Guinea Pig Skin Collagen Research Articles

Collagen in its fibrillar state is protected from glycation

To assess the impact collagen structures may have on glycation, the effects of glucose upon bovine serum albumin, guinea pig skin collagen, rat tail tendon and monomeric collagen were compared under near physiological conditions. Proteins were incubated with or without 50 mM glucose for 64 d in pH 7.4 50 mM phosphate buffer, followed by reduction, acid/alkaline hydrolysis, and analysis. Yields of non-reducible fructose-lysine, in the form of the acid-degradation products furosine and pyridosine, were significantly higher from skin collagen when compared to albumin. Yields of reducible fructose-lysine, in the form of glucitol- and mannitol-lysine, were conversely much greater for albumin, while tail tendon reported intermediate values. Fructose-lysine and unmodified lysine within collagen fibres prior to incubation was therefore protected by the tight packing of the collagen helices, where milling of tail tendon to increase the surface area exposed much of it to reduction protocols. Together with an analysis of pentosidine formation and other products, these results have shown that the interior of the tightly packed skin collagen fibres is protected from both glycation and reduction, and that glycation products differ depending on the protein incubated. Amino acid analysis then showed that our glycated skin collagen was similar to human diabetic skin collagen. Significant quantities of glucose-independent unknowns form in control incubations; their composition again being protein-dependent. The four compound Ks as previously reported were found to be unique to glycated rat tail tendon and soluble collagen, while another glycation product detected in collagen but not albumin may be attributable to carboxymethyl-arginine.

The gelatinolytic activity of rat uterus collagenase.

The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism.

Demonstration of collagenase activity in adult Strongyloides ratti (Nematoda:Strongyloididae) and its absence in the infective larvae.

Larvae and adults of Strongyloides ratti were examined for collagenolytic activity on 14C proline-labelled, native, guinea-pig skin collagen substrate. The activity was measured by determining either the amount of hydroxyproline released or the amount of radioactivity in the solubilized fraction of the collagen substrate. Bacterial collagenase was used for enzyme control and trypsin served as substrate control. No collagenolytic activity was found in living larvae, their extracts or metabolites. The collagenolytic activity of the metabolites of adult worms appeared weak, whereas that of the extracts of the adults was pronounced. It is suggested that collagenase is active in the adult females at the time of migration in the intestinal mucosa during oviposition.

The nonenzymatic glycosylation of collagen

Calf skin acid-soluble collagen, containing about 34 residues of lysine plus hydroxylysine per 100,000 dalton polypeptide chain, was treated with [ 14C]glucose in the presence or absence of NaCNBH 3. In 144 h, under the conditions employed, the presence of NaCNBH 3 increased the extent of glycosylation from 8 to 15% of the total residues of lysine plus hydroxylysine. The extent of glycosylation was estimated, using acid hydrolysates of the protein, by isolation and determination of reduced adducts (1-lysinohexitols) employing a system of paper chromatography followed by chromatography on an amino acid analyzer. By those means the difficulties of using specific color reactions such as that with thiobarbituric acid were obviated. Identification of the reduced adducts as forms of 1-lysinohexitol was made by comparison of that substance prepared by treatment of polylysine with [ 14C]glucose in the presence of NaCNBH 3. Of interest is the fact that treatment of the polymer with glucose for 144 h under conditions similar to those used for the collagen, resulted in an increase of extent of glycosylation from 3 to about 50% of the total lysine residues when NaCNBH 3 was present in the incubation medium. The greater degree of glycosylation of lysine residues in polylysine as compared with collagen (15 versus about 50%) may be ascribed to the different orders of macromolecular structure in the protein that could sequester certain of the residues from reaction with glucose. 1-Lysinohexitol was also identified in hydrolysates of neutral salt-soluble guinea pig skin collagen that had been reacted with glucose and then treated with NaB 3H 4. The glycosylated collagens were fragmented by treatment with CNBr, and modified lysine residues were found to occur along the entire length of the collagen chains. The use of NaCNBH 3 in the manner described above permits measurement of both aldimine and ketoamine forms of the adducts made with glucose. The possible physiological significance of the reversibility of the ketoamine form of adduct is discussed briefly.

Collagenase in keloid biopsies and fibroblasts.

