Insights into O-linked N-acetylglucosamine ([0-9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates.
Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.
Department of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada.
David L
DL
Shen
Tracey M
TM
Gloster
Scott A
SA
Yuzwa
David J
DJ
Vocadlo
D000117:Acetylglucosamine / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4 / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011499:Protein Processing, Post-Translational; D011484:Protein-Arginine N-Methyltransferases / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D001619:beta-N-Acetylhexosaminidases / Q000235:genetics / Q000378:metabolism*; D016875:tau Proteins / Q000235:genetics / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D011506:Proteins; D011994:Recombinant Proteins; C486300:TAB1 protein, human; C049312:nuclear pore protein p62; D016875:tau Proteins; D012694:Serine; D011484:Protein-Arginine N-Methyltransferases; C119966:coactivator-associated arginine methyltransferase 1; D017351:N-Acetylglucosaminyltransferases; C063057:UDP-N-acetylglucosamine-peptide beta-N-acetylglucosaminyltransferase; D054734:Calcium-Calmodulin-Dependent Protein Kinase Type 4; C110628:hexosaminidase C; D001619:beta-N-Acetylhexosaminidases; D000117:Acetylglucosamine