Properties of radiolabeled type I, II, and III collagens related to their use as substrates in collagenase assays

Calf skin and rat tendon type I, bovine cartilage type II, and human amnion type III collagens have been radiolabeled by reaction with [3H]acetic anhydride, [3H]formaldehyde, and succinimidyl 2,3-[3H]propionate. All three reactions produce collagens with high specific activities that are suitable for use as substrates in collagenase assays. The identity of the radiolabel and the labeling indices do not alter the molecular weights or thermal stabilities of the collagens or the solubilities of the collagens or gelatins in dioxane-water mixtures at 4 degrees C. However, in contrast to native or sparsely labeled collagens, those with 40 or more lysine + hydroxylysine residues labeled per molecule do not undergo fibrillogenesis in the presence of 0.2-0.4 M NaCl in the 4-35 degree C temperature range. Thus, the modification reactions not only serve to introduce the radiolabel, but also to keep the collagens soluble over a wide range of temperatures and concentrations. The TCA, TCB fragments produced on partial reaction of each collagen type with tissue collagenases can be selectively denatured by a 10-minute incubation under specific conditions and the intact collagens selectively precipitated by addition of 50% v/v dioxane. This serves as the basis for soluble collagenase assays. The effect of labeling index on the properties of the collagens has been investigated and the results establish the range of conditions over which these collagens can be used as substrates for soluble versus fibrillar collagenase assays.     

Kinetics of hydrolysis of type I, II, and III collagens by the class I and II Clostridium histolyticum collagenases.

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Kinetics of hydrolysis of type I,II, and III collagens by the class I and IIClostridium histolyticum collagenases

The kinetics of hydrolysis of rat tendon type I, bovine nasal septum type II, and human placental type III collagens by class I and class IIClostridium histolyticum collagenases (CHC) have been investigated. To facilitate this study, radioassays developed previously for the hydrolysis of these [³H]acetylated collagens by tissue collagenases have been adapted for use with the CHC. While the CHC are known to make multiple scissions in these collagens, the assays are shown to monitor the initial proteolytic events. The individual kinetic parametersk _cat andK _M have been determined for the hydrolysis of all three collagens by both class I and class II CHC. The specific activities of these CHC toward fibrillar type I and III collagens have also been measured. In contrast to human tissue collagenases, neither class of CHC exhibits a marked specificity toward any collagen type either in solution or in fibrillar form. The values of the kinetic parametersk _cat andK _M for the CHC are similar in magnitude to those of the human enzymes acting on their preferred substrates. Thus, the widely held view that the CHC are more potent collagenases is not strictly correct. As with the tissue collagenases, the local collagen structure at the cleavage sites is believed to play an important role in determining the rates of the reactions studied.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I,II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I, II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the 1(I), 2(I), 1(II), and 1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

The characterization of type I and type III collagens from human peripheral nerve.

The normal chemical features of peripheral nerve collagens were determined on postmortem, histologically normal adult human femoral nerve. 1. Genetically distinct type I, [alpha1(I)2]alpha2, and type III, [alpha1(III)]3, were isolated by differential salt precipitation and the component subunit chains, alphal(I), alpha2 and alphal(III) were obtained by ion-exchange chromatography and gel filtration. 2. The molecular weight of alphal(I) and alpha2 of type I collagen was 95 000 and that for type III was 280 000. Reduction of type III with dithiothreitol yielded expected alpha1(III) chains of 95 000 molecular weight. 3. The amino acid composition of the three collagen chains, alpha1(I), alpha2, and alpha1(III), was the same as previously reported values for the corresponding chains from human skin except for slightly elevated hydroxylysine content. 4. Peripheral nerve collagen was found to contain 81% type I collagen and 19% type III. These results indicate that peripheral nerve collagen characteristics closely simulate that of human skin and differ from that of human aorta and other parenchymal organs. These data will permit a chemical analysis for possible abnormalities of peripheral nerve collagen in various neurogenic disorders.     

