Tissue transglutaminase (TG) is an enzyme that stabilizes the structure of tissues by covalently ligating extracellular matrix molecules. Expression and localization of TG are not well established during wound healing. We performed punch biopsy wounds on anesthetized rats and monitored the wound healing process by histological and immunohistochemical methods. The TG antigen and activity are expressed at sites of neovascularization in the provisional fibrin matrix within 24 h of wounding. Endothelial cells, macrophages, and skeletal muscle cells expressed TG throughout the healing process. The TG antigen within the wound was active in vivo based on the detection of isopeptide bonds. The TG antigen increased four- to fivefold by day 3 postwounding and was proteolytically degraded. TG expression occurred in association with TGF-beta, TNF-alpha, IL-6, and VEGF production in the wound. Recombinant TG increased vessel length density (a measure of angiogenesis) when applied topically in rat dorsal skin flap window chambers. We have established that TG is an important tissue stabilizing enzyme that is active during wound healing and can function to promote angiogenesis.

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.

Tissue transglutaminase (tTG) exhibits a magnesiumdependent GTP/ATPase activity that is involved in the regulation of the cell cycle and cell receptor signaling. The portion of the molecule involved in GTP/ATP hydrolysis is unknown. We expressed and purified a series of C-terminal truncation mutants of human tTG as glutathione S-transferase fusion proteins (⌬S538, ⌬E447, ⌬P345, ⌬C290, ⌬V228, and ⌬F185) to determine the effect on GTP/ATPase activity. The truncation of the C terminus did not change significantly the apparent K m value for either GTP or ATP. In contrast, the K cat value for GTP was increased by 4.6-and 3-fold for the ⌬S538 and ⌬E447 mutants, respectively. The ⌬P345 mutant had the highest hydrolysis activity with a 34-fold increase. The hydrolysis activity then declined to 8.1-, 8.7-, and 1.9-fold for the ⌬C290, ⌬V228, and ⌬F185 mutants, respectively. The K cat for ATP changed in parallel with the GTPase results. Thin layer chromatography analysis of the hydrolysis reaction products revealed that ATP was rapidly converted to ADP followed by a much slower conversion of ADP to AMP when incubated with wild type tTG or the ⌬P345 mutant. There was a substantial decrease in the calcium-dependent TGase activity when the last 149 amino acid residues were deleted from the C terminus. Less than 5% of the TGase activity was detected for the ⌬S538 and ⌬E447 mutants. In conclusion, we have located the ATP and GTP hydrolytic domain to amino acid residues 1-185. The C terminus functions to inhibit the expression of endogenous GTP/ATPase activity of tTG, and the potential role of the C terminus in modulating this activity is discussed.Tissue transglutaminase (tTG) 1 is a unique member of the transglutaminase gene family in that it exhibits two distinct enzyme activities (1-3). The calcium-dependent transglutaminase activity (TGase) catalyzes the covalent modification of proteins by the formation of ␥-glutamyl-⑀-lysine bonds between proteins or polyamines (1, 2). The TGase activity is considered to be an important intracellular and extracellular reaction during apoptosis (4, 5), bone ossification (6), tissue repair (7), and tumor growth (8). TGase activity requires a calcium binding site and active site cysteine to form a thioester bond with the glutamine substrate (1, 2). The active site of human tTG is located at Cys-277, and the putative calcium binding site is located between amino acids 446 and 453 based on sequence homology to the calcium binding site in the factor XIII A chains (9).The tTG will selectively modify a group of protein-bound glutamine residues that exist in proteins found in the extracellular matrix (ECM) including vitronectin, fibronectin, osteonectin, and nidogen (1, 2). When tTG is released into plasma or ECM it binds to fibronectin and retains TGase activity (1, 2). Fibronectin binding functions to localize tTG to sites of fibronectin expression and deposition and limits the availability of the enzyme for cross-linking other substrates. The fibronectin binding site is located in the N-termi...