Binding of latent rheumatoid synovial collagenase to collagen fibrils.

Collagenase released from rheumatoid synovial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24 degrees C and 37 degrees C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibrils can be degraded. Binding was not inhibited by the presence of alpha2 macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding to substrate, and that activation by proteases such as plasmin initiates collagen breakdown.     

Binding of latent rheumatoid synovial collagenase to collagen fibrils

Collagenase released from rheumatoid synivial cells in culture is in a latent form. Subsequently, it may be activated by limited proteolysis. This study was designed to determine whether latent enzyme could bind to collagen fibrils and await activation. The data showed that latent collagenase bound to fibrils equally well at 24°C and 37°C, but that this represented little more than half the binding achieved by active enzyme at temperatures lower than that at which fibril can be degraded. Binding was not inhibited by the presence of α₂ macroglobulin, the principal proteinase inhibitor of plasma which cannot complex with inactive or latent collagenase but readily complexes with active species of enzyme. The data support the hypotheses that inactive forms of collagenase accumulate in tissues by binding the substrate, and that activation by proteases such as plasmin intiates collagen breakdown.     

Action of rheumatoid synovial collagenase on cartilage collagen. Different susceptibilities of cartilage and tendon collagen to collagenase attack.

The action of purified rheumatoid synovial collagenase on purified cartilage collagen, alpha-1(II)-3, in solution at 25 degrees C has been characterised. The enzyme attacked cartilage collagen in solution producing a 58% reduction in specific viscosity and resulting in the appearance of two reaction products which represented approximately three-quarter and one-quarter fragments of the intact molecule as shown by disc electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. The alpha-chain fragments which comprised each of these components corresponded to molecular weights of approximately 74000 and 21000. Electron microscopy of segment-long-spacing crystallites of the reaction products revealed three-quarter (TC-a) and one-quarter (TC-b) length fragments, and permitted accurate localization of the cleavage locus between bands 41 and 42 (I-41). This cleavage site and the formation of TC-a and TC-b reaction products are very similar to those found for type-I collagen substrates. Cartilage collagen in solution was found to be more resistant to collagenase attack than tendon collagen, the rate of cartilage collagen degradation being six times slower than that for tendon collagen, as judged by viscometry. The mid-point melting temperatures (T-m) for lathyritic cartilage and tendon collagen were 40.5 and 41.5 degrees C, and for the collagenase-produced reaction products 38.5 and 37.5 degrees C, respectively. The significance of these findings is discussed in relation to the structure of type I and II collagens.     

A model for biased diffusion of collagenase along collagen fibrils

We present a model to describe the biased diffusion of a collagenase along collagen fibrils. Based on the structures of collagen fibril and collagenase, the interaction is described by a one-dimensional potential that is symmetric in the region of no cleavage and asymmetric in the cleavage region. We show that the mean velocity of the unidirectional diffusion of the collagenase depends on the three parameters: the asymmetric ratio of the local potential in the cleavage region, the chemical reaction rate of proteolysis and the jumping rate of collagenase between two neighboring tracks. We calculate the correlation function and the mean transport velocity for both wild-type and mutant collagenases along collagen fibrils, the results of which are consistent with the previous experiments.     

A handy assay for collagenase using reconstituted fluorescein-labeled collagen fibrils

This report describes an assay for measuring the activity of collagenases using reconstituted fibrils of fluorescein-labeled soluble collagen as substrate. The labeling of commercially available material is easy and not expensive. The assay is very sensitive, reproducible, and is linear with collagenase concentration up to 100% consumption of the reconstituted fibrils. Readings of collagenolytic activity can be determined directly by measuring the fluorescence of the released labeled peptides without further processing of the data.     

Purification of rheumatoid synovial collagenase and its action on soluble and insoluble collagen.

1. The neutral collagenase released into the culture medium by explants of ehrumatoid synovial tissue has been purified by ultrafiltration and column chromatography, utilising Sephadex G-200, Sephadex QAE A-50 and Sephadex G-100 superfine. 2. The final collagenase preparation had a specific activity against thermally reconstituted collagen fibrils of 312 mug collagen degraded min-1 mg enzyme protein-1, representing more than a 1000-fold increase over that of the active culture medium. 3. Electrophoresis in polyacrylamide disc-gels with and without sodium dodecyl sulphate showed the enzyme to migrate as a single protein band. Elution experiments from polyacrylamide gels and chromatography columns have provided no evidence for the existence of more than one collagenase. 4. The molecular weight of the enzyme, as determined by dodecylsulphate-polyacrylamide gel electrophoresis, was 33000. 5. Data obtained from sutdies with the ion-exchange resin and from gel electrophoresis in acid and alkaline buffer systems suggested a basically charged enzyme. 6. It did not hydrolyse the synthetic collagen peptide Pz-Pro-Leu-Gly-Pro-D-Arg and non-specific protease activity was absent. 7. The collagenase attacked undenatured collagen in solution at 25 degrees C resulting in a 58% loss of viscosity and producing the two characteristic products TCA(3/4) and TCB(1/4). 8. At 37 degrees C and pH 8.0 both reconstituted collagen fibrils and gelatin were degraded to peptides of less than 10000 molecular weight. 9. As judged by the release of soluble hydroxyproline peptides and electron microscopic appearances the enzyme degraded human insoluble collagens derived from tendon and soft juxta-articular tissues although rates of attack were less than with reconstituted fibrils. 10. The data suggests that pure rheumatoid synovial collagenase at 37 degrees C and neutral pH can degrade gelatin, reconstituted fibrils and insoluble collagens without the intervention of non-specific proteases. 11. The different susceptibilities of various collagenous substrates to collagenase attack are discussed.     