FBJ virus-induced osteosarcoma contains type I, type I trimer, type III as well as type V collagens

The FBJ osteosarcoma (a virus-induced osteosarcoma named after its discoverers, Finkel, Biskis, and Jinkins) contains an extensive extracellular matrix. Collagens were extracted by digestion with pepsin in dilute acetic acid from tumors grown in lathyritic mice and fractionated by differential salt precipitation, yielding five fractions. Fraction 1 (precipitated at acidic 0.7 M and neutral 2.0 M NaCl) gave rise mainly to alpha 1(III) chain on phosphocellulose column chromatography. The alpha 1(III) chain was identified by its typical behavior on interrupted electrophoresis and analysis of the CNBr-cleaved peptides. The alpha 1(III) chain of the FBJ tumor had a high content of hydroxylysine and neutral saccharide. Fraction 2 (precipitated at acidic 0.7 M and neutral 4.5 M NaCl) yielded alpha 1(I) and alpha 2(I) chains on the phosphocellulose column from which alpha 1(I) was eluted as a broad peak, conceivably reflecting a high content of hydroxylysine and neutral saccharide. Fraction 4 (precipitated at acidic 1.2 M and neutral 4.5 M NaCl) yielded type V collagen, which also featured an exceptionally high content of neutral saccharide (Yamagata, S., et al. (1982) Biochem. Biophys. Res. Commun. 105, 1208-1214). The proportions of type I, type I trimer, type III, and type V collagens extracted by pepsin digestion from FBJ tumor were calculated to be 33, 29, 26, and 12%, respectively. The FBJ tumor is free from invasion by blood vessels, shows no deposition of calcium, and thus has the appearance of cartilage. But type II collagen, a specific gene product of cartilage, could not be identified in any of the fractions analyzed. Contrary to its appearance, collagen type analyses indicate that FBJ osteosarcoma is literally induced from osteogenic cells.     

Characterization of type I,III and V collagens in high-density cultured tenocytes by triple-immunofluorescence technique

The purpose of this study is to examine the intracellular distribution of collagen types I, III and V in tenocytes using triple-label immunofluorescence staining technique in high-density tenocyte culture on Filter Well Inserts (FWI). The tenocytes were incubated for 4 weeks under monolayer conditions and for 3 weeks on FWI. At the end of the third week of high-density culture, we observed tenocyte aggregation followed by macromass cluster formation. Immunofluorescence labeling with anti-collagen type I antibody revealed that the presence of collagen type I was mostly around the nucleus. Type III collagen was more diffused in the cytoplasm. Type V collagen was detected in fibrillar and vesicular forms in the cytoplasm. We conclude that, the high-density culture on FWI is an appropriate method for the production of tenocytes without loosing specialized processes such as the synthesis of different collagen molecules. We consider that the high-density culture system is suitable for in vitro applications which affect tendon biology and will improve our understanding of the biological behavior of tenocytes in view of adequate matrix structure synthesis. Such high-density cultures may serve as a model system to provide sufficient quantities of tenocytes to prepare tenocyte-polymer constructs for tissue engineering applications in tendon repair.     

Accurate, quantitative assays for the hydrolysis of soluble type I, II, and III 3H-acetylated collagens by bacterial and tissue collagenases

Accurate and quantitative assays for the hydrolysis of soluble 3H-acetylated rat tendon type I, bovine cartilage type II, and human amnion type III collagens by both bacterial and tissue collagenases have been developed. The assays are carried out at any temperature in the 1-30 degrees C range in a single reaction tube and the progress of the reaction is monitored by withdrawing aliquots as a function of time, quenching with 1,10-phenanthroline, and quantitation of the concentration of hydrolysis fragments. The latter is achieved by selective denaturation of these fragments by incubation under conditions described in the previous paper of this issue. The assays give percentages of hydrolysis of all three collagen types by neutrophil collagenase that agree well with the results of gel electrophoresis experiments. The initial rates of hydrolysis of all three collagens are proportional to the concentration of both neutrophil or Clostridial collagenases over a 10-fold range of enzyme concentrations. All three assays can be carried out at collagen concentrations. that range from 0.06 to 2 mg/ml and give linear double reciprocal plots for both tissue and bacterial collagenases that can be used to evaluate the kinetic parameters Km and kcat or Vmax. The assay developed for the hydrolysis of rat type I collagen by neutrophil collagenase is shown to be more sensitive by at least one order of magnitude than comparable assays that use rat type I collagen fibrils or gels as substrate.     

