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Molecular & Cellular Proteomics 7:2229-2245, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Identification of Structural and Functional O-Linked N-Acetylglucosamine-bearing Proteins in Xenopus laevis Oocyte^*,S

Vanessa Dehennaut^,,¶, Marie-Christine Slomianny^,||, Adeline Page^||,**, Anne-Sophie Vercoutter-Edouart, Catherine Jessus, Jean-Claude Michalski, Jean-Pierre Vilain, Jean-François Bodart and Tony Lefebvre^,

From the UMR-CNRS 8576, Unité de Glycobiologie Structurale et Fonctionnelle, EA 4020, Laboratoire de Régulation des Signaux de Division, and ^** Centre Commun de Mesures de Spectrométrie de Masse, Université des Sciences et Technologies de Lille, IFR 147, 59655 Villeneuve d'Ascq, France and UMR-CNRS 7622, Laboratoire de Biologie du Développement, Equipe Biologie de l'Ovocyte, Université Pierre et Marie Curie, 75252 Paris, France

O-Linked N-acetylglucosaminylation (O-GlcNAcylation) (or O-linked N-acetylglucosamine (O-GlcNAc)) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have described previously the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G₂/M transition, and we have demonstrated that the inhibition of O-GlcNAc-transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we identified proteins that are O-GlcNAc-modified during the G₂/M transition. Because of a low expression of O-GlcNAcylation in Xenopus oocyte, classical enrichment of O-GlcNAc-bearing proteins using O-GlcNAc-directed antibodies or wheat germ agglutinin lectin affinity were hard to apply, albeit these techniques allowed the identification of actin and erk2. Therefore, another strategy based on an in vitro enzymatic labeling of O-GlcNAc residues with azido-GalNAc followed by a chemical addition of a biotin alkyne probe and by enrichment of the tagged proteins on avidin beads was used. Bound proteins were analyzed by nano-LC-nano-ESI-MS/MS allowing for the identification of an average of 20 X. laevis oocyte O-GlcNAcylated proteins. In addition to actin and β-tubulin, we identified metabolic/functional proteins such as PP2A, proliferating cell nuclear antigen, transitional endoplasmic reticulum ATPase, aldolase, lactate dehydrogenase, and ribosomal proteins. This labeling allowed for the mapping of a major O-GlcNAcylation site within the 318–324 region of β-actin. Furthermore immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomally bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification both on microtubules and on chromosomes remains to be determined. However, the mapping of the O-GlcNAcylation sites will help to underline the function of this post-translational modification on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.

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