“Activation of tissue tranglutsaminase by removal of carboxyl-terminal peptides”

Abstract

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.