A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications.
We report a new chemoenzymatic strategy for the rapid and sensitive detection of O-GlcNAc posttranslational modifications. The approach exploits the ability of an engineered mutant of beta-1,4-galactosyltransferase to selectively transfer an unnatural ketone functionality onto O-GlcNAc glycosylated proteins. Once transferred, the ketone moiety serves as a versatile handle for the attachment of biotin, thereby enabling chemiluminescent detection of the modified protein. Importantly, this approach permits the rapid visualization of proteins that are at the limits of detection using traditional methods. Moreover, it bypasses the need for radioactive precursors and captures the glycosylated species without perturbing metabolic pathways. We anticipate that this general chemoenzymatic strategy will have broad application to the study of posttranslational modifications.
Khidekel
Nelly
N
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125, USA.
Arndt
Sabine
S
Lamarre-Vincent
Nathan
N
Lippert
Alexander
A
Poulin-Kerstien
Katherine G
KG
Ramakrishnan
Boopathy
B
Qasba
Pradman K
PK
Hsieh-Wilson
Linda C
LC
D000117:Acetylglucosamine / Q000032:analysis / Q000378:metabolism*; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D001710:Biotin; D017362:Cyclic AMP Response Element-Binding Protein / Q000032:analysis / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis / Q000378:metabolism*; D006031:Glycosylation; D006735:Horseradish Peroxidase / Q000737:chemistry / Q000378:metabolism; D008163:Luminescent Measurements; D009237:N-Acetyllactosamine Synthase / Q000737:chemistry / Q000378:metabolism*; D038201:alpha-Crystallins / Q000032:analysis / Q000378:metabolism
D001426:Bacterial Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D006023:Glycoproteins; C058865:Strep-avidin conjugated horseradish peroxidase; D038201:alpha-Crystallins; D001710:Biotin; D006735:Horseradish Peroxidase; D009237:N-Acetyllactosamine Synthase; D000117:Acetylglucosamine