Lm. Atley et al., Proteolysis of human bone collagen by cathepsin K: Characterization of thecleavage sites generating the cross-linked N-telopeptide neoepitope, BONE, 26(3), 2000, pp. 241-247
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 acti
ve 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 yi
eld of K, respectively. The matrix metalloproteinases that were tested, str
omelysin, collagenase 3, or matrilysin, did not produce any immunoreactivit
y. Cathepsin K also acted on demineralized bone matrix, releasing NTx epito
pe and completely dissolving the bone particles in 24-18 h. Proteolytic cle
avage 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 iden
tified bonds in the N-telopeptide and helical cross-linking domains adjacen
t to the cross-linking residues at which cathepsin K cleaved in bone collag
en, The sites were consistent with the known substrate specificity of cathe
psin K, which prefers a hydrophobic residue or proline in the critical P2 p
osition. The NTs peptides generated by cathepsin K were of low molecular we
ight, in the range previously found in human urine. Because cathepsin K app
ears to be essential for the normal resorption of mineralized bone matrix b
y osteoclasts, these findings help explain the specificity and responsivene
ss of NTx as a marker of osteoclastic bone resorption in vivo. (C) 2000 by
Elsevier Science Inc. All rights reserved.