title: Trypsin-2 degrades human type II collagen and is expressed and activated in mesenchymally transformed rheumatoid arthritis synovitis tissue.
abstract: 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 alpha(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.
authors.affiliation: Department of Anatomy, Institute of Biomedicine, Helsinki, Finland. mathias.stenman@helsinki.fi
authors.forename: Mathias
authors.initials: M
authors.lastname: Stenman
authors.forename: Mari
authors.initials: M
authors.lastname: Ainola
authors.forename: Leena
authors.initials: L
authors.lastname: Valmu
authors.forename: Anders
authors.initials: A
authors.lastname: Bjartell
authors.forename: Guofeng
authors.initials: G
authors.lastname: Ma
authors.forename: Ulf-Håkan
authors.initials: UH
authors.lastname: Stenman
authors.forename: Timo
authors.initials: T
authors.lastname: Sorsa
authors.forename: Reijo
authors.initials: R
authors.lastname: Luukkainen
authors.forename: Yrjö T
authors.initials: YT
authors.lastname: Konttinen
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001172:Arthritis, Rheumatoid / Q000378:metabolism* / Q000473:pathology; D001483:Base Sequence; D002417:Cattle; D018929:Cell Culture Techniques; D002478:Cells, Cultured; D024043:Collagen Type II / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005063:Europium; D005260:Female; D005470:Fluorometry; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013058:Mass Spectrometry; D020784:Matrix Metalloproteinase 8 / Q000494:pharmacology; D008875:Middle Aged; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013582:Synovial Fluid / Q000166:cytology / Q000378:metabolism; D013583:Synovial Membrane / Q000378:metabolism / Q000473:pathology; D014357:Trypsin / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification / Q000494:pharmacology* / Q000652:urine; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000032:analysis / Q000302:isolation & purification / Q000494:pharmacology* / Q000652:urine; D014362:Trypsinogen / Q000302:isolation & purification / Q000494:pharmacology* / Q000652:urine
chemical_list: D000911:Antibodies, Monoclonal; D024043:Collagen Type II; D012333:RNA, Messenger; C064831:PRSS2 protein, human; D005063:Europium; D014359:Trypsin Inhibitor, Kazal Pancreatic; D014362:Trypsinogen; D014357:Trypsin; D020784:Matrix Metalloproteinase 8