Trypsin-activated collagenase activity was analyzed in media from keloid biopsies as well as keloid fibroblasts using radiolabeled guinea pig skin collagen as a substrate for collagenase digestion. Collagenase activity per tissue dry weight and DNA was similar in keloid and normal dermal biopsies; however, it was significantly elevated in keloid compared to normal dermal fibroblast media. In two paired cell lines where keloid and normal dermal fibroblasts were isolated from the same patient, relative collagen synthesis and trypsin-activated collagenase were increased to a similar extent in the keloid cell lines compared to normals. The biopsy data are in agreement with other findings, and further confirm the hypothesis that increased collagen deposition in keloids does not result from decreased collagen degradation. In vitro, however, the data presented here indicate that in some cell lines increased collagen synthesis may be associated with increased trypsin-activated collagenase.

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.

Post-proline cleaving enzyme. Purification of this endopeptidase by affinity chromatography.

The endopeptidase, post-proline cleaving enzyme, has been purified 10,500-fold in an overall yield of 18% from lamb kidney. The enzyme possesses a specific activity of 45 mumol/mg/min as tested with the substrate Z-Gly-Pro-Leu-Gly (Km = 6.0 X 10(-5)), has a molecular weight of 115,000, is comprised of two subunits with a molecular weight of 57,000, and exhibits maximal activity at pH 7.5 to 8.0. With the exception of the -Pro-Pro linkage, the -Pro-X-peptide bond (X equals L- and D-amino acid residues) located internally in the peptide sequence can be hydrolyzed (cleavage occurs faster when X = lipophilic side chain as compared to X = acidic side chain). The appropriate -Pro-X- bonds in zinc-free porcine insulin, oxytocin, arginine vasopressin, angiotensin II, bradykinin-potentiating factor were cleaved. Human gastrin, adrenocorticotropic hormone, denatured guinea pig skin collagen, and ascaris cuticle collagen were not degraded. Dipeptides with the structure Z-Pro-LD-X competitively inhibit post-proline cleaving enzyme.

Maturation of collagenous tissue: Specific [formula omitted] proteolytic cleavage of only α1(I) chains

A partially cleaved α1(I) chain, α1 χ, has been isolated from earlier synthesized or older (acid-extracted) guinea pig skin collagen. The α1 χ component is shown to be absent from the newly synthesized (neutral salt-extracted) collagen. This degradation is a result of specific in vivo proteolytic sission of α1(I) chain since the soluble collagen has no corresponding product from the α2 chain. The in vivo proteolytic cleavage is believed to result from processes related to natural physiological maturation of collagenous tissue.

Pepsin-solubilized collagen of human nucleus pulposus and annulus fibrosus

Human nucleus pulposus and annulus fibrosus, obtained at autopsy from patients 7–30 years of age, were extracted with 2 M guanidine HCl (pH 5.82) to remove proteoglycans, then stirred with pepsin in 0.5 M acetic acid, followed by three 24-h extractions with 1 M NaCl (pH 7.5) and one 24-h extraction with 2 M KSCN (potassium thiocyanate) (pH 7.2). Pepsin and NaCl solubilized an average of about 30% of nucleus pulposus collagen and 18% of annulus fibrosus collagen. KSCN extracted a further 34% of nucleus pulposus collagen and only 4% of annulus fibrosus collagen. CM-cellulose chromatography of nucleus and annulus collagen purified from the pepsin, NaCl and KSCN supernatants consistently revealed only one peak, always appearing slightly ahead of the α 1 position for rat tail tendon type I collagen. Polyacrylamide and SDS-gel electrophoresis consistently revealed only one band with the mobility of α 1 chains. Amino acid composition of collagen from nucleus and annulus is comparable to those of mammalian and avian cartilage type II collagen, and distinctly different from those of rat tail tendon and guinea pig skin type I collagens. Periodate oxidation of nucleus and annulus collagens showed that 81% and 67%, respectively, of the hydroxylysine residues survive treatment, compared to 71% for bovine articular cartilage collagen and 17% for guinea pig skin collagen. Total hexose analysis revealed 1.8 μM and 2.0 μM hexose per μM periodate-stable hydroxylysine in nucleus and annulus collagens, respectively. Ion exchange chromatography showed the presence of glucose and galactose in a ratio of 0.92:1 in nucleus collagen and 1.07:1 in annulus collagen. Pepsin-solubilized, NaCl-extracted collagen from nucleus and annulus formed native-type fibrils in vitro. The banding patterns of ATP-induced segment-long-spacing precipitates of nucleus and annulus collagens were identical to each other and indistinguishable from those of cartilage (type II) collagen, but distinctly different from those of rat tail tendon (type I) collagen. These data suggest that the collagen which can be extracted after limited pepsin attack of human nucleus and annulus is of the form [ α 1 ( II)] 3.

