Human cathepsin K cleaves native type I and II collagens at the N-terminal end of the triple helix.
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.
Kafienah
W
W
Department of Human Metabolism and Clinical Biochemistry, and Institute for Bone and Joint Medicine, University of Sheffield Medical School, Beech Hill Road, Sheffield S10 2RX, UK.
Brömme
D
D
Buttle
D J
DJ
Croucher
L J
LJ
Hollander
A P
AP
D000595:Amino Acid Sequence; D000818:Animals; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism*; D002417:Cattle; D003094:Collagen / Q000737:chemistry*; D017364:Collagenases / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K; C534133:Ctsk protein, rat; D017364:Collagenases