Fibroblast behavior on gels of type I, III, and IV human placental collagens

Various collagens were extracted and purified from human placenta after partial pepsin digestion. We prepared type III + I (57:43), enriched type I, type III, and type IV collagens on an industrial level, and studied their biological properties with MRC5 fibroblast cells. Using the process of contraction of a hydrated collagen lattice described by Bell, we found tha the contraction rate was dependent on collagen type composition. The contraction was faster and more pronounced with pepsinized type I collagen than with pepsinized type III + I (57:43) collagen; the lowest rate was obtained with the pepsinized type III collagen. Using a new technique of collagen cross-linking, a gel was made with type IV collagen. This cross-linking procedure, based on partial oxidation of sugar residues and hydroxylysine by periodic acid, followed by neutralization, resulted in an increased number of natural cross-link bridges between oxidized and nonoxidized collagen molecules, without internal toxic residues. The fibroblasts were unable to contract type IV/IVox collagen gels. The type IV/IVox collagen gel was transparent and its amorphous ultrastructure lacked any visible striated fibrils. Fibroblast cells exhibited atypical behavior in these type IV/IVox collagen gels as evidenced by optical and electron microscopy. The penetration of fibroblasts could be measured. Fibroblasts penetrated faster in type IV/IVox collagen gels than in untreated type III + I collagen gels. The lowest rate of penetration was obtained with cross-linked type III + I gels. Fibroblast proliferation was similar on untreated or cross-linked type III + I collagen gels and slightly increased on type IV/IVox collagen gels, suggesting that this cross-linking procedure was not toxic.     

Susceptibility of cartilage collagens type II, IX, X, and XI to human synovial collagenase and neutrophil elastase

The action of purified rheumatoid synovial collagenase and human neutrophil elastase on the cartilage collagen types II, IX, X and XI was examined. At 25 degrees C, collagenase attacked type II and type X (45-kDa pepsin-solubilized) collagens to produce specific products reflecting one and at least two cleavages respectively. At 35 degrees C, collagenase completely degraded the type II collagen molecule to small peptides whereas a large fragment of the type X molecule was resistant to further degradation. In contrast, collagen type IX (native, intact and pepsin-solubilized type M) and collagen type XI were resistant to collagenase attack at both 25 degrees C and 35 degrees C even in the presence of excess enzyme. Mixtures of type II collagen with equimolar amounts of either type IX or XI did not affect the rate at which the former was degraded by collagenase at 25 degrees C. Purified neutrophil elastase, shown to be functionally active against soluble type III collagen, had no effect on collagen type II at 25 degrees C or 35 degrees C. At 25 degrees C collagen types IX (pepsin-solubilized type M) and XI were also resistant to elastase, but at 35 degrees C both were susceptible to degradation with type IX being reduced to very small peptides. Collagen type X (45-kDa pepsin-solubilized) was susceptible to elastase attack at 25 degrees C and 35 degrees C as judged by the production of specific products that corresponded closely with those produced by collagenase. Although synovial collagenase failed to degrade collagen types IX and XI, all the cartilage collagen species examined were degraded at 35 degrees C by conditioned culture medium from IL1-activated human articular chondrocytes. Thus chondrocytes have the potential to catabolise each cartilage collagen species, but the specificity and number of the chondrocyte-derived collagenase(s) has yet to be resolved.     

