modulation of o-glcnac glycosylation during xenopus oocyte maturation

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  • Journal of Cellular Biochemistry 93:9991010 (2004)

    Modulation of O-GlcNAc Glycosylation DuringXenopus Oocyte Maturation

    Tony Lefebvre,1* Frederic Baert,2 Jean-Francois Bodart,2 Stephane Flament,3 Jean-Claude Michalski,1

    and Jean-Pierre Vilain2

    1Unite Mixte de Recherches 8576 du CNRS, Glycobiologie Structurale et Fonctionnelle, IFR 118, USTL,Batiment C9, 59655 Villeneuve dAscq, France2UPRES-EA, 1033, Laboratoire de Biologie du Developpement, IFR 118, USTL, Batiment SN3,59655 Villeneuve dAscq, France3UPRES-EA, 3442, Genetique, Signalisation, Differenciation, Faculte des sciences, Universite de Nancy I,BP 239, 54506 Vandoeuvre-le`s-Nancy cedex, France

    Abstract O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which isbelieved antagonises phosphorylation. We have studied the O-GlcNAc level duringXenopus oocyte meiotic resumption,taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation ofimmature stage VI oocytes using progesterone was followed by a 4.51 0.32 fold increase in the GlcNAc content,concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase ofO-GlcNAc for the 97 kDa protein, which we identified as b-catenin was partly related to its accumulation duringmaturation, as was demonstrated by the use of the protein synthesis inhibitorcycloheximide. Microinjection of freeGlcNAc, which inhibits O-glycosylated proteinslectins interactions, delayed the progesterone-induced maturationwithout affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate proteinproteininteractions required for the cell cycle kinetic. J. Cell. Biochem. 93: 9991010, 2004. 2004 Wiley-Liss, Inc.

    Key words: oocyte; Xenopus; maturation; O-GlcNAc; beta-catenin

    O-linked N-acetylglucosamine (O-GlcNAc)glycosylation is the major glycosylation typefound within the nucleus and the cytosolic com-partment of eukaryota [for review, see Wells

    et al., 2001]. O-GlcNAc consists in the additionof a single residue ofN-acetylglucosamine to thehydroxyl groups of serine and threonine. Thistype of glycosylation shares similar featureswith protein phosphorylation [Haltiwangeret al., 1997], and the occurrence of an antagon-ism between phosphorylation and O-GlcNAcglycosylation was demonstrated. The existenceof a direct competition between O-GlcNAc andphosphate to occupy the same sites on proteinswas reported [Haltiwanger et al., 1998; Griffithand Schmitz, 1999; Lefebvre et al., 1999].The emergence of an O-GlcNAc/phosphoryla-tion balance leads to the question of its biologi-cal significance: if the role of phosphorylation iswell documented and generally contributesto the regulation of the protein activity, theparticular role of O-GlcNAc glycosylation is stillnot well understood. For example, whereasthe reciprocity between O-GlcNAc and O-Phos-phate on the carboxyl terminal domain (CTD) ofRNA polymerase II was studied in detail, thephosphorylation of the CTD is associated with

    2004 Wiley-Liss, Inc.

    Abbreviations used: O-GlcNAc, O-linked N-acetylglucosa-mine; GVBD, germinal vesicle breakdown; PNGase F,peptide N-glycosidase F; WGA, wheat germ agglutinin;PNA, peanut agglutinin; TBS, tris buffered saline; BSA,bovine serum albumin; AMP, adenosine 50-monophosphate;MPF, maturation promoting factor; MAPK, mitogen acti-vated protein kinase; CHX, cycloheximide.

    Tony Lefebvre and Frederic Baert are co-authors.

    Grant sponsor: Genopole of Lille; Grant sponsor: CentreNational de la Recherche Scientifique; Grant sponsor:Universite des Sciences et Technologies de Lille; Grantsponsor: Comite du Nord de la Ligue contre le Cancer.

    *Correspondence to: Tony Lefebvre, UGSF/CNRS, UMR8576, cite scientifique, 59655 Villeneuve dAscq, France.E-mail: tony.lefebvre@univ-lille1.fr

    Received 28 August 2002; Accepted 11 June 2004

    DOI 10.1002/jcb.20242

  • promoter clearance, stabilization of elongationcomplexes and with the involvement of themRNA processing machinerythe function ofO-GlcNAc glycosylation of the CTD remainsspeculative [Comer and Hart, 2001].Changes in the level of glycosylation in

    several nuclear and cytoplasmic proteins wereobserved during the mitogenic activation ofboth T-lymphocytes and T-cell hybridoma. InT-lymphocytes, O-GlcNAc levels change rapidlyafter stimulation to enter the cell cycle [KearseandHart, 1991]. In thehuman colon cell lineHT29,mitotic arrestwith either okadaic acid or themicrotubule destabilizing agent nocodazole,causes an increase in the O-GlcNAc levels ofkeratins [Haltiwanger and Philipsberg, 1997].These observations suggest that O-GlcNAcglycosylation might be involved in the cell cycleregulation, while no other data corroboratedthis assumption. Many of the nuclear porecomplexes, which are modified by single seriesof O-GlcNAc, are phosphorylated in a cell cyclespecific manner, though the levels of O-GlcNAcremain constant [Miller et al., 1999]. Otherexperimentswere led to investigate into the roleof O-GlcNAc during the cell division processthrough the microinjection of galactosyltrans-ferase (GalT) into Xenopus oocytes arrested inprophase I.However, injectedGalTwas toxic foroocytes stimulated to resumemeiosis [Fang andMiller, 2001].Xenopus oocyte offers opportunities to study

