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(American Journal of Pathology. 2005;167:1119-1124.)
© 2005 American Society for Investigative Pathology

Trypsin-2 Degrades Human Type II Collagen and Is Expressed and Activated in Mesenchymally Transformed Rheumatoid Arthritis Synovitis Tissue

Mathias Stenman^*, Mari Ainola^*, Leena Valmu, Anders Bjartell, Guofeng Ma, Ulf-Håkan Stenman^¶, Timo Sorsa^||, Reijo Luukkainen^** and Yrjö T. Konttinen

From the Department of Anatomy,^* Institute of Biomedicine, Helsinki, Finland; the Institute of Biotechnology, Helsinki, Finland; Orton Orthopedic Hospital of the Invalid Foundation, Helsinki, Finland; the Departments of Clinical Chemistry^¶ and Internal Medicine/Invärtes Medicin, Helsinki University Central Hospital, Department of Oral and Maxillofacial Diseases, Helsinki, Finland; the Institute of Dentistry,|| University of Helsinki, Helsinki, Finland; the Department of Rheumatology,^** Rauma Regional Hospital, Rauma, Finland; COXA Hospital for the Joint Replacement, Tampere, Finland; and the Department of Urology, Malmö University Central Hospital, Malmö, Sweden

It has traditionally been believed that only the human collagenases (matrix metalloproteinase-1, -8, and -13) are capable of initiating the degradation of collagens. Here, we show that human trypsin-2 is also capable of cleaving the triple helix of human cartilage collagen type II. We purified human trypsin-2 and tumor-associated trypsin inhibitor by affinity chromatography whereas collagen type II was purified from cartilage extracts using pepsin digestion and salt precipitation. Degradation of type II collagen and gelatin by trypsin-2 was demonstrated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, zymography, and mass spectrometry, and tumor-associated trypsin inhibitor specifically inhibited this degradation. Although human trypsin-2 efficiently digested type II collagen, bovine trypsin did not. Furthermore, immunohistochemical staining detected trypsin-2 in the fibroblast-like synovial lining and in stromal cells of human rheumatoid arthritis synovial membrane. These findings were confirmed by reverse transcriptase-polymerase chain reaction and nucleotide sequencing. Trypsin-2 alone and complexed with ₁-proteinase inhibitor were also detected in the synovial fluid of affected joints by time-resolved immunofluorometric assay, suggesting that trypsin-2 is activated locally. These results are the first to assess the ability of human trypsin to cleave human type II collagen. Thus, trypsin-2 and its regulators should be further studied for use as markers of prognosis and disease activity in rheumatoid arthritis.