HUMAN GRANZYME-B DEGRADES AGGRECAN PROTEOGLYCAN IN MATRIX SYNTHESIZEDBY CHONDROCYTES

Cartilage degradation, a hallmark of rheumatoid arthritis, is attribut ed to serine and metalloproteases secreted by neutrophils, synovial li ning cells, macrophages, and chondrocytes. A large proportion of synov ial fluid lymphocytes contains the granule-associated serine proteases granzymes A and B. We report that lysates of IL-2-stimulated lymphocy tes contain an enzymatic activity (ECMase; cartilage extracellular mat rix S-35 release assay; extracellular matrix degrading activity) that solubilizes matrix synthesized by chondrocyte monolayers. ECMase activ ity is inactivated by the serine protease inhibitor diisopropylfluorop hosphate, is stored in dense granules and cleaves aggrecan proteoglyca ns but not free glycosaminoglycans, hyaluronic acid, or type II collag en. ECMase is mediated by a cationic protein with biochemical properti es identical to granzyme B, inasmuch as it preferentially hydrolyzes t he substrate Boc-Ala-Ala-Asp-SBzl, immunochemically cross-reacts with an antibody that binds to a conserved amino-terminal region of lymphoi d-myeloid serine proteases, and has amino-terminal sequence identity w ith human Q31 granzyme B. Using an agarose gel electrophoresis techniq ue to assess cleavage of the rat sarcoma aggrecan, the catalytic effic iency of granzyme B for the digestion of aggrecan (catalytic efficienc y = 1.7 x 10(7) M-1 s-1) was 425-fold faster than the catalytic effici ency reported for human stromelysin-1 at pH 7.5 (catalytic efficiency 4000 M-1 s-1) and 3200-fold faster than granzyme A. Based on these obs ervations, we propose that granzyme B, secreted from cytotoxic lymphoc ytes within the rheumatoid joint, may contribute to cartilage loss by degrading resident aggrecan.