Human Skin Collagen Research Articles

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.

Enhancement of platelet response to immune complexes and IgG aggregates by lipid A-rich bacterial lipopolysaccharides.

The effect of the common lipid moiety of bacterial LPS on secretion from washed human platelets has been studied. The lipid A-rich LPS of S. minnesota R595 and a lipid A preparation both potentiated platelet serotonin secretion in response to IgG aggregates or immune complexes up to 50-fold but had little effect in the absence of IgG. Lipid A has been shown to bind immune aggregates, raising the possibility that its mechanism of action involved effective enlargement or insolubilization of the aggregates. IgG aggregates of dimer to tetramer size were shown to be platelet simuli, equivalent on a weight basis to larger soluble aggregates. The effect of both sizes of aggregates on platelets were equally enhanced by the LPS, indicating that increased size of aggregates alone could not account for the effect of LPS. Similarly, because lipid A-rich LPS enhanced platelet response to already insoluble immune complexes, its mechanism of action cannot simply be insolubilization of immune aggregates. These LPS did not enhance platelet stimulation by antiplatelet antibody, monosodium urate crystals, or thrombin and only slightly enhanced stimulation by insoluble human skin collagen. This indicates some stimulus specificity in the ability of LPS to increase platelet secretion. The enhancement of cell response to immune complexes by the common lipid region of LPS may represent a mechanism for the diverse effects of LPS in vivo and in vitro.

Age-related changes in human skin collagen galactosyltransferase and collagen glucosyltransferase activities

Collagen galactosyltransferase and collagen glucosyltransferase activities were assayed in human skin specimens of about 100 mg wet weight. The assay of the glucosyltransferase activity was found to be highly specific. The assay of the galactosyltransferase activity was somewhat less specific, but there was no difference in specificity between the foetal and adult human skin samples.The activities of the two collagen glycosyltransferases in human skin extract were found to vary with age, being highest in foetal skin, and higher in the skin of young children than in that of adults. The galactosyltransferase and glucosyltransferase activities in foetal skin were respectively about 4 times and 6 times those in adult skin. The magnitudes of the changes with age in the two collagen glycosyltransferase activities were smaller than those occurring in the activities of the two other intracellular enzymes of collagen biosynthesis, namely prolyl and lysyl hydroxylase. This difference suggests that the four intracellular enzyme activities of collagen biosynthesis are not regulated in an identical manner.

Comparative Study of Carbohydrate-Protein Complexes II. Determination of Hydroxylysine and Its Glycosides in Human Skin and Scar Collagens by an Improved Method

A modification of the existing methods for measuring hydroxylysine, galactosylhydroxylysine, and glucosylgalactosylhydroxylysine is described. The method is based on analysis with an automated amino acid analyzer using a conventional separation system for basic amino acids. The prior removal of acidic and neutral amino acids was necessary. This was achieved by passing an alkaline hydrolysate of collagen through a column of Amberlite CG-120, Type II (H+) and washing the column with 8% aqueous pyridine. A basic fraction containing the hydroxylysine compounds was then recovered from the column by elution with 3 M NH4OH. Model experiments showed that hydroxylysine and its glycosides could be analyzed with an hour and that recoveries exceeded 90%. This method was applied to human tissues to investigate whether the dermal scar is different in collagen composition from normal skin. With the limited number of samples analyzed, the data suggested that long-standing scar tissues reverted to a composition similar to that of normal skin. The composition of hydroxylysine-linked carbohydrate units is also discussed on the basis of the age-related change.

Collagenase inhibition by cationic proteins derived from cartilage and aorta

Collagenolytic activity from normal human skin and human hypertrophic scar tissue are inhibited by protease inhibitors isolated by immobilized trypsin affinity chromatography from bovine cartilage and bovine aorta. The collagenase inhibitors are of low molecular weight (approximately 11,000) and are cationic proteins. Other trypsin inhibitors such as the bovine basic pancreatic trypsin inhibitor (Kunitz) as Trasylol R and soybean trypsin inhibitor show only minimal effects on collagenase activities. Human skin collagenase is inhibited by a protein (protease-inhibitor) obtained from human cartilage using human skin collagen as substrate. The inhibitors described may function in the physiological regulation of collagenase activity in connective tissues.

