title: Purification and characterization of lymphocyte chymase I, a granzyme implicated in perforin-mediated lysis.
abstract: One mechanism of killing by cytotoxic lymphocytes involves the exocytosis of specialized granules. The released granules contain perforin, which assembles into pores in the membranes of cells targeted for death. Serine proteases termed granzymes are present in the cytotoxic granules and include several chymases (with chymotrypsin-like specificity of cleavage). One chymase is selectively reactive with an inhibitor, Biotinyl-Aca-Aca-Phe-Leu-PheP(OPh)2, that blocks perforin lysis. We report the purification and characterization of this chymase, lymphocyte chymase I, from rat natural killer cell (RNK)-16 granules. Lymphocyte chymase I is 30 kDa with a pH 7.5 to 9 optimum and primary substrate preference for tryptophan, a preference distinct from rat mast cell chymases. This chymase also reacts with other selective serine protease inhibitors that block perforin pore formation. It elutes by Cu2+-immobilized metal affinity chromatography with other granzymes and has the N-terminal protein sequence conserved among granzymes. Chymase I reduces pore formation when preincubated with perforin at 37 degrees C. In contrast, addition of the chymase without preincubation had little effect on lysis. It should be noted that the perforin preparation contained 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 perforin during similar preincubation. Identification of the natural substrate of chymase I will help resolve how it regulates perforin-mediated pore formation.
authors.affiliation: Department of Microbiology, School of Medicine, University of Nevada, Reno 89557, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Woodard
authors.forename: S A
authors.initials: SA
authors.lastname: Fraser
authors.forename: U
authors.initials: U
authors.lastname: Winkler
authors.forename: D S
authors.initials: DS
authors.lastname: Jackson
authors.forename: C M
authors.initials: CM
authors.lastname: Kam
authors.forename: J C
authors.initials: JC
authors.lastname: Powers
authors.forename: D
authors.initials: D
authors.lastname: Hudig
mesh_terms: D000818:Animals; D053818:Chymases; D003602:Cytotoxicity, Immunologic; D007694:Killer Cells, Natural / Q000201:enzymology*; D008562:Membrane Glycoproteins / Q000502:physiology*; D054353:Perforin; D052899:Pore Forming Cytotoxic Proteins; D011817:Rabbits; D051381:Rats; D011916:Rats, Inbred F344; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology
chemical_list: D008562:Membrane Glycoproteins; D052899:Pore Forming Cytotoxic Proteins; D054353:Perforin; D012697:Serine Endopeptidases; D053818:Chymases