An Ehrlich chromogen in collagen cross-links.

A well-characterized three-chain peptide [(Col1)2 X T9] from human type III collagen was a rich source of Ehrlich chromogen. The corresponding two-chain peptide [(Col1)2] was not, implying that the Ehrlich chromogen is a trifunctional cross-link. (Col1)2 X T9 also contained pyridinoline, which is not an Ehrlich chromogen. The 7S domain of type IV collagen also contained an Ehrlich chromogen.     

Properties of pig synovial collagenase.

1. Properties of a purified chemically activated form of pig synovial collagenase were examined and compared with a spontaneously active form of the enzyme. 2. The active enzyme has a specific activity of 53 000 units (microgram/min)/mg, a mol.wt. of 44 000 (by sodium dodecyl sulphate/polyarcylamide-gel electrophoresis in 2-mercaptoethanol) and pI 5.2 (by isoelectric focusing in polyacrylamide gels). 3. The activity has the characteristics of a metalloproteinase that degrades types I and III soluble or insoluble collagens in preference to type II, at an optimum pH of 6.5-8.5. 4. There is no detectable difference in these properties between the chemically activated and spontaneously active form of collagenase.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

Purification of pig synovial collagenase to high specific activity.

1. Pig synovium in tissue culture secretes a specific collagenase in a latent form. 2. The latent enzyme was concentrated by (NH4)2SO4 precipitation and activated with 4-aminophenylmercuric acetate, and the active enzyme was purified by chromatography on Ultrogel AcA44, DEAE-cellulose, heparin-Sepharose and a zinc-chelate medium to a specific activity of 53 400 units/mg. of protein. 3. The enzyme was shown to be essentially homogeneous by polyacrylamide-gel electrophoresis. 4. The purified collagenase digested collagen to give the characteristic three-quarter and one-quarter pieces.     

Some properties of latent collagenase from human synovial fluid

Latent collagenase has been isolated in pure form from the rheumatoid synovial fluid. The final preparation, activated by trypsin, yielded a collagenase of specific activity 2,227 units/mg. Electrophoresis in sodium dodecyl sulfate polyacrylamide gels revealed a protein doublet of 54 and 50 kDa. Trypsin or HgCl2 activation resulted in disappearance of the doublet and emergence of a new doublet of 47 and 43 kDa. The latent collagenase could also be activated by leucocyte cathepsin G or plasmin. Neither the latent nor the active collagenase from synovial fluid showed any cross-reactivity with the antibodies against leucocyte collagenase. The trypsin activated collagenase degraded collagen type I, II, III giving typical cleavage products but did not degrade type IV and V collagen.     

Chemistry of the collagen cross-links. Nature of the cross-links in the polymorphic forms of dermal collagen during development.

Both the type I and type III collagens present in embryonic dermis are stabilized by the intermolecular cross-link, hydroxylysino-5-oxonorleucine, derived from hydroxylysine-aldehyde, although the type I collagen possesses a significant proportion of dehydrohydroxylysinonorleucine. However, concurrent with the change in the proportion of the two types of collagen during postnatal development there is a change-over with both type I and III collagens to the labile cross-link, dehydrohydroxylysinonorleucine, derived from lysine aldehyde. The results indicate that the change in the nature of the cross-link with development is determined primarily by the change in the extent of hydroxylation of the lysine residues in the terminal non-helical regions rather than being due to the change in the type of collagen.     

Hyperchromic effect of collagen induced by human collagenase

Loss of the highly ordered triple-helix structure of native collagen on denaturation or enzymatic degradation involves a helix-to-coil transition, which can be seen as an increase at 227 nm in its ultraviolet difference absorption spectrum. We report here the successful use of this hyperchromic effect to quantify collagen in solution and to follow up the time-course of collagen degradation catalyzed by collagenase. Using 14C-labelled collagen substrate we show the excellent correlation between enzyme-induced increase in ultraviolet difference absorption and formation of specific cleavage products. The novel method was found to be suitable to characterize the enzymatic properties of human leukocyte collagenase. Activation of latent collagenase to the active enzyme could be followed continuously and an activation lag estimated.