Modulation of the in Situ Activity of Tissue Transglutaminase by Calcium and GTP

Abstract

Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the posttranslational modification of proteins by transamidation of specific polypeptide-bound glutamine residues. Previous in vitro studies have demonstrated that the transamidating activity of tTG requires calcium and is inhibited by GTP. To investigate the endogenous regulation of tTG, a quantitative in situ transglutaminase (TG) activity assay was developed. Treatment of human neuroblastoma SH-SY5Y cells with retinoic acid (RA) resulted in a significant increase in tTG levels and in vitro TG activity. In contrast, basal in situ TG activity did not increase concurrently with RA-induced increased tTG levels. However, stimulation of cells with the calcium-mobilizing drug maitotoxin (MTX) 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 combination of MTX and tiazofurin resulted in significantly less in situ TG activity compared with treatment with MTX alone. This raised the possibility of calcium-dependent proteolysis due to the effects of tiazofurin, because in vitro GTP protects tTG against proteolysis by trypsin. Studies with a selective membrane permeable calpain inhibitor indicated that tTG is likely to be an endogenous substrate of calpain, and that depletion of GTP increases tTG degradation after elevation of intracellular calcium levels. TG activity was also increased in response to activation of muscarinic cholinergic receptors, which increases intracellular calcium through inositol 1,4,5-trisphosphate generation. The results of these experiments demonstrate that selective changes in calcium and GTP regulate the activity and levels of tTG in situ.

Role of transglutaminase 2 in PAC1 receptor mediated protection against hypoxia-induced cell death and neurite outgrowth in differentiating N2a neuroblastoma cells

“…5-HT2A receptor-mediated incorporation of 5-HT into the small GTPases RhoA and Rab4 has also been observed in platelets (Walther et al, 2003). It was suggested that 5-HT2A and muscarinic receptor-mediated release of Ca 2+ from intracellular Ca 2+ stores may be responsible for triggering TG transamidating activity (Zhang et al, 1998;Walther et al, 2003). Our recent studies have shown that the Gi/o-protein coupled A1 adenosine receptor stimulates TG2 activation in H9c2 cardiomyocyte-like cells (Vyas et al, 2016).…”

“…Previous studies have shown that release of Ca 2+ from intracellular Ca 2+ stores or influx of extracellular Ca 2+ is linked to TG activation in response to GPCR stimulation (Zhang et al, 1998;Walther et al, 2003;Vyas et al, 2016 et al, 2011).…”

“…Examples in the literature include muscarinic receptor-mediated increases in TG2 activity in SH-SY5Y cells (Zhang et al, 1998) and 5-HT2A receptor-mediated transamidation (TG-catalyzed) of the small G-protein Rac1 in the rat cortical cell line A1A1v (Dai et al, 2008). The study by Zhang et al (1998) measured in situ TG2 activity (polyamine incorporation) triggered by the non-selective muscarinic agonist carbachol, whereas, Dai et al (2008) reported TG2 catalysed incorporation of 5-hydroxytryptamine into Rac1. 5-HT2A receptor-mediated incorporation of 5-HT into the small GTPases RhoA and Rab4 has also been observed in platelets (Walther et al, 2003).…”

“…However, the regulation of intracellular TG2 following stimulation of GPCRs is not well understood. Examples include muscarinic receptor-mediated increases in TG2 activity in SH-SY5Y cells (Zhang et al, 1998), 5-HT2A receptor-mediated transamidation of Rac1 in the rat A1A1v cortical cell line (Dai et al, 2008) and A1 adenosine receptor-mediated increases in TG2 transamidase activity in rat H9c2 cardiomyoblasts (Vyas et al, 2016).…”

Tissue transglutaminase links TGF-β, epithelial to mesenchymal transition and a stem cell phenotype in ovarian cancer

“…Dose-response experiments demonstrated that the effects of TGF-b1 on TG2 expression occur from concentrations of 0.5-5 ng/ml of TGF-b1 (Supplementary Figure 1e), ranging within physiological limits. The enzymatic function of TG2 was also induced by TGF-b1, as measured by amine incorporation into total cellular proteins (Zhang et al, 1998). Increased TG2 activity was observed in OC cells (Hey and OVCA#16), but not in non-transformed LP9 or NOSE cells (Supplementary Figure 2).…”