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

• Published in: The FEBS journal 2009-12, Vol.276 (23), p.7083-7096
• Main Authors: Király, Róbert, Csősz, Éva, Kurtán, Tibor, Antus, Sándor, Szigeti, Krisztián, Simon-Vecsei, Zsófia, Korponay-Szabó, Ilma Rita, Keresztessy, Zsolt, Fésüs, László
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino acids; Binding Sites; Biochemistry; Calcium; Calcium - chemistry; Calcium - metabolism; calcium binding; celiac epitope; Circular Dichroism; Enzymes; GTP-Binding Proteins - chemistry; GTP-Binding Proteins - genetics; GTP-Binding Proteins - metabolism; GTPase activity; Humans; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Sequence Alignment; transglutaminase 2 (tissue transglutaminase); transglutaminase activity; Transglutaminases - chemistry; Transglutaminases - genetics; Transglutaminases - metabolism
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/j.1742-4658.2009.07420.x

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.