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

Abstract

Transglutaminase 2 (TG2) is a multifunctional protein with diverse catalytic activities and biological roles. Its best studied function is the Ca2+-dependent transamidase activity leading to formation of γ-glutamyl-ε-lysine isopeptide crosslinks between proteins and γ-glutamyl-amine derivatives. TG2 has a poorly studied isopeptidase activity cleaving these bonds. We have developed and characterised TG2 mutants which are significantly deficient in transamidase activity while have normal or increased isopeptidase activity (W332F) and vice versa (W278F). The W332F mutation led to significant changes of both the K m and the V max kinetic parameters of the isopeptidase reaction of TG2 while its calcium and GTP sensitivity was similar to the wild-type enzyme. The W278F mutation resulted in six times elevated amine incorporating transamidase activity demonstrating the regulatory significance of W278 and W332 in TG2 and that mutations can change opposed activities located at the same active site. The further application of our results in cellular systems may help to understand TG2-driven physiological and pathological processes better and lead to novel therapeutic approaches where an increased amount of crosslinked proteins correlates with the manifestation of degenerative disorders.

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Acknowledgments

The authors are grateful to Dr. Máté Demény for the critical reading of the manuscript. This work was supported by the Research University grant from University of Debrecen (RH/885/2013), the Hungarian Scientific Research Fund (OTKA NK 105046), the New Hungary Development Plan via the TÁMOP-4.2.2.A-11/1/KONV-2012-0023 “VÉD-ELEM” project co-financed by the European Social Fund, the European Union Framework Programme 7 TRANSCOM IAPP 251506 and TRANSPATH ITN 289964 and the European Union and the State of Hungary, co-financed by the European Social Fund in the framework of the TÁMOP 4.2.4. A/2-11-1-2012-0001 ‘National Excellence Programme’ which provided personal support to R.K.

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Correspondence to László Fésüs.

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László Fésüs and Martin Griffin have contributed equally to this study.

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Király, R., Thangaraju, K., Nagy, Z. et al. Isopeptidase activity of human transglutaminase 2: disconnection from transamidation and characterization by kinetic parameters. Amino Acids 48, 31–40 (2016). https://doi.org/10.1007/s00726-015-2063-5

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Keywords

  • Human transglutaminase 2
  • Isopeptidase activity
  • γ-Glutamyl-hydrolase
  • Transamidation
  • Regulation of activities
  • Moonlighting enzyme