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.
Publication types
-
Research Support, Non-U.S. Gov't
MeSH terms
-
Acylation
-
Alkynes / chemistry
-
Amines / chemistry
-
Amino Acid Sequence
-
Animals
-
Azides / chemistry
-
Biocatalysis
-
Biotin / analogs & derivatives
-
Biotin / chemistry
-
Cadaverine / metabolism
-
Cell Membrane / metabolism
-
Click Chemistry*
-
Cross-Linking Reagents / chemistry
-
Cycloaddition Reaction
-
Enzyme Inhibitors / chemistry
-
Fluorescein-5-isothiocyanate / metabolism
-
Fluorescent Dyes / metabolism
-
GTP-Binding Proteins / antagonists & inhibitors
-
GTP-Binding Proteins / chemistry*
-
HSP20 Heat-Shock Proteins / chemistry
-
HeLa Cells
-
Humans
-
Mice
-
Peptide Fragments / chemistry
-
Permeability
-
Protein Glutamine gamma Glutamyltransferase 2
-
Staining and Labeling
-
Transglutaminases / antagonists & inhibitors
-
Transglutaminases / chemistry*
Substances
-
Alkynes
-
Amines
-
Azides
-
Cross-Linking Reagents
-
Enzyme Inhibitors
-
Fluorescent Dyes
-
HSP20 Heat-Shock Proteins
-
HSPB6 protein, human
-
Peptide Fragments
-
5-(biotinamido)pentylamine
-
Biotin
-
Protein Glutamine gamma Glutamyltransferase 2
-
Transglutaminases
-
GTP-Binding Proteins
-
Fluorescein-5-isothiocyanate
-
Cadaverine