TISSUE TRANSGLUTAMINASE IS AN IN-SITU SUBSTRATE OF CALPAIN - REGULATION OF ACTIVITY

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catal yzes the transamidation of specific polypeptide-bound glutamine residu es, a reaction that is inhibited by GTP. There is also preliminary evi dence that, in situ, calpain and GTP may regulate tTG indirectly by mo dulating its turnover by the calcium-activated protease calpain. In th e present study, the in vitro and in situ proteolysis of tTG by calpai n, and modulation of this process by GTP, was examined. tTG is an exce llent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by t rypsin. In a similar manner, guanosine-5'-O-(3-thiotriphosphate) prote cts tTG against proteolysis by calpain. Treatment of SH-SY5Y cells wit h 1 nM maitotoxin, which increases intracellular calcium levels, resul ted in a significant increase in in situ TG activity, with only a slig ht decrease in tTG protein levels. In contrast, when GTP levels were d epleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an similar to 50% decrease in tTG protein levels, and a s ignificant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tT G in response to the combined treatment of maitotoxin acid tiazofurin and resulted in a significant increase in in situ TG activity. These s tudies indicate that tTG is an endogenous substrate of calpain and tha t GTP selectively inhibits the degradation of tTG by calpain.