Glycobiologyvol.19no.8pp.816–828,2009 doi:10.1093/glycob/cwp066 AdvanceAccesspublicationonMay9,2009 REVIEW ∗ Protein O-mannosylation: Conserved from bacteria to humans MarkLommel2 andSabineStrahl1,2 O-glycosylation, glycans get attached via an O-glycosidic linkage to the hydroxy group of hydroxy amino acids. All D 2DepartmentVCellChemistry,HeidelbergInstituteforPlantSciences, o hydroxy amino acids (serine (Ser), threonine (Thr), tyrosine, w UniversityofHeidelberg,ImNeuenheimerFeld360,69120Heidelberg, n Germany hydroxyproline, and hydroxylysine) have been implicated in lo a O-linked glycosylation, although the most commonly modi- d R20e0ce9ivedonMarch18,2009;revisedonMay6,2009;acceptedonMay6, fimeidnaolnseusgaarrereSseirduaensdcTahnrbreesOid-ugelsy.cAosivdairciaeltlyyoafttraecdhuecdintog tSeer-r ed from PfurnogteiinanOd-manainmnaolssy.laDtiioffneriesntanfroemssenmtioasltmotohdeirfictaytpioens oinf Nan-dacTehtyr,lgsluucchosaasmNi-naece(GtyllcgNalAacct)o,sxaymloinsee,,gfuaclaocsteos(eFu(Gc)a,lg),luacroabsei-, https O-glycosylation, protein O-mannosylation is initiated in nose,andmannose(Man)(reviewedinSpiro2002). ://a c the endoplasmic reticulum by the transfer of mannose In this review, we particularly address protein O- ad from dolichol monophosphate-activated mannose to ser- mannosylation, which for a long time was believed to be re- em ine and threonine residues of secretory proteins. In recent stricted to fungi. However, it is now clear that this type of ic.o years, it has emerged that even bacteria are capable of O-glycosylationisconservedthroughoutthefungalandanimal up O-mannosylation and that the biosynthetic pathway of O- kingdomsandthatbacteriaarealsocapableofO-mannosylation. .co mannosyl glycans is conserved between pro- and eukary- Mainly focusing on protein O-mannosyltransferases, we sