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.

Cathepsin K Activity-dependent Regulation of Osteoclast Actin Ring Formation and Bone Resorption

“…1), which was in a similar range to the inhibition found after the addition of the synthetic control GRGDS peptide. Triple helical type I collagen which contains in its ␣1 and ␣2 chains a total of 7 RGD motifs, is cleaved by catK at multiple sites and generates low molecular weight fragments (39,40), which may contain small soluble RGD containing peptides. The presence of soluble RGD-motif containing peptides was further supported by the loss of inhibitory effect when catK-digested collagen fragments were further degraded by trypsin before addition to cells (Fig.…”

Targeting subchondral bone for treating osteoarthritis: what is the evidence?

“…Transgenic mouse models have provided evidence supporting its important role in arthritis. Interestingly, cathepsin K is one of the few non-collagenase enzymes capable of degrading native fibrillar collagen types I and II [148]. Owing to the lack of adequate cross reactivity between the rat and human isoenzymes, animal models using cathepsin K inhibitors have been developed mostly in monkeys, and data have shown that inhibiting cathepsin K results in the prevention of bone loss.…”

Characterization of a Novel Subtilisin-like Protease Myroicolsin from Deep Sea Bacterium Myroides profundi D25 and Molecular Insight into Its Collagenolytic Mechanism

“…Among the collagenolytic proteases, several mammalian matrix metalloproteinases (MMPs, peptidase family M10), serine proteases isolated from Uca pugilator (family S1), and cathepsins K and L from animals (family C1) have been extensively studied, and their collagen degradation mechanisms have been explored (11)(12)(13)(14)(15)(16)(17)(18)20). Some bacterial extracellular metalloproteases in the M9 family, primarily from the Vibrio (21) and Clostridium strains (22,23), also show strong collagenolytic activity.…”