The diagnostic application of specific anti-procollagen sera: I. Analysis of skin biopsies

Antisera were prepared in rabbits against mature calf skin collagen (type I) and a polypeptide characteristic for calf skin procollagen (type I). The latter peptide is attached to the aminoterminal end of the α-chains and cleaved off during normal maturation by a specific enzyme, procollagen peptidase. For the present study procollagen was isolated from the skin of calves with an inborn deficiency of procollagen peptidase, a disease called dermatosparaxis. The antisera to mature collagen and procollagen were rendered specific by appropriate immunoadsorption procedures. These sera show a strong cross-reaction with human skin collagen and procollagen. While anti-collagen sera produced indirect immunofluorescent staining of the whole dermis, anti-procollagen sera only react with the uppermost dermal layer (stratum papillare) of unfixed frozen sections of normal human skin biopsies. Frozen unfixed sections of skin biopsies from 50 patients with various diseases were analyzed in indirect immunofluorescence tests with anti-procollagen sera. In 19 cases, material from uninvolved skin areas were also available for study. Skin from patients with “collagen diseases” (systematic lupus erythematosus, dermatomyositis, scleroderma) reacts like normal skin. Skin from patients with psoriasis vulgaris and lichen planus shows broad subepithelial zones of procollagen fluorescence. Keloids display very weak or no procollagen immunofluorescence in several instances. In three studied cases with prolonged local corticoid treatment a normal procollagen immunofluorescence pattern reappeared. Biopsies from one case of Wilson's disease under prolonged d-penicillamine therapy and one case of epidermolysis bullosa also lacked reactivity with anti-procollagen sera. Uninvolved skin of all patients revealed normal procollagen immunofluorescence. It is concluded that immunofluorescence studies with specific antisera to procollagen provide a simple and exact tool for the localized analysis of collagen metabolism.

Collagen-and collagen peptide-induced chemotaxis of human blood monocytes.

The ability of collagen and collagen-derived peptides to act as chemotactic stimuli was investigated by in vitro chemotaxis assays. Native human and chick skin collagen (type I) and alpha-chains obtained from purified chick skin collagen were each chemotactic for human peripheral blood monocytes. In addition, smaller peptides obtained either by digesting native collagen with bacterial collagenase or by degrading purified alpha-chains with cyanogen bromide or pepsin were also chemotactic for monocytes. In contrast, native collagen, alpha-chains, and smaller collagen-derived peptides were not chemotactic for human neutrophils. Since collagen is degraded at sites of tissue damage and inflammation, our findings suggest the possibility that such collagen-derived degradation products might directly serve as chemotactic stimuli for human peripheral blood monocytes in vivo.

Age-related variations in glycosylation of hydroxylysine in human and rat skin collagens

The extent of glycosylation of hydroxylysine in human skin collagen rapidly decreased during maturation and then gradually increased in proportion to the age. This decrease of glycosylation observed during maturation was also confirmed in whole, soluble and insoluble collagens from rat skin. These findings may contribute to the investigations on the functional role of glycosylation and also on the mechanism of maturational as well as senile processes.

The effects of cyclophosphamide and azathioprine on collagen in skin and granulation tissue in rats, and the effects of cyclophosphamide on collagen in human skin.

Granulation tissue was produced in rats by subcutaneous implantation of cellulose sponges. The effect of daily intraperitoneal injections of cyclophosphamide or azathioprine on rat skin and granulation tissue was examined after 14 and 42 days. Skin biopsies from patients with glomerulonephritis were analyzed before and after 42 days of treatment with cyclophosphamide or placebo. In the rats, cytostatic treatment caused an increase in the dry weight of the skin, and azathioprine increased the dry weight and the protein content of the granulomas. The increase in the dry weight was accompanied by a decrease in water percentage. The alpha-amino nitrogen/OH-proline ratio in purified acid soluble collagen from skin and granulation tissue increased with the dose and duration of cytostatic treatment. No effect on the aldehyde content was observed. Cyclophosphamide caused a decrease in the alpha/beta ratio in acid soluble collagen from granulation tissue, but not in the collagen from the skin. Salt soluble collagen was increased in the skin after 14 days of cytostatic treatment, but remained unchanged in the granulation tissue. In human skin cyclophosphamide caused no statistically significant changes in the amount of salt soluble or total collagen. It is concluded, that daily treatment of rats with cyclophosphamide or azathioprine from 14 to 42 days seems to inhibit the catabolic processes in the skin and granulation tissue, to decrease the hydroxylation of proline in collagen, and to inhibit the intermolecular cross-linking in collagen.?222

Proteins of the periodontium. Biochemical studies on the collagen and noncollagenous proteins of human gingivae.

