MODULATION OF THE IN-SITU ACTIVITY OF TISSUE TRANSGLUTAMINASE BY CALCIUM AND GTP

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catal yzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro st udies have demonstrated that the transamidating activity of tTG requir es calcium and is inhibited by GTP, To investigate the endogenous regu lation of tTG, a quantitative in situ transglutaminase (TG) activity a ssay was developed, Treatment of human neuroblastoma SH-SY5Y cells wit h retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did n ot increase concurrently with RA-induced increased tTG levels. However , stimulation of cells with the calcium-mobilizing drug maitotoxin (MT X) resulted in increases in in situ TG activity that correlated (r(2) = 0.76) with increased tTG levels. To examine the effects of GTP on in situ TG; activity, tiazofurin, a drug that selectively decreases GTP levels, was used. Depletion of GTP resulted in a significant increase in in situ TG activity; however, treatment of SH-SY5Y cells with a com bination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone, This raised the po ssibility of calcium-dependent proteolysis due to the effects of tiazo furin, because in vitro GTP protects tTG against proteolysis by trypsi n. Studies with a selective membrane permeable calpain inhibitor indic ated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of int racellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases int racellular calcium through inositol 1,4,5-trisphosphate generation. Th e results of these experiments demonstrate that selective changes in c alcium and GTP regulate the activity and levels of tTG in situ.