The Order of Cyanogen-Bromide Peptides and Location of Carbohydrate in the alpha2 Chain of Guinea-Pig Skin Collagen

The alpha2 chain of guinea pig skin collagen contains two additional methionyl residues in comparison with the alpha2 chain of other vertebrate species. The order of the three CNBr peptides unique to the alpha2 chain was established on the basis of the homology of their primary structures to sequences of previously ordered regions in the alpha1 and alpha2 chains of other colagens. The two larger peptides, 4A + 4B, were found to correspond to the region homologous to alpha2-CB4 of other species, while the smaller peptide, 3A, was homologous to the NH2-terminal portion of alpha2-CB3. Thus, the order of the peptides in the alpha2 chain of this collagen is 1-O-4A-4B-2-3A-3B-5. Periodate oxidation and alkaline or acid hydrolysis of the CNBr fragments showed that all of the hydroxlysine-linked carbohydrate in the alpha2 chain was present in alpha2-CB4B. Carbohydrate analyses were most consistent with the existence of single monosaccharide and disaccharide units in this region.

The effect of chemical or enzymatic modifications upon the ability of collagen to form multimers and to initiate platelet aggregation

The previous paper (1) presented some correlations between collagen multimer formation and platelet aggregation. The present study investigates the effect of chemical or enzymic modification upon its melting temperature and rate of multimerization, and therefore upon platelet aggregation. Digestion of collagen with tadpole or bacterial collagenase led to a decreased melting point of the collagen fragments and prevented their initiation of platelet aggregation. In contrast, the absence of interchain crosslinks, in lathyritic collagen, had no effect on these parameters and thus these crosslinks are not required for collagen multimer formation and induction of platelet aggregation. Oxidation of the galactoses alone with galactose oxidase, or of all the sugars by periodate, led to a perturbed melting curve featuring two melting points, ∼ 28° and 37°. These treated collagen preparations were thus unable to form multimers at platelet aggregation (> 29°) temperatures, and they could not initiate the aggregation of platelets. Reduction with sodium borohydride regenerated a normal melting curve, multimerization, and platelet aggregation initiation. Thus the carbohydrate residues, and a majority of the intact tropocollagen chains, are required for the maintenance of native structure and of multimerization at 37° which, in turn, are necessary for initiation of platelet aggregation by guinea pig skin collagen.

The effect of the conformation of collagen on its ability to aggregate platelets

The interaction between platelets and collagen, which marks the initiation of hemostasis, was examined as a function of the conformation and the multimerization of soluble guinea pig skin collagen. Both the rate and extent of collagen multimerization, as measured by the intensity of scattered light, are dependent on the time of incubation and on the temperature of the system. Compounds such as arginine or penicillamine, which at low concentrations (< 10 −2 M) inhibit collagen crosslinking, in higher quantities (> 2 × 10 −2 M) decrease the ability of collagen to form multimers and hence to initiate platelet aggregation. By these techniques we have been able to correlate the initiation of platelet aggregation by collagen with the extent of preformed multimers, the lag time approaching zero as the multimerization of the initiating collagen increases.

Comparison of the cyanogen bromide peptides of insoluble guinea-pig skin and scar collagen

Insoluble guinea-pig skin collagen and insoluble dermal scar collagen were cleaved with CNBr and the peptides derived from the α 1- chain were separated by ion-exchange and molecular-sieve chromatography. Comparison of the peptides from scar collagen with those from skin collagen showed that the former contained more hydroxylysine. Separation of the CNBr peptides showed that this increase in hydroxylysine was found not only in the non-helical regions, but was also seen down the helical portion of the molecule. Separation of the peptides revealed the presence of more peptides in digests of skin collagen than those of scar, and these have been attributed to the presence of Type III collagen in skin, but no evidence was found for the presence of this Type III collagen in dermal scar tissue.

