Detection of Transglutaminase Activity Using Click Chemistry

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.