Abstract. Insoluble collagen was prepared from gingivae of full term, stillborn infants (SI) and from that of patients exhibiting gingival hyperplasia that was secondary to diphenylhydantoin therapy (DPH). During the preparations only 4 % of the collagen from DPH was extracted and 17 % from SI gingivae was solubilized, by neutral salt and dilute acid extractants. Subsequent amino acid analysis indicated the presence of a relatively large quantity of the noncollagenous protein(s) in the DPH collagen preparation. After collagen samples were converted to soluble cyanogen bromide (CNBr) pep‐tides, the noncollagenous protein(s) proved to be insoluble in the dilute citrate buffer routinely used for initial chromotographic separation of the CNBr peptides. This fraction, whether from DPH or SI gingivae, had essentially the same composition. The noncollagenous protein fraction comprised about 7 and 20 %, respectively, of the dry weights of the SI and DPH insoluble collagen preparations.CNBr peptides from gingival collagen have been purified and shown to be identical with those reported for human skin collagen. Thus, the collagen of gingivae is principally of the type with the chain structure [αl(I)]2α2. Preliminary data strongly suggest the presence in gingivae of a lower level of collagen of the [al (III)]s type.

Isolation, characterization and comparative studies of the N-terminal peptides from soluble pig skin collagen

Collagen is an ubiquitous structural protein, which constitutes about 25-30% by weight of all proteins in the mammalian body. It functions as an important supporting element in vertebrate tissues. Using the method of cleaving the protein chains at the methionyl residues with cyanogen bromide (CNBr), many species of collagen from different sources have been isolated and characterized, e.g. rat skin [l ,2] , chick skin [3] , calf skin [4,5] and human skin collagen [6] . Pig skin collagen seems to be interesting from the point of view, that is, its applicability in the medical sector [7,11,12] , due to its poor or non-antigenic reactivity. We reported previously about the isolation and characterization of the large CB-peptides derived from both oiland cw2-chain of the pig skin collagen [8]. This report deals with the isolation, characterization and comparison of the N-terminal CB-peptides of soluble pig skin collagen with the corresponding peptides derived from rat skin, calf skin and human skin collagen.

The Distribution of Carbohydrate Groups in Rat Skin Collagen

Abstract Glycopeptides from soluble rat skin collagen were prepared by sequential digestion with collagenase and trypsin. The isolation of the major glycopeptides was accomplished with gel filtration and ion exchange chromatography. The glycohexapeptide, Gly-Met-Hyl (Gal-Glc)-Gly-His-Arg, has previously been characterized in digests of various vertebrate collagens studied in this and other laboratories. Two other glycopeptides known to be present in digests of human skin collagen, Gly-Phe-Hyl(Gal-Glc)-Gly-Ile-Arg and Gly-Pro-Hyl(Gal) were also isolated from digests of rat skin collagen. Three additional glycopeptides which had not been previously reported in other vertebrate collagens were purified and their structures analyzed. Two of them were hexapeptides, Gly-Phe-Hyl(Gal)-Gly-Ile-Arg and Gly-Pro-Hyl(Gal)-Gly-Glu-Leu, while the other was a tripeptide, Gly-Met-Hyl(Gal). Characterization of the glycopeptides obtained from the purified α1 and α2 components of rat skin collagen and hexose analysis of the cyanogen bromide (CNBr) peptides derived from whole collagen as well as from both chains indicate the presence of only four major sites of glycosylation in the molecule. The evidence presented is consistent with the existence of an identical site in each α1 chain, corresponding to a disaccharide unit near the NH2 terminus of the α1-CB5 cyanogen bromide peptide. The other two sites are located in the CNBr peptide α2-CB4 derived from the α2 chain. There are, in addition, other minor sites of glycosylation in the molecule. The limited number of sites of glycosylation and the location of all such sites in the same general area of the α chains near the NH2 terminus suggest a specific but unknown functional role in synthesis, fibril formation, or other basic aspects of collagen metabolism in skin.

