title: Detection of transglutaminase activity using click chemistry.
abstract: Transglutaminase 2 (TG2) is a Ca(2+)-dependent enzyme able to catalyze the formation of ε(γ-glutamyl)-lysine crosslinks between polypeptides, resulting in high molecular mass multimers. We have developed a bioorthogonal chemical method for the labeling of TG2 glutamine-donor proteins. As amine-donor substrates we used a set of azide- and alkyne-containing primary alkylamines that allow, after being crosslinked to glutamine-donor proteins, specific labeling of these proteins via the azide-alkyne cycloaddition. We demonstrate that these azide- and alkyne-functionalized TG2 substrates are cell permeable and suitable for specific labeling of TG2 glutamine-donor substrates in HeLa and Movas cells. Both the Cu(I)-catalyzed and strain promoted azide-alkyne cycloaddition proved applicable for subsequent derivatization of the TG2 substrate proteins with the desired probe. This new method for labeling TG2 substrate proteins introduces flexibility in the detection and/or purification of crosslinked proteins, allowing differential labeling of cellular proteins.
authors.affiliation: Department of Biomolecular Chemistry, Nijmegen Centre for Molecular Life Sciences, Institute for Molecules and Materials and Netherlands Proteomics Centre, Radboud University, Nijmegen, The Netherlands.
authors.forename: Remon
authors.initials: R
authors.lastname: van Geel
authors.forename: Marjoke F
authors.initials: MF
authors.lastname: Debets
authors.forename: Dennis W P M
authors.initials: DW
authors.lastname: Löwik
authors.forename: Ger J M
authors.initials: GJ
authors.lastname: Pruijn
authors.forename: Wilbert C
authors.initials: WC
authors.lastname: Boelens
mesh_terms: D000215:Acylation; D000480:Alkynes / Q000737:chemistry; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000737:chemistry; D055162:Biocatalysis; D001710:Biotin / Q000031:analogs & derivatives / Q000737:chemistry; D002103:Cadaverine / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D057930:Click Chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D061565:Cycloaddition Reaction; D004791:Enzyme Inhibitors / Q000737:chemistry; D016650:Fluorescein-5-isothiocyanate / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry*; D050886:HSP20 Heat-Shock Proteins / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D010446:Peptide Fragments / Q000737:chemistry; D010539:Permeability; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D013194:Staining and Labeling; D011503:Transglutaminases / Q000037:antagonists & inhibitors / Q000737:chemistry*
chemical_list: D000480:Alkynes; D000588:Amines; D001386:Azides; D003432:Cross-Linking Reagents; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D050886:HSP20 Heat-Shock Proteins; C494382:HSPB6 protein, human; D010446:Peptide Fragments; C076022:5-(biotinamido)pentylamine; D001710:Biotin; D000091345:Protein Glutamine gamma Glutamyltransferase 2; D011503:Transglutaminases; D019204:GTP-Binding Proteins; D016650:Fluorescein-5-isothiocyanate; D002103:Cadaverine