title: Using carboxyfluorescein diacetate succinimidyl ester to monitor intracellular protein glycation.
abstract: Protein glycation is a ubiquitous process involved in vascular complications observed in diabetes. Glyoxal (GO), an intracellular reactive oxoaldehyde that is one of the most potent glycation agents, readily reacts with amines present on proteins to produce the lysine-derived adduct carboxymethyllysine, which is a prevalent advanced glycation end-product (AGE). Our group previously showed that cell exposure to GO leads to an alteration in the cell contractile activity that could occur as a result of the glycation of various proteins regulating the cell contractile machinery. Here, we measured the extent of glycation on three functionally distinct proteins known to participate in cell contraction and cytoskeletal organization-Rho-kinase (ROCK), actin, and gelsolin (GSN)-using an assay based on the reaction of the cell membrane-permeable fluorescent probe carboxyfluorescein diacetate succinimidyl ester (CFDA-SE), which reacts with primary amine groups of proteins. By combining CFDA-SE fluorescence and Western blot detection, we observed (following GO incubation) increased glycation of actin and ROCK as well as an increased interaction between actin and GSN as observed by co-immunoprecipitation. Thus, we conclude that the use of the fluorescent probe CFDA-SE offers an interesting alternative to perform a comparative analysis of the extent of intracellular protein glycation in live cells.
authors: Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors: Julie
authors: J
authors: Boucher
authors: Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors: Élie
authors: É
authors: Simard
authors: Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors: Ulrike
authors: U
authors: Froehlich
authors: Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada.
authors: Pedro
authors: P
authors: D'Orléans-Juste
authors: Department of Pharmacology, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Electronic address: michel.grandbois@usherbrooke.ca.
authors: Michel
authors: M
authors: Grandbois
mesh_terms: D000199:Actins / Q000032:analysis / Q000378:metabolism; D015153:Blotting, Western; D002460:Cell Line; D005452:Fluoresceins / Q000032:analysis / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism*; D018260:Gelsolin / Q000032:analysis / Q000378:metabolism; D006031:Glycosylation; D006037:Glyoxal / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D013388:Succinimides / Q000032:analysis / Q000378:metabolism*; D054460:rho-Associated Kinases / Q000032:analysis / Q000378:metabolism
chemical_list: C087165:5-(6)-carboxyfluorescein diacetate succinimidyl ester; D000199:Actins; D005452:Fluoresceins; D005456:Fluorescent Dyes; D018260:Gelsolin; D013388:Succinimides; D006037:Glyoxal; D054460:rho-Associated Kinases
keywords: Actin; Carboxyfluorescein diacetate succinimidyl ester (CFDA–SE); Cellular protein glycation; Gelsolin; Glyoxal; Rho-kinase