Degradation of monomeric and fibrillar type III collagens by human skin collagenase. Kinetic constants using different animal substrates

Human skin collagenase activity was examined against type III collagens, in both soluble and fibrillar form, from different animal species. In either form, human, dog, and cat type III were degraded 10- to 30-fold faster than was that from guinea pig and nearly 100-fold more readily than chick type III. These differences in susceptibility were mirrored by essentially identical differences in the rate of trypsin cleavage of the same substrates. Human, dog, and cat type III were cleaved most rapidly by trypsin, guinea pig III more slowly, and chick III was completely resistant to the serine protease. Arrhenius plots, relating enzyme activity to temperature, revealed differences in the various type III substrates consistent with their collagenase and trypsin susceptibilities. Human, dog, and cat type III collagens yielded nonlinear plots, with accompanying activation energies which decreased at temperatures above 26 degrees C; guinea pig type III displayed a plot which deviated only slightly from linearity while the plot for chick type III was completely linear. These data strongly suggest that type III collagens display substantial variability in the stability of the helix at or near the collagenase cleavage site. The susceptibility of these type III substrates as reconstituted fibrils was also examined. The relative rates of degradation of these substrates by collagenase, and by trypsin, were the same as those observed in solution. The absolute rates of degradation of collagen in fibrillar form, however, were massively lower than predicted by extrapolation from solution values. This reduction in rate is even greater for type III than for type I collagens. Thus, whereas in solution type III substrates are cleaved much faster than type I collagens, in fibrillar form these differences are less than 2-fold. These data, together with values for activation energies and deuterium isotope effects on type III fibrillar substrates, reinforce the concept that helical integrity near the collagenase cleavage site is a major specifier of the rate of collagenase activity. Furthermore, the data suggest that the exclusion of water accompanying the tight packing of monomers into fibrils presents a major energy barrier to collagenase activity, which is particularly large for type III collagen.     

Distribution of collagens type I,type III and type V in the pancreas of rat,dog, pig and man

The presence of collagens type I, type III and type V was determined immunohistochemically in pancreatic tissue of rat, pig, dog and man. The reaction to anti-collagen type I is weak (pig, dog) or moderate (rat, man) in the peri-insular region and in the lobar, lobular and acinar septa, whereas the reaction to anti-collagen type III is well developed. In rat and dog, the latter reaction deposit on the lobar and acinar septa is prominent. These elements only show a moderate reaction intensity in pig and man. The peri-insular region displays a weak (rat, dog, man) or very weak (pig) reaction against collagen type III. Anti-collagen type V reacts moderately (rat, dog, man) or weakly (pig) in the lobar and lobular septa. The acinar septa show a moderate (rat, dog, man) or very weak (pig) reaction. This information regarding the types and distribution of the collagenous compounds in pancreatic extracellular matrix could lead to differentiated enzymatic pancreas dissociation and, ultimately, increased islet yield and improved reproducibility of pancreatic islet isolation procedures for transplantation purposes.     

The second von Willebrand type A domain of cochlin has high affinity for type I, type II and type IV collagens

Cochlin is colocalized with type II collagen in the extracellular matrix of cochlea and has been suggested to interact with this collagen. Here we show that the second von Willebrand type A domain of cochlin has affinity for type II collagen, as well as type I and type IV collagens whereas the LCCL-domain of cochlin has no affinity for these proteins. The implications of these findings for the mechanism whereby cochlin mutations cause the dominant negative DFNA9-type hearing loss are discussed.

Structured summary

MINT-6796048:

type I collagen (uniprotkb:P02452) binds (MI:0407) to cochlin-vWA2 uniprotkb:O43405) by surface plasmon resonance (MI:0107)

MINT-6796166:

type III collagen (uniprotkb:P02462) binds (MI:0407) to cochlin-vWA2 (uniprotkb:O43405) by surface plasmon resonance (MI:0107)

MINT-6796062:

type II collagen (uniprotkb:P02458) binds (MI:0407) to cochlin-vWA2 (uniprotkb:O43405) by surface plasmon resonance (MI:0107)

     

Separation of the alpha chains of type I and III collagens by SDS-polyacrylamide gel electrophoresis.

A method for the separation of type III collagen from type I collagen by SDS-polyacrylamide gel electrophoresis has been developed. This is based on the observation that the presence of 3-4 M urea decreases the mobility of the alpha 1 [III] chain to a greater extent than those of the alpha 1[I] and alpha 2 chains, although the alpha 1[I] and alpha 1[III] chains move at the same rate in the absence of urea. An attempt to separate the alpha 1[II] chain of type II collagen from the alpha 1[I] chain was unsuccessful under the experimental conditions employed.