title: FRET-based detection of isozyme-specific activities of transglutaminases.
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.
authors: Cellular Biochemistry Lab., Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-0814, Japan. htatsukawa@ps.nagoya-u.ac.jp.
authors: Hideki
authors: 0000-0003-0059-9677
authors: H
authors: Tatsukawa
authors: Cellular Biochemistry Lab., Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-0814, Japan.
authors: Hong Hong
authors: HH
authors: Liu
authors: Cellular Biochemistry Lab., Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-0814, Japan.
authors: Shota
authors: S
authors: Oba
authors: Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Fukuoka, 819-0395, Japan.|Division of Biotechnology, Center for Future Chemistry, Kyushu University, Fukuoka, 819-0388, Japan.
authors: Noriho
authors: N
authors: Kamiya
authors: Department of Biological Mechanisms and Functions, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Japan.
authors: Yoichi
authors: Y
authors: Nakanishi
authors: Cellular Biochemistry Lab., Department of Basic Medicinal Sciences, Graduate School of Pharmaceutical Sciences, Nagoya University, Furo-cho, Chikusa, Nagoya, 464-0814, Japan.
authors: Kiyotaka
authors: K
authors: Hitomi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D001681:Biological Assay; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D015335:Molecular Probes / Q000138:chemical synthesis / Q000378:metabolism*; D019151:Peptide Library; D010455:Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D007527:Isoenzymes; D015335:Molecular Probes; D019151:Peptide Library; D010455:Peptides; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; C105804:transglutaminase 1; D019204:GTP-Binding Proteins
keywords: Activity; Förster resonance energy transfer; Substrate peptide; Transglutaminase