FRET-based detection of isozyme-specific activities of transglutaminases

Hideki Tatsukawa, Hong Hong Liu, Shota Oba, Noriho Kamiya, Yoichi Nakanishi, Kiyotaka Hitomi

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2 Citations (Scopus)

Abstract

Transglutaminases (TGs) comprise a protein family in which the members catalyze the formation of isopeptide bonds between glutamine and lysine residues in various proteins. Expression studies on its three major members, FXIII, TG1, and TG2, have been performed in a relatively large number of mammalian tissues in comparison with those on the other isozymes. We previously identified a highly reactive substrate peptide, including glutamine, for each isozyme from a phage display library and developed a method for detecting isozyme-specific activities by incorporating a labeled substrate peptide into lysine residues of proteins. Here, we describe genetically encoded Förster resonance energy transfer (FRET)-based probes composed of each fluorescence protein (Cerulean and EVenus) fused with substrate peptides. The probe pairs, designated as Trac-MTG (His-CerΔ11-LQ/EV-K-His) containing linker and substrate peptide sequence for microbial TG (MTG), increased the EVenus:Cerulean fluorescence intensity ratio by more than 1.5-fold. Furthermore, we demonstrated that Trac-TG1 (His-CerΔ11-K5) and Trac-TG2 (His-CerΔ11-T26) containing substrate peptide sequence for mammalian TGs successfully detected the isozyme-specific activity of TG1 and TG2, respectively. In this study, we developed a rapid and convenient experimental system for measuring the isozyme-specific activity of TGs. The application of these probes for analyses in cells and tissues will be helpful for elucidating the physiological and pathological functions of TGs.

Original languageEnglish
Pages (from-to)615-623
Number of pages9
JournalAmino Acids
Volume49
Issue number3
DOIs
Publication statusPublished - 2017

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Clinical Biochemistry
  • Organic Chemistry

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  • Cite this

    Tatsukawa, H., Liu, H. H., Oba, S., Kamiya, N., Nakanishi, Y., & Hitomi, K. (2017). FRET-based detection of isozyme-specific activities of transglutaminases. Amino Acids, 49(3), 615-623. https://doi.org/10.1007/s00726-016-2322-0