The 55 kDa tissue transglutaminase cross-linking active isoform TG induces cell death.

Transamidations are calcium-dependent reactions catalyzed by transglutaminase enzymes. Tissue transglutaminase (TG2 or TGC) is a multifunctional protein with a controversial role in apoptosis. The cross-linking function of transglutaminase enzymes has been shown to play a role in cell death. Human breast cancer cell lines (MCF7 and T47D), which express low endogenous levels of transglutaminase, were transiently transfected with the cross-linking 55 kDa active TG isoform or its precursor the 80 kDa full-length TGC. The increased frequency of apoptosis correlated with the increase in transglutaminase expression and the highest rates of apoptosis were found in cells transfected with the potent TG isoform as compared to the full length TGC. The calcium ionophores A231827 and maitotoxin, which are known to induce transamidation, were found to promote apoptosis, whereas cystamine, an active transglutaminase inhibitor, blocked apoptosis due to the over-expression of the active TG isoform. This is the first time that TG has been used in cellular transfections and the results presented show that TG is a potent inducer of cell death. This finding may help to clarify the conflicting functions of TG in the induction of cell death. The TG-dependent irreversible cross-linking of intracellular proteins, a function previously assigned to TGC, represents an important biochemical event in the induction of the structural changes present in cells during apoptosis.

Induction and translocation of tissue transglutaminase isoforms increased phosphorylation in retinoic acid treated erythroleukemia cells.

Tissue transglutaminase (TGC, TG2, 80 kDa) is inactive in cross-linking reactions and is converted in vitro and in vivo to the TG (55 kDa) active isoform (Fraij in J Cell Biochem 112:2469-2489, 2011). Two isoforms of human TGC were cloned from human erythroleukemia (HEL) cells induced with retinoic acid (RA) and termed TGH, 63 kDa (Fraij et al. in J Biol Chem 267:22616-22673, 1992) and TGH2, 37 kDa. The purified TGC isoforms exhibited GTPase activity and TGH and TGH2 showed higher activities than the native TGC protein. In all normal cells examined, TGC was found in membrane fractions several fold higher than the supernatant fractions; however, in the natural tumor cell line HEL the TGC cellular distribution was reversed. Although TGC is the major enzyme in normal human erythrocytes, its expression level was significantly decreased in HEL cells. RA treatment induced a sevenfold increase in the level of TGC protein in HEL cells and was accompanied by its translocation to cell membranes. When isolated membrane and supernatant fractions from normal human foreskin (CF3), normal human embryonic lung (WI-38), and HEL cells treated with or without RA were incubated with [(32)P]-ATP at 37 °C for 1 h, more radio-labeled proteins were detected in the membrane fractions than the cytosolic fractions. More labeled protein bands were detected in RA treated HEL cells in comparison to control HEL cell extracts. Radio labeled proteins coimmunoprecipitated with the TGC isoforms in RA treated HEL membrane fractions thereby confirming that the radio-labeled material consists of endogenous proteins associated with TGC isoforms. Protein phosphorylation is related to the induction and translocation of the isoforms in RA treated cells. These results show that the TGC isoforms complexes with proteins in vivo and that the phosphorylation of these proteins is catalyzed directly by TGC kinase activity or indirectly by the TGC phosphorylation of other protein kinases.

"Activation of tissue tranglutsaminase by removal of carboxyl-terminal peptides".

Tissue transglutaminase (TGC or TG2) functions as transglutaminase (cross-linking), deamidase, kinase, and disulfide isomerase and its activities are implicated in the pathogenesis of several human diseases. Proteolytic activation of zymogens in the transglutaminase family is not unusual. Plasma transglutaminase (FXIIIa), epidermal transglutaminase (TG 3), transglutaminase-5, and microbial transglutaminase (MTG) can be subjected to proteolysis from specific proteases to generate the active functional enzyme. In the present study, calcium or GTP was essential for activation of TGC cross-linking activity by trypsin in membrane fractions from human RBC and was accompanied by the conversion of TGC (80 kDa) to a smaller TG form (55 kDa). While bacterially expressed TGC showed no activity, bacterial expression of C-terminal domain deletion constructs with carboxy-terminal ends ranging from lysine 464 (TG464) to glycine 480 (TG480) produced enzymes that were highly active in cross-linking activity. The product of a construct with a coding region ended at proline 446 (TG446), which interrupted the calcium-binding domain, exhibited weak cross-linking activity. TG480 and TG512 were characterized by about 80% and 10%, respectively, of the cross-linking activities of TG464. This may indicate that the longer the peptide after the calcium binding domain, the less the enzymatic activity expressed, possibly because the folding of such peptide which interfere with the calcium binding site or the catalytic site. Western analysis of MCF7 and T47D human breast cancer cells transfected with TGC showed TGC as a major protein and TG as a minor fragment. Incubation of lysate from transfected cells with serum resulted in the conversion of the TGC to TG, a condition that may be comparable to injury or wounds that lead to rapid enzymatic transamidation activation.