Recombinant human granzyme A induces cell death more potently than granzyme A isolated from lymphokine-activated killer or NK cells. (A) Four sources of GzmA were added with a sublytic concentration of perforin to K562 cells at the indicated concentrations. Percent specific cytotoxicity was calculated by 51Cr release after 4 hours. Background cytolysis of cells treated with perforin without GzmA was subtracted. GzmA was either rGzmA or native GzmA purified from human lymphokine-activated killer (LAK) cells or NK92 cells either by us (JL) or from NK92 cells by the Froelich laboratory (CF). (B) The 4 sources of GzmA were also compared for in vitro cleavage of recombinant SET protein using 2-fold serial dilutions at concentrations ranging from 31nM to 1000nM. Reactions were incubated for 1 hour at 37°C. Enzymatically inactive rGzmA in which the active site Ser is mutated to Ala (S-A) was an additional negative control. (C) Cytotoxicity by rGzmA was compared with cytotoxicity induced by native GzmB purified from an NK-cell line (YT-Indy) and native GzmA from NK92 cells. 51Cr release assay performed as in panel A.