Cyanogen bromide cleavage of guinea pig skin collagen. Isolation and characterization of peptides from the 1 and 2 chains.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCyanogen bromide cleavage of guinea pig skin collagen. Isolation and characterization of peptides from the α1 and α2 chainsC. C. Clark and Paul BornsteinCite this: Biochemistry 1972, 11, 8, 1468–1474Publication Date (Print):April 1, 1972Publication History Published online1 May 2002Published inissue 1 April 1972https://doi.org/10.1021/bi00758a022RIGHTS & PERMISSIONSArticle Views59Altmetric-Citations20LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (727 KB) Get e-Alerts Get e-Alerts

The collagen-like triple helix to random-chain transition: Experiment and theory

The thermal stabilities in solution of guinea pig skin collagen and three synthetic polytripeptide models for collagen, poly( l-prolyl- l-seryl-glycyl), poly( l-prolyl- l-alanyl-glycyl), and poly( l-prolyl-glycyl- l-prolyl), were monitored by observing the temperature-dependence of the circular dichroism spectra. Analysis of the melting temperatures for these polytripeptide sequences suggested a number of factors which should be included in a description of the thermodynamic stability of the collagen triple helix. Among these contributions are: (a) the primary sequence; (b) the molecular weight and polydispersity of the polymer; and (c) the extent of polymer-solvent interaction. A semi-empirical equation incorporating these factors was derived to describe the two-state triple helix to random-chain transition. The enthalpy and entropy parameters employed in the equation, with the exception of those involving polymer-solvent interactions, were calculated by theoretical conformational analysis. In order to evaluate the polymer-solvent interaction energies, it was necessary to calibrate the equation for each solvent system. The predicted versus observed melting temperatures were compared for the synthetic polymers in two solvents of different polarities, water and 1,3-propanediol.

Synthesis and structural studies of two collagen analogues: Poly ( l-prolyl- l-seryl-glycyl) and poly ( l-prolyl- l-alanyl-glycyl)

Poly ( l-prolyl- l-seryl-glycyl) and poly ( l-prolyl- l-alanyl-glycyl), two synthetic polytripeptide analogues of the non-polar regions of collagen, were synthesized by polymerization of the tripeptide p-nitrophenyl esters with C -terminal glycine residues. Circular dichroism studies of the polymers indicated the presence of an ordered collagen-like structure for each polymer in solution. This ordered structure was shown to be solvent-dependent and also dependent on the molecular weight and concentration of the polymer in solution. Both polytripeptides were investigated in a variety of solvents ranging in relative hydrogen-bonding strengths from 1,3-propanediol to hexafluoroisopropanol, and an increase in the amount of ordered structure with decreasing hydrogen-bonding strength of the solvent was observed. In 1,3-propanediol, the solvent system in which the polymers exhibited the most ordered structure at room temperature, both polymers exhibited circular dichroism spectra with identical positions of the maxima and minima and similar rotatory strengths to those observed for guinea pig skin collagen. The slow attainment of stable circular dichroism spectra of heated solutions, coupled with the demonstrated concentration dependence of the spectra, shows that aggregation of the polymers takes place in solution. Molecular weight studies of both polytripeptides in their single-chain random conformation versus the presumed triple-helical associated form showed the expected threefold association of the peptide chains in solution. The threefold change in molecular weight, coupled with the similarity between the circular dichroism spectra of the polytripeptides and collagen, suggests that the observed optical properties arise from the collagen-like triple-helical structure found for both polymers in the solid state.

