Immunochemical characterization of assay for carboxyterminal telopeptide of human type I collagen: Loss of antigenicity by treatment with cathepsin K

The assay for the cross-linked carboxyterminal telopeptide of type I collag en (ICTP) has been shown to reflect increased type I collagen degradation i n such pathological conditions as bone metastases and rheumatoid arthritis, but to be rather insensitive to the changes in physiological bone collagen turnover (e.g., induced by estrogen or bisphosphonate treatment). To deter mine the reasons for this discrepancy we localized the antigenic determinan t recognized by the ICTP assay and studied the effects of two major osteocl astic proteinases, cathepsin K (EC 3.4.22.38) and matrix metalloproteinase- 9 (MMP-9; gelatinase B; EC 3.4.24.35), on immunoreactivity. The antigenic d eterminant was shown to reside within the hydrophobic phenylalanine-rich re gions of the carboxyterminal telopeptides of the two alpha 1 chains of huma n type I collagen, situated between the triple helical domain and the lysin e-derived trivalent cross-link. This conclusion was based on differences be tween the amino acid sequences and cross reactivities of the corresponding human and bovine antigens before and after proteolytic treatments with chym otrypsin, A trivalent cross-link is necessary for providing such a structur e, because the divalently cross-linked and monomeric natural and synthetic peptides from the same region, but containing only one phenylalanine-rich s equence, showed poor immunoreaction. Recombinant human cathepsin K cleaved the trivalently cross-linked ICTP structure at two sites between the phenyl alanine-rich region and the crosslink, destroying the reactivity with ICTP antibodies. On the contrary, the treatment of isolated ICTP by the matrix m etalloproteinases MMP-9 (gelatinase B), MMP-1 (collagenase 1), or MMP-13 (c ollagenase 3) had no effect on the immunoreaction. Our results indicate tha t the increased circulating concentrations of ICTP found in several clinica l situations are most likely produced by matrix metalloproteinases, whereas cathepsin K-mediated, osteoclastic bone resorption destroys ICTP antigenic ity. (Bone 26:367-373; 2000) (C) 2000 by Elsevier Science Inc. All rights r eserved.