Identification of structural and functional O-GlcNAc-bearing proteins in Xenopus laevis oocyte. Export

@article{citeulike:2997405, abstract = {O-GlcNAcylation (or O-GlcNAc) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have previously described the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G2/M transition and we have demonstrated that the inhibition of the O-GlcNAc transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we have identified proteins that are O-GlcNAc modified during the G2/M transition. Due to 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 GalNAz (i.e. 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 analysed by nanoLC-nanoESI-MS/MS allowing for the identification of an average of twenty Xenopus laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, PCNA, TER ATPase, aldolase, lactate dehydrogenase and ribosomal proteins. This labelling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore, immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomal-bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification (PTM) 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 PTM on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle.}, address = {B\^{a}timent C9, UMR-CNRS 8576, Villeneuve d'Ascq 59655.}, author = {Dennaut, Vanessa and Slomianny, Marie-Christine C. and Page, Adeline and Vercoutter-Edouart, Anne-Sophie S. and Jessus, Catherine and Michalski, Jean-Claude C. and Vilain, Jean-Pierre P. and Bodart, Jean-Fran\c{c}ois F. and Lefebvre, Tony}, citeulike-article-id = {2997405}, citeulike-linkout-0 = {http://dx.doi.org/10.1074/mcp.M700494-MCP200}, citeulike-linkout-1 = {http://view.ncbi.nlm.nih.gov/pubmed/18617508}, citeulike-linkout-2 = {http://www.hubmed.org/display.cgi?uids=18617508}, day = {9}, doi = {10.1074/mcp.M700494-MCP200}, issn = {1535-9484}, journal = {Molecular \& cellular proteomics : MCP}, keywords = {cell-cycle, glyco}, month = {July}, posted-at = {2008-07-13 17:32:34}, priority = {2}, title = {Identification of structural and functional O-GlcNAc-bearing proteins in Xenopus laevis oocyte.}, url = {http://dx.doi.org/10.1074/mcp.M700494-MCP200}, year = {2008} }

TY - JOUR ID - citeulike:2997405 L3 - citeulike-article-id:2997405 N2 - O-GlcNAcylation (or O-GlcNAc) is an abundant and reversible glycosylation type found within the cytosolic and the nuclear compartments. We have previously described the sudden O-GlcNAcylation increase occurring during the Xenopus laevis oocyte G2/M transition and we have demonstrated that the inhibition of the O-GlcNAc transferase (OGT) blocked this process, showing that the O-GlcNAcylation dynamism interferes with the cell cycle progression. In this work, we have identified proteins that are O-GlcNAc modified during the G2/M transition. Due to 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 GalNAz (i.e. 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 analysed by nanoLC-nanoESI-MS/MS allowing for the identification of an average of twenty Xenopus laevis oocyte O-GlcNAcylated proteins. In addition to actin and beta-tubulin, we identified metabolic/functional proteins such as PP2A, PCNA, TER ATPase, aldolase, lactate dehydrogenase and ribosomal proteins. This labelling allowed for the mapping of a major O-GlcNAcylation site within the 318-324 region of beta-actin. Furthermore, immunofluorescence microscopy enabled the direct visualization of O-GlcNAcylation and OGT on the meiotic spindle as well as the observation that chromosomal-bound proteins were enriched in O-GlcNAc and OGT. The biological relevance of this post-translational modification (PTM) 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 PTM on each identified protein and will provide a better understanding of O-GlcNAcylation in the control of the cell cycle. JF - Molecular & cellular proteomics : MCP SN - 1535-9484 AD - Bâtiment C9, UMR-CNRS 8576, Villeneuve d'Ascq 59655. TI - Identification of structural and functional O-GlcNAc-bearing proteins in Xenopus laevis oocyte. KW - cell-cycle KW - glyco AU - Dennaut, Vanessa AU - Slomianny, Marie-Christine AU - Page, Adeline AU - Vercoutter-Edouart, Anne-Sophie AU - Jessus, Catherine AU - Michalski, Jean-Claude AU - Vilain, Jean-Pierre AU - Bodart, Jean-François AU - Lefebvre, Tony PY - 2008/07/09/ UR - http://dx.doi.org/10.1074/mcp.M700494-MCP200 ER -