Proteolysis of human bone collagen by cathepsin K: Characterization of the cleavage sites generating the 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 Ii is highly active in releasing the NTx neoepitope in 100% yield from hone 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 24-18 h. Proteolytic cleavage of a G-L peptide bond in the alpha 2(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 NTs 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. (C) 2000 by Elsevier Science Inc. All rights reserved.