title: Recombinant human progranzyme 3 expressed in Escherichia coli for analysis of its activation mechanism.
abstract: Gr3 is reported to play an important role in defense against viral infection. Although it is known that Gr3 is synthesized as a proenzyme and activated in the cytotoxic granules of NK cells and CTL, the activation mechanism is not clearly understood. In an attempt to analyze the activation mechanism of human Gr3, a recombinant pro-Gr3 was expressed in the periplasm of E. coli and purified to homogeneity. On SDS-PAGE the recombinant pro-Gr3 showed a slightly higher molecular weight than the enzymatically active Gr3, because the former possesses a small propeptide at its N-terminal. The recombinant pro-Gr3 was enzymatically inactive. It could be activated by treatment with cathepsin C, which concomitantly decreased the molecular weight to that of active Gr3. The proteolytic reaction of cathepsin C did not continue after one dipeptide had been removed, indicating that the recombinant pro-Gr3 had the native conformation without any refolding process. The recombinant pro-Gr3 would be a valuable tool for analyzing the activation mechanism and exploring other activating enzymes besides cathepsin C.
authors.affiliation: Department of Hygiene and Public Health, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan. yuki-hir@nms.ac.jp
authors.forename: Yukiyo
authors.initials: Y
authors.lastname: Hirata
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Inagaki
authors.forename: Tomoyuki
authors.initials: T
authors.lastname: Kawada
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D020789:Cathepsin C / Q000378:metabolism; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D053804:Granzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004792:Enzyme Precursors; D011994:Recombinant Proteins; D020789:Cathepsin C; C088546:GZMK protein, human; D053804:Granzymes