PURIFICATION AND CHARACTERIZATION OF LYMPHOCYTE CHYMASE-I, A GRANZYMEIMPLICATED IN PERFORIN-MEDIATED LYSIS

One mechanism of killing by cytotoxic lymphocytes involves the esocyto sis of specialized granules, The released granules contain perforin, w hich assembles into pores in the membranes of cells targeted for death . Serine proteases termed granzymes are present in the cytotoxic granu les and include several chymases (with chymotrypsin-like specificity o f cleavage). One chymase is selectively reactive with an inhibitor, Bi otinyl-Aca-Aca-Phe-Leu-Phe(P)(OPh)(2), that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocy te chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocy te chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrat e preference for tryptophan, a preference distinct from rat mast cell chymases, This chymase also reacts with other selective serine proteas e inhibitors that block perforin pore formation, It elutes by Cu2+-imm obilized metal affinity chromatography with other granzymes and has th e N-terminal protein sequence conserved among granzymes. Chymase I red uces pore formation when preincubated with perforin at 37 degrees C. I n contrast, addition of the chymase without preincubation had little e ffect on lysis, It should be noted that the perforin preparation conta ined sufficient residual chymase activity to support lysis, Thus, the reduction of lysis may represent an effect of excess prolytic chymase I or a means to limit perforin lysis of bystander cells. In contrast, other chymases and granzyme K were without effect when added to perfor in during similar preincubation, Identification of the natural substra te of chymase I will help resolve how it regulates perforin-mediated p ore formation.