title: Real-time kinetic method to monitor isopeptidase activity of transglutaminase 2 on protein substrate.
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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary.
authors.forename: Kiruphagaran
authors.initials: K
authors.lastname: Thangaraju
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary.
authors.forename: Beáta
authors.initials: B
authors.lastname: Biri
authors.affiliation: MTA-ELTE Research Group of Peptide Chemistry, Hungarian Academy of Sciences-Eötvös Loránd University, 1117 Budapest, Hungary.
authors.forename: Gitta
authors.initials: G
authors.lastname: Schlosser
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary.
authors.forename: Bence
authors.initials: B
authors.lastname: Kiss
authors.affiliation: Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary.
authors.forename: László
authors.initials: L
authors.lastname: Nyitray
authors.affiliation: 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.
authors.forename: László
authors.initials: L
authors.lastname: Fésüs
authors.affiliation: Department of Biochemistry and Molecular Biology, Faculty of Medicine, University of Debrecen, 4012 Debrecen, Hungary. Electronic address: kiralyr@med.unideb.hu.
authors.forename: Róbert
authors.initials: R
authors.lastname: Király
mesh_terms: D019759:Carbon-Nitrogen Lyases / Q000737:chemistry / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000737:chemistry; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000737:chemistry; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013997:Time Factors; D011503:Transglutaminases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase
keywords: Fluorescence anisotropy; Isopeptidase activity; S100A4; Transglutaminase 2