Functional significance of five noncanonical Ca²⁺‐binding sites of human transglutaminase 2 characterized by site‐directed mutagenesis

Abstract

The multifunctional tissue transglutaminase 2 (TG2) has a four‐domain structure with several Ca2+‐regulated biochemical activities, including transglutamylation and GTP hydrolysis. The structure of the Ca2+‐binding form of the human enzyme is not known, and its Ca2+‐binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca2+‐binding residues of epidermal transglutaminase and factor XIIIa (S1–S3), and two regions with negative surface potentials (S4 and S5). CD spectroscopy, antibody‐binding assay and GTPase activity measurements indicated that the amino acid substitutions did not cause major structural alterations. Calcium‐45 equilibrium dialysis and isothermal calorimetric titration showed that both wild‐type and active site‐deleted enzymes (C277S) bind six Ca2+. Each of the S1–S5 mutants binds fewer than six Ca2+, S1 is a strong Ca2+‐binding site, and mutation of one site resulted in the loss of more than one bound Ca2+, suggesting cooperativity among sites. All mutants were deficient in transglutaminase activity, and GTP inhibited remnant activities. Like those of the wild‐type enzyme, the GTPase activities of the mutants were inhibited by Ca2+, except in the case of the S4 and S5 mutants, which exhibited increased activity. TG2 is the major autoantigen in celiac disease, and testing the reactivity of mutants with autoantibodies from celiac disease patients revealed that S4 strongly determines antigenicity. It can be concluded that five of the Ca2+‐binding sites of TG2 influence its transglutaminase activity, two sites are involved in the regulation of GTPase activity, and one determines antigenicity for autoantibodies in celiac patients.

Early tissue transglutaminase–mediated response underlies K562(S)-cell gliadin-dependent agglutination

“…Therefore, we investigated the effects of increasing Ca 2+ concentrations in the culture medium on K562(S) agglutination. In fact, TG2 is activated by a concentration of Ca 2+ higher than 1-2 mmol/l (21,22). As shown in Figure 3a, Ca 2+ concentrations below 1 mmol/l were not able to induce K562(S) agglutination, whereas Ca 2+ levels higher than 2 mmol/l induced massive agglutination.…”

Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters

“…The earlier prepared Gly 224 containing recombinant human TG2 variant and its mutants were expressed in N-terminally (His)6-tagged form (pET-30 Ek/LIC-TG2) and purified by Ni-NTA affinity chromatography as described previously (Király et al 2009). There were no differences in expression level, yield and stability of the mutant proteins as compared to the wild type TG2.…”

Metastasis-associated S100A4 is a specific amine donor and an activity-independent binding partner of transglutaminase-2

“…The TG2 mutants Trp241Phe, Trp241Ala and Cys277Ser were made by QuickChange site directed mutagenesis protocol (Stratagene), the Megaprimer method [15], and as described previously [18], respectively.…”