    the M-phase regulation that is highly control-led by specific kinases and phosphatases. Full-grown Xenopus stage VI oocytes are arrestedat the prophase of the first division of meiosis ina G2-like state. In response to progesterone,oocytes undergo germinal vesicle breakdown(GVBD), chromosomes condensation, and spin-dle formation. The end of the first division ofmeiosis is attested by the extrusion of the firstpolar body. The second division resumes, but itis arrested at metaphase [Hausen and Riebe-sell, 1991]. This process, also calledmaturation,is characterized by the simultaneous activationof two pathways. On one hand, the M-phasePromoting Factor (MPF; p34Cdc2/Cyclin B)that regulates both the entry and exit of theM-phase is activated through the dephosphor-ylation of its catalytic subunit by a dual-specificity phosphatase Cdc25 [Masui, 2001].On the other hand, the activation of the Mos/MEK/Xp42Mpk1 Mitogen Activated ProteinKinases (MAPK) cascade depends upon Mos

    accumulation; and the effects of this pathwayare mainly mediated through p90Rsk [Gotohand Nishida, 1995; Gross et al., 2000].Since phosphorylation is well known as

    means to regulate the cell cycle progressionand since O-GlcNAc could antagonize phos-phorylation, we assumed that O-GlcNAc varia-tions could also occur during the meiosisresumption and that O-glycosylated proteinslectins interactions might influence the G2/Mtransition in Xenopus laevis oocytes. Thus, westudied the O-GlcNAc level during Xenopusoocytematuration, takingadvantage of thehighand natural synchrony of this model [Ferrell,1999]. Moreover, we observed the O-GlcNAcglycosylation of several proteins during matu-ration. Among them, we identified b-cateninwhich is phosphorylated by the Glycogensynthase kinase-3 (GSK3) and lead to its de-gradation. In return, the stabilization of b-catenin through the inactivation of GSK3 isessential for the dorsal determination in Xeno-pus embryos [Heasman et al., 1994]. Moreover,we have shown that the microinjection of freeGlcNAc delayed progesterone-induced matura-tion, thereby suggesting a role for O-GlcNAcmediated interaction in the cell cycle.

    MATERIALS AND METHODS

    Handling of Oocytes

    Stage VI Xenopus oocytes were isolated andprepared as previously described [Flamentet al., 1996]. Briefly, prophase I-arrestedoocytes were obtained from animals that werenot primed with any gonadotropins. Defollicu-lated oocytes were kept in ND96 medium(96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mMMgCl2, and 5 mM HEPES, pH 7.5). Progest-erone was added at a final concentration of10 mM. Percentages of maturation weremarkedby the appearance of a white spot at the animalpole of the oocyte and GVBD was confirmedvia microscopically examination after the dis-section of heat-fixed oocytes. For biochemicalanalysis, oocytes were homogenized in b gly-cerophosphate, 60 mM; paranitrophenylpho-sphate, 15 mM; MOPS, 25 mM; EGTA, 15 mM;MgCl2, 15 mM; DTT, 2 mM; sodium orthov-anadate, 1 mM; NaF, 1 mM and proteasesinhibitors; pH 7.2 [Flament et al., 1996] andcentrifuged at 10,000 g for 10 min to eliminateyolk and membranous pellet. All the experi-ments were performed at least in duplicate.

    1000 Lefebvre et al.

  • Enucleation

    Immature oocytes were pricked at the animalpole and placed in ND96 diluted four times.Under such conditions, the germinal vesiclethat is located in the animal hemisphere wentout of the cell. Enucleated oocytes were thenreplaced in normal ND96 for recovery, at least1 h prior to treatment.To check the efficiency of oocytes enucleation,

    we used an antibody raised against p62 (sc-1916, Santa Cruz Biotechnology, Santa Cruz,CA), a nuclear pore specific protein. Afternitrocellulose saturation with milk, the anti-body was used at a dilution of 1:1,000 overnightat 48C, and the horseradish peroxidase-coupledanti-goat secondary antibody was used at adilution of 1:2,000 for 1 h (Dako, Glostrop,Denmark).

    Cytological Analysis

    Oocytes were fixed overnight in Smithsfixative, dehydrated and embedded in paraffin.Sections (7 mm thick) were stained with nuclearred to detect nuclear structures and chromo-somes and with picroindigo-carmine, whichreveals cytoplasmic and microtubular struc-tures. This method is precise enough to detectspindles and condensed chromosomes, even ifthey are not located near the plasmamembrane[Flament et al., 1996].

    Western Blotting

    Analysis of the O-GlcNAc content. Theproteins were run on a 10% SDSPAGE andwere electrophoretically transferred onto nit

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