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  3. Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate

  • Anal Biochem. 2016 Jul 15;505:36-42. doi: 10.1016/j.ab.2016.04.012.
Kiruphagaran Thangaraju 1,  Beáta Biri 2,  Gitta Schlosser 3,  Bence Kiss 2,  László Nyitray 2,  László Fésüs 4,  Róbert Király 5
Affiliations

Affiliations

  • 1 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary.
  • 2 Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary.
  • 3 MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences-Eötvös Loránd University, 1117 Budapest, Hungary.
  • 4 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary; MTA-DE Stem Cell, Apoptosis, and Genomics Research Group of Hungarian Academy of Sciences, University of Debrecen, 4012 Debrecen, Hungary.
  • 5 Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary. Electronic address: kiralyr@med.unideb.hu.
PMID: 27131890 DOI: 10.1016/j.ab.2016.04.012
Abstract

Transglutaminase 2 (TG2) is a ubiquitously expressed multifunctional protein with CA(2+)-dependent transamidase activity forming protease-resistant N(ε)-(γ-glutamyl) lysine crosslinks between proteins. It can also function as an isopeptidase cleaving the previously formed crosslinks. The biological significance of this activity has not been revealed yet, mainly because of the lack of a protein-based method for its characterization. Here we report the development of a novel kinetic method for measuring isopeptidase activity of human TG2 by monitoring decrease in the fluorescence polarization of a protein substrate previously formed by crosslinking fluorescently labeled glutamine donor FLpepT26 to S100A4 at a specific lysine residue. The developed method could be applied to test mutant Enzymes and compounds that influence isopeptidase activity of TG2.

Keywords

Fluorescence anisotropy; Isopeptidase activity; S100A4; Transglutaminase 2.

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