Abstract
Modification of proteins at serine or threonine residues with N-acetylglucosamine, termed O-GlcNAcylation, plays an important role in most eukaryotic cells. To understand the molecular mechanism by which O-GlcNAcylation regulates the entry of Caenorhabditis elegans into the non-aging dauer state, we performed proteomic studies using two mutant strains: the O-GlcNAc transferase-deficient ogt-1(ok430) strain and the O-GlcNAcase-defective oga-1(ok1207) strain. In the presence of the dauer pheromone daumone, ogt-1 showed suppression of dauer formation, whereas oga-1 exhibited enhancement of dauer formation. Consistent with these findings, treatment of wild-type N2 worms with low concentrations of daumone and the O-GlcNAcase inhibitor O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc) enhanced dauer formation, which was dependent on intact O-GlcNAcylation metabolism. We also found that the treatment of daumone enhanced O-GlcNAcylation in vivo. Seven proteins, identified by coupled two-dimensional electrophoresis/liquid chromatography-mass spectroscopy (LC-MS) analysis, were differentially expressed in oga-1(ok1207) worms compared with wild-type N2 worms. The identities of these proteins suggest that O- GlcNAcylation influences stress resistance, protein folding, and mitochondrial function. Using O-GlcNAc labeling with fluorescent dye combined with two-dimensional electrophoresis/LC-MS analysis, we also identified five proteins that were differentially O-GlcNAcylated during dauer formation. Analysis of these candidate O-GlcNAcylated proteins suggests that O-GlcNAcylation may regulate cytoskeleton modifications and protein turnover during dauer formation.
Highlights
OGA contains a hexominidase domain, which is responsible for removing O-GlcNAc from the target proteins, 2 The abbreviations used are: OGT, O-GlcNAc transferase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; GalT, galactosyltransferase; liquid chromatography-mass spectroscopy (LC-MS), liquid chromatography-mass spectrometry; MS/MS, tandem mass spectrometry; IGF, insulin-like growth factor; OGA, OGlcNAcase; NGM, nematode growth medium; Dauer Formation Constitutive (Daf-c), dauer formation constitutive; STZ, streptozotocin; TAMRA, 6-carboxytetramethylrhodamine; MAPK, mitogen-activated protein kinase; Gene Ontology (GO), gene ontology; RNAi, RNA interference; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; GFP, green fluorescent protein
Because various post-translational modifications have been implicated in the regulation of the dauer process, it is reasonable to suppose that O-GlcNAcylation might be among the posttranslational modifications that contribute to dauer formation
1) Does O-GlcNAcylation affect dauer formation? 2) If so, at what step during dauer formation does O-GlcNAcylation act? 3) What kinds of proteins are modified by O-GlcNAcylation? 4) What are the physiological roles of O-GlcNAcylation during dauer formation? Here, we report a proteomic view of O-GlcNAcylation-mediated formation of the non-aging dauer state in C. elegans
Summary
Strains and General Handling—Methods used for the maintenance and handling of C. elegans were as described previously [16]. For PUGNAc treatment in either standard or suboptimal daumone assays, a stock solution of PUGNAc (50 mM PUGNAc in H2O:EtOH (1:1)) was added to NGM plates at the same time that daumone was added. These plates, containing a final PUGNAc concentration of 0.5 mM, were used for subsequent daumone assays. Measuring Dauer Formation in PUGNAc-treated Dauer Formation Constitutive (Daf-c) Mutants—In these assays, NGM plates containing a final PUGNAc concentration of 0.5 mM were used. Tagging of O-GlcNAcylated Proteins with TAMRA—Dauer worms were prepared by collecting eggs from gravid adults as described [20] and plating them on standard daumone plates.
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