Calcium Regulates S-Nitrosylation, Denitrosylation, and Activity of Tissue Transglutaminase

Abstract

Nitric oxide (NO) and related molecules play important roles in vascular biology. 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 expressed by endothelial cells and secreted into the extracellular matrix (ECM) where it is bound to fibronectin. Tissue TG exhibits a Ca(2+)-dependent transglutaminase activity (TGase) that cross-links proteins involved in wound healing, tissue remodeling, and ECM stabilization. Since tTG is in proximity to sites of NO production, has 18 free cysteine residues, and utilizes a cysteine for catalysis, we investigated the factors that regulated NO binding and tTG activity. We report that TGase activity is regulated by NO through a unique Ca(2+)-dependent mechanism. Tissue TG can be poly-S-nitrosylated by the NO carrier, S-nitrosocysteine (CysNO). In the absence of Ca(2+), up to eight cysteines were nitrosylated without modifying TGase activity. In the presence of Ca(2+), up to 15 cysteines were found to be nitrosylated and this modification resulted in an inhibition of TGase activity. The addition of Ca(2+) to nitrosylated tTG was able to trigger the release of NO groups (i.e. denitrosylation). tTG nitrosylated in the absence of Ca(2+) was 6-fold more susceptible to inhibition by Mg-GTP. When endothelial cells in culture were incubated with tTG and stimulated to produce NO, the exogenous tTG was S-nitrosylated. Furthermore, S-nitrosylated tTG inhibited platelet aggregation induced by ADP. In conclusion, we provide evidence that Ca(2+) regulates the S-nitrosylation and denitrosylation of tTG and thereby TGase activity. These data suggest a novel allosteric role for Ca(2+) in regulating the inhibition of tTG by NO and a novel function for tTG in dispensing NO bioactivity.

A protein microarray-based analysis of S -nitrosylation

“…Other similar examples have recently been uncovered, including the preferential reactivity of GSNO (versus CysNO) with E. coli OxyR (43), and the stereoselective inhibition of T-type calcium channels by l-versus d-CysNO (44). NaAD ϩ -dependent Snitrosylation of Qns1p points further to the importance of allostery in control of S-nitrosylation, and it is notable that S-nitrosylation of hemoglobin (45), serum albumin (46), tissue transglutaminase (47), and the ryanodine receptor (48) are also modulated allosterically (by O 2 , fatty acid, Ca 2ϩ and Ca 2ϩ -calmodulin, respectively). More generally, allosteric effectors are shown to have profound consequences on efficiency of Snitrosylation and thus may be underappreciated determinants of SNO reactivity.…”

Assessment of nitric oxide signals by triiodide chemiluminescence

“…SNO-proteins in vivo would be exposed to the effects of proteolytic enzymes (30) and calcium, which is released from intracellular stores. We have previously shown that tissue transglutaminase is regulated by poly-S-nitrosylation and that Ca 2ϩ determines the stoichiometry of S-nitrosylation (28). As shown in Fig.…”

“…Accordingly, bond dissociation energies of RSNO are reported to vary from Ϸ22 to 32 kcal⅐mol Ϫ1 (6,26), and the dissociation constants of FeNO can vary by a factor of Ͼ10 6 (13,23,24), translating to intrinsic FeNO/SNO lifetimes ranging from seconds to years. Environmental factors that have been reported to influence SNO stability and reactivity, directly or through elicited conformational changes in proteins, include pH (low and high) (5,6,20,26), metal ions (Ca, Mg, Cu, and Fe) (6,14,20,27,28), nucleophiles (ascorbate, thiolate, and amine) (6,13), local hydrophobicity (denaturants) (29), oxidants and reductants (6,19), proteolytic enzymes (30), alkylators (31), O 2 tension (5,32), and various intramolecular interactions (H-bonding, S-, N-, O-coordination, and aromatic residue interactions) (6,16,20,22,(33)(34)(35)(36). Many of these factors also affect FeNO stability (17,23,24).…”

Roles of transglutaminases in cardiac and vascular diseases

“…Both TG2 and FXIII transglutaminase activities are also regulated by nitric oxide. Of the 18 free cysteine residues in TG2, 15 can be nitrosylated and denitrosylated in a calcium-dependent manner, resulting in enzymatic inhibition or activation, respectively (97). Factor XIIIa is similarly inhibited by NO (98).…”