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

Róbert Király, Éva Cssz, T. Kurtán, S. Antus, Krisztián Szigeti, Zsófia Simon-Vecsei, Ilma Rita Korponay-Szabó, Zsolt Keresztessy, L. Fésüs

Research output: Article

52 Citations (Scopus)

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 Ca 2+-binding form of the human enzyme is not known, and its Ca 2+-binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca 2+-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 Ca 2+. 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.

Original languageEnglish
Pages (from-to)7083-7096
Number of pages14
JournalFEBS Journal
Volume276
Issue number23
DOIs
Publication statusPublished - dec. 2009

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Mutagenesis
Site-Directed Mutagenesis
Transglutaminases
GTP Phosphohydrolases
Binding Sites
Celiac Disease
Guanosine Triphosphate
Autoantibodies
Catalytic Domain
Enzymes
Factor XIIIa
Dialysis
Autoantigens
Amino Acid Substitution
Titration
Abdomen
Hydrolysis
Assays
Spectrum Analysis
Substitution reactions

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis. / Király, Róbert; Cssz, Éva; Kurtán, T.; Antus, S.; Szigeti, Krisztián; Simon-Vecsei, Zsófia; Korponay-Szabó, Ilma Rita; Keresztessy, Zsolt; Fésüs, L.

In: FEBS Journal, Vol. 276, No. 23, 12.2009, p. 7083-7096.

Research output: Article

Király, Róbert ; Cssz, Éva ; Kurtán, T. ; Antus, S. ; Szigeti, Krisztián ; Simon-Vecsei, Zsófia ; Korponay-Szabó, Ilma Rita ; Keresztessy, Zsolt ; Fésüs, L. / Functional significance of five noncanonical Ca2+-binding sites of human transglutaminase 2 characterized by site-directed mutagenesis. In: FEBS Journal. 2009 ; Vol. 276, No. 23. pp. 7083-7096.
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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 Ca 2+-binding form of the human enzyme is not known, and its Ca 2+-binding sites have not been fully characterized. By mutagenesis, we have targeted its active site Cys, three sites based on homology to Ca 2+-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 Ca 2+. 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.",
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AU - Szigeti, Krisztián

AU - Simon-Vecsei, Zsófia

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