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

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