Calcium regulates S-nitrosylation, denitrosylation, and activity of tissuetransglutaminase

Nitric oxide (NO) and related molecules play important roles in vascular bi ology. NO modifies proteins through nitrosylation of free cysteine residues , and such modifications are important in mediating NO's biologic activity. Tissue transglutaminase (tTG) is a sulfhydryl rich protein that is express ed by endothelial cells and secreted into the extracellular matrix (ECM) wh ere it is bound to fibronectin. Tissue TG exhibits a Ca2+-dependent transgl utaminase activity (TGase) that cross-links proteins involved in wound heal ing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cyst eine for catalysis, we investigated the factors that regulated NO binding a nd tTG activity. We report that TGase activity is regulated by NO through a unique Ca2+-dependent mechanism. Tissue TG can be poly-S-nitrosylated by t he NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca2+, up to eig ht cysteines were nitrosylated without modifying TGase activity. In the pre sence of Ca2+, up to 15 cysteines were found to be nitrosylated and this mo dification resulted in an inhibition of TGase activity. The addition of Ca2 + to nitrosylated tTG was able to trigger the release of NO groups (i.e. de nitrosylation). tTG nitrosylated in the absence of Ca2+ was 6-fold more sus ceptible to inhibition by Mg-GTP. When endothelial cells in culture were in cubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitr osylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation in duced by ADP. In conclusion, we provide evidence that Ca2+ regulates the S- nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca2+ in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.