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Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix.

ABSTRACT: Cathepsin K (EC 3.4.22.38) is a recently described enzyme that has been shown to cleave type I collagen in its triple helix. The aim of this study was to determine if it also cleaves type II collagen in the triple helix and to identify the helical cleavage site(s) in types I and II collagens. Soluble human and bovine type II collagen, and rat type I collagen, were incubated with cathepsin K before the reaction was stopped with trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Analysis by SDS/PAGE of the collagen digests showed that optimal activity of cathepsin K against native type II collagen was between pH 5.0 and 5.5 and against denatured collagen between pH 4.0 and 7.0. The enzyme cleaved telopeptides as well as the alpha1(II) chains, generating multiple fragments in the range 90-120 kDa. The collagenolytic activity was not due to a contaminating metalloenzyme or serine proteinase as it was not inhibited by 1,10-phenanthroline, EDTA or 3,4-dichloroisocoumarin. Western blotting with anti-peptide antibodies to different regions of the alpha1(II) chain suggested that cathepsin K cleaved native alpha1(II) chains in the N-terminal region of the helical domain rather than at the well-defined collagenase cleavage site. This was confirmed by N-terminal sequencing of one of the fragments, revealing cleavage at a Gly-Lys bond, 58 residues from the N-terminus of the helical domain. By using a similar approach, cathepsin K was found to cleave native type I collagen close to the N-terminus of its triple helix. These results indicate that cathepsin K could have a role in the turnover of type II collagen, as well as type I collagen.

SUBMITTER: Kafienah W

PROVIDER: S-EPMC1219411 | biostudies-other | 1998 May

REPOSITORIES: biostudies-other

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Project description:BackgroundThe vitreoretinal interface is the border of the cortical vitreous and the inner surface of the retina. The adhesion of the cortical vitreous to the ILM, namely vitreoretinal adhesion, involves a series of complex molecular adhesion mechanisms and has been considered as an important pathogenic factor in many vitreoretinal diseases. The presence of type VI collagen at the vitreoretinal interface and its possible interaction with collagens and glycoproteins indicates that type VI collagen may contribute to the vitreoretinal adhesion.PurposeTo clarify the ultrastructural location of type VI collagen and its relationship to type II and IV collagens at the vitreoretinal interface.MethodsThe ultrastructural localization of type II, IV and VI collagens in the adult human vitreoretinal interface of five donor eyes was evaluated by transmission electron microscopy using immunogold labeling.ResultsIn the pre-equatorial region, we observed densely packed vitreous lamellae with a partly intraretinal course containing type II and VI collagens, reticular structures containing type IV and VI collagens and a thin inner limiting membrane (ILM) containing type IV and VI collagens in a linear distribution pattern. From the anterior to the posterior retina, the linear pattern of type IV and VI collagen labeling gradually became more diffusely present throughout the entire thickness of the ILM.ConclusionsThe presence of type VI collagen in vitreous lamellae penetrating the ILM into the superficial retina suggests that type VI collagen may be involved in the organization of vitreous fibers into lamellae and in the adhesion of the vitreous fibers to the retina. The close relation of type VI to type IV collagen in the ILM suggests that type VI collagen is an important collagen type in the ILM. The topographic variations of type IV and VI collagens in the different regions of the ILM suggest a regional heterogeneity of the ILM. The reticular labeling pattern of type IV and VI collagens observed in the anterior vitreous are highly similar to labeling patterns of blood vessel walls. In the anterior vitreous, they may represent remnants of the regressed embryonic hyaloid blood vessel system. Their presence is in support of the theory on interactive remodeling of the developing vitreous as opposed to the main stream theory of displacement and compression of the primary by the secondary vitreous.

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Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix.

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