Bacterial glycoproteins: Identification and properties of glycoprotein glycosyltransferases in Escherichia coli

Nucleoside diphosphate [ 14C] monosaccharides were efficient precursors for the transfer of [ 14C]monosaccharides onto endogenous glycoproteins by 0.1% Triton X-100 homogenates of Escherichia coli. The order of incorporation was UDP-[ 14C]-glucose > GDP-[ 14C]mannose > UDP-N-[ 14 C] acetylglucosamine > UDP-[ 14C]galactose > GDP-[ 14C]fucose > UDP-N-[ 14 C] acetylgalactosamine . E. coli glycosyltransferases were found to transfer radioactivity from nucleoside diphosphate [ 14C]monosaccharides onto a variety of mammalian glycoprotein acceptors. Five E. coli glycosyltransferases were characterized using exogenous acceptors. The acceptors for the respective nucleoside diphosphate monosaccharides were as follows: Fetuin minus N-acetylneuraminic acid:UDP- N-acetylgalactosamine; fetuin minus N-acetylneuraminic acid, galactose, N-acetylglucosamine:UDP- N-acetylglucosamine; bovine submaxillary mucin minus N-acetylneuraminic acid:UDP-galactose; guinea pig skin collagen minus glucose:UDP-glucose; fetuin minus N-acetylneuraminic acid, galactose:GDP-mannose. The E. coli glycosyltransferases utilizing exogenous acceptors were purified 49–226-fold with recoveries of 9–49% by centrifugation and successive column chromatography on Sephadex G-100 and G-200. This purification scheme separated the transferases entirely from their endogenous acceptors. These purified E. coli glycosyltransferases had optimum temperatures between 35° and 40° and pH between 7 and 8. Hydrolysis of the radioactivity transfered and chromatography of the product revealed that the monosaccharides were transferred without isomerization or epimerization. The enzymes were absolutely dependent on divalent cations for activity, Mn 2+, Mg 2+, and Co 2+ being the most effective activators. Hg 2+, Fe 2+, Ba 2+, and Cd 2+ inhibited the E. coli glycoprotein glycosyltransferases. The transferases had an absolute dependence on the macromolecular glycoprotein acceptor, being specific for terminal monosaccharide and carbohydrate and amino acid sequence of the acceptor. Mono- and disaccharides were ineffective as acceptors.

Immunochemical studies on collagen—II Antigenic differences between guinea pig skin collagen and gelatin

Immunochemical studies on collagen—II Antigenic differences between guinea pig skin collagen and gelatin

Immunochemical studies on collagen—II Antigenic differences between guinea pig skin collagen and gelatin

Immunization of rabbits with guinea pig skin collagen elicited antibody with specificity for α2-CB1, a cyanogen bromide derived peptide from the amino-terminal region of α2, whereas, the α1 chain and peptides derived from it by cleavage with cyanogen bromide were essentially devoid of antigenic activity. Immunization with gelatin (denatured collagen) resulted in the formation of antibodies with specificities for α2-CB1 as well as to a portion of the α1 chain. Antiserum prepared against native collagen contained antibodies which reacted with antigens detected only in gelatin. The denaturation accompnying the transformation of collagen to gelatin is believed to expose immunogenic and antigenic sites previously hidden in the native molecule.

A Comparative Study of Glycopeptides Derived from Selected Vertebrate Collagens: A POSSIBLE ROLE OF THE CARBOHYDRATE IN FIBRIL FORMATION

Abstract Glycopeptides from carp swim bladder, guinea pig skin, and human skin collagens were prepared by sequential collagenase-trypsin digestion. Gel filtration on Sephadex G-25 permitted the isolation of a low molecular weight fraction containing most of the hydroxylysyl glycosides present in the parent collagen. Application of this fraction to a Dowex 50X-8 ion exchange column consistently resulted in distinctive glycopeptide patterns characteristic of each collagen. The glycohexapeptide, Gly-Met-Hyl (Gal-Glc)-Gly-His-Arg, which had been previously characterized in digests of soluble guinea pig skin collagen, was found in significant quantities in all three collagens examined, but the carp and human collagens each revealed an additional major glycopeptide. The subtractive Edman procedure was used in establishing amino acid sequences, and alkaline hydrolysis was used to confirm the composition and mode of attachment of the carbohydrate side chains. The predominant glycopeptide isolated from carp swim bladder collagen was Gly-Ile-Hyl(Gal-Glc)-Gly-His-Arg, while the structure of the unique glycopeptide isolated from human skin collagen was Gly-Phe-Hyl(Gal-Glc)-Gly-Ile-Arg. A possible role of the carbohydrate side chains in the organization of fibrils is discussed.