Recombinant human progranzyme 3 expressed in Escherichia coli for analysis of its activation mechanism.
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.
Hirata
Yukiyo
Y
Department of Hygiene and Public Health, Nippon Medical School, Bunkyo-ku, Tokyo 113-8602, Japan. yuki-hir@nms.ac.jp
Inagaki
Hirofumi
H
Kawada
Tomoyuki
T
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
D004792:Enzyme Precursors; D011994:Recombinant Proteins; D020789:Cathepsin C; C088546:GZMK protein, human; D053804:Granzymes