type I and type III collagen mRNAs were observed in the various mesenchymal tissues that constitute the tooth germ. High concentration for pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were found within the osteoblasts. We found that the cellular content of type I collagen mRNAs in the odontoblasts varies throughout the tooth formation: whereas mRNA concentration for pro-alpha 1(I) collagen decreases and that of pro-alpha 2(I) increases, during postnatal development. Moreover, different amounts of pro-alpha 1(I) and pro-alpha 2(I) collagen mRNAs were observed in crown and root odontoblasts, respectively. Type III collagen mRNAs were detected in most of the mesenchymal cells, codistributed with type I collagen mRNAs, except in odontoblasts and osteoblasts. Finally, this study reports differential accumulation of collagen mRNAs during mouse tooth development and points out that type I collagen gene expression is regulated by distinct mechanisms during odontoblast differentiation process. These results support the independent expression of the collagen genes under developmental tissue-specific control.