Corticosteroid-induced inhibition of the biosynthesis of human skin collagen

The effects of hydrocortisone acetate, fluocinolone acetonide, fluclorolone acetonide, betamethasone-17-valerate, fluprednyliden-21-acetate and flumethasone pivalate on the biosynthesis of human skin collagen were studied in vitro. Skin specimens were incubated in a medium containing a test substance and radioactive proline, and the formation of radioactive hydroxyproline in nondialysable proteins was taken as an index of the rate of collagen biosynthesis. Hydroxyproline formation was inhibited by all the corticosteroids tested in concentrations of 30 μg/ml or higher. The effect on hydroxyproline formation was smallest with hydrocortisone acetate and most pronounced with betamethasone-17-valerate in all concentrations used. In general, the corticosteroid-induced inhibition of collagen biosynthesis was found to be dose-dependent. The differences in the degree to which collagen formation is inhibited by the different corticosteroids may have relevance to the extent of the local side-effects, such as atrophy of skin, reported to be produced by fluorinated corticosteroids.

Comparative solubility of bone and skin collagen in primates including man.

Man and the chimpanzee were shown to have less soluble bone collagen than the baboon and rhesus monkey in the tibia, iliac crest, and vertebral body. The DNA content of these three bones was significantly less in man than in the other primates and the rat; the monkey and the baboon had lower values than did the rat. In man, the cell content was approximately the same in the tibia, iliac crest and vertebral body while it was 3-4 times greater in the vertebral body in the monkey and baboon. Soluble collagen in human skin was less than in all other species; all primates had relatively less acid-soluble collagen than the rat. Collagen biosynthesis in vitro in baboon skin and bone was greater than that in man and less than that in the rat. The data suggest significant differences between the primate species in the biosynthesis and turnover of collagen although structural differences cannot be ruled out.

Connective tissue in scleroderma. A biochemical study on the correlation of fractionated glycosaminoglycans and collagen in human skin

No abstract available

Peptide mapping of partial acid hydrolysates of human dentin and bovine skin collagens by thin-layer methods.

Human dentin and bovine skin collagens, partially hydrolyzed with acid, were compared by peptide mapping on thin-layer plates. The patterns, which showed 20 to 25 spots, were similar for the two types of collagen. No major differences in the structural characteristics of dentin and skin collagens can be predicted from these results.

Identification of three genetically distinct collagens by cyanogen bromide cleavage of insoluble human skin and cartilage collagen

Cyanogen bromide cleavage of insoluble newborn human skin and cartilage in 70% formic acid liberates in good yield peptides derived from collagen. Characterization of these peptides and comparison with those previously derived from soluble human skin collagen permit the identification of an additional type of collagen in skin and yet another type in cartilage. The results further indicate that the new types of collagen are comprised of chains homologous to the α1 chain of soluble human skin collagen.

Isolation and Characterization of the Peptides Derived from Soluble Human and Baboon Skin Collagen after Cyanogen Bromide Cleavage

Abstract The α1 and α2 chains of soluble collagen from human and baboon skin were separated and cleaved with cyanogen bromide, and the resultant peptides were separated by ion exchange and molecular sieve chromatography. Eight peptides from the α1 chain and six from the α2 chain accounted for the weight and amino acid content of both collagens. Only small differences of questionable significance could be detected between the collagens of the two primate species by amino acid analysis or molecular weight of the peptides. The amino-terminal cross-link region of both the α1 and α2 chain is identical in both species. The peptides are very similar to those previously isolated from the rat and the chick, demonstrating a close homology in the helical portion of the molecule and, to a lesser extent, in the amino-terminal crosslink region.

Solubility of skin collagen in normal human subjects and in patients with generalised scleroderma

Solubility of human skin collagen was studied by extracting skin specimens with o.14 MNaCl solution. The extraction conditions were studied by varying the time and number of extractions. A method combining pooled supernatants from three successive extractions of 24 h was chosen for standard procedure of extracting the neutral salt-soluble fraction from human skin collagen. Using this method, the solubility of skin collagen in biopsies taken from 70 control subjects of various ages and from 27 patients with generalised scleroderma was determined. No changes in the degree of solubility during ageing could be observed, whereas the solubility in the skin of patients with scleroderma was increased.

Isolation and characterization of the cyanogen bromide peptides from the alpha 1 and alpha 2 chains of human skin collagen.

Isolation and characterization of the cyanogen bromide peptides from the alpha 1 and alpha 2 chains of human skin collagen.