J S Mort1, F Beaudry2, K Théroux3, A A Emmott4, H Richard3, W D Fisher5, E R Lee6, A R Poole5, S Laverty3. 1. Genetics Unit, Shriners Hospitals for Children, Montreal, Québec, Canada; Department of Surgery, McGill University, Montreal, Québec, Canada. Electronic address: jmort@shriners.mcgill.ca. 2. Groupe de Recherche en Pharmacologie Animal du Québec (GREPAQ), Département de Biomédecine Vétérinaire, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada. 3. Comparative Orthopaedic Research Laboratory, Département de Sciences Cliniques, Faculté de Médecine Vétérinaire, Université de Montréal, Saint-Hyacinthe, Québec, Canada. 4. Genetics Unit, Shriners Hospitals for Children, Montreal, Québec, Canada. 5. Department of Surgery, McGill University, Montreal, Québec, Canada. 6. Genetics Unit, Shriners Hospitals for Children, Montreal, Québec, Canada; Department of Surgery, McGill University, Montreal, Québec, Canada.
Abstract
OBJECTIVE: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equine articular osteoarthritic cartilage. DESIGN: Equine type II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and human osteoarthritic cartilage. RESULTS: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equine type II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovine type II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or human osteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. CONCLUSIONS: Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.
OBJECTIVE: To characterize the initial events in the cleavage of type II collagen mediated by cathepsin K and demonstrate the presence of the resulting products in human and equinearticular osteoarthritic cartilage. DESIGN:Equinetype II collagen was digested with cathepsin K and the cleavage products characterized by mass spectrometry. Anti-neoepitope antibodies were raised against the most N-terminal cleavage products and used to investigate the progress of collagen cleavage, in vitro, and the presence of cathepsin K-derived products in equine and humanosteoarthritic cartilage. RESULTS: Six cathepsin K cleavage sites distributed throughout the triple helical region were identified in equinetype II collagen. Most of the cleavages occurred following a hydroxyproline residue. The most N-terminal site was within three residues of the previously identified site in bovinetype II collagen. Western blotting using anti-neoepitope antibodies showed that the initial cleavages occurred at the N-terminal sites and this was followed by more extensive degradation resulting in products too small to be resolved by SDS gel electrophoresis. Immunohistochemical staining of cartilage sections from equine or humanosteoarthritic joints showed staining in lesional areas which was not observed in non-arthritic sites. CONCLUSIONS:Cathepsin K cleaves triple helical collagen by erosion from the N-terminus and with subsequent progressive cleavages. The liberated fragments can be detected in osteoarthritic cartilage and may represent useful biomarkers for disease activity.