Proteolysis of human bone collagen by cathepsin K: characterization of the cleavage sites generating by cross-linked N-telopeptide neoepitope.
An immunoassay for cross-linked N-telopeptides of type I collagen (NTx) in urine or serum has proven to give a sensitive index of osteoclast-mediated bone resorption. We show that recombinant human cathepsin K is highly active in releasing the NTx neoepitope in 100% yield from bone type I collagen. Cathepsins S, L, and B were also active but at 57%, 36%, and 27% of the yield of K, respectively. The matrix metalloproteinases that were tested, stromelysin, collagenase 3, or matrilysin, did not produce any immunoreactivity. Cathepsin K also acted on demineralized bone matrix, releasing NTx epitope and completely dissolving the bone particles in 24-48 h. Proteolytic cleavage of a G-L peptide bond in the alpha2(I)N-telopeptide was shown to be required for recognition by monoclonal antibody 1H11. Peptide analysis identified bonds in the N-telopeptide and helical cross-linking domains adjacent to the cross-linking residues at which cathepsin K cleaved in bone collagen. The sites were consistent with the known substrate specificity of cathepsin K, which prefers a hydrophobic residue or proline in the critical P2 position. The NTx peptides generated by cathepsin K were of low molecular weight, in the range previously found in human urine. Because cathepsin K appears to be essential for the normal resorption of mineralized bone matrix by osteoclasts, these findings help explain the specificity and responsiveness of NTx as a marker of osteoclastic bone resorption in vivo.
Atley
L M
LM
Orthopaedic Research Laboratories, University of Washington, Seattle 98195-6500, USA. latley@u.washington.edu
Mort
J S
JS
Lalumiere
M
M
Eyre
D R
DR
D000328:Adult; D000595:Amino Acid Sequence; D001842:Bone and Bones / Q000378:metabolism*; D056657:Cathepsin K; D002403:Cathepsins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D024042:Collagen Type I; D000939:Epitopes / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D008297:Male; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D014556:Urine / Q000737:chemistry
D024042:Collagen Type I; D000939:Epitopes; D010455:Peptides; C052929:collagen type I trimeric cross-linked peptide; D003094:Collagen; D002403:Cathepsins; C534130:CTSK protein, human; D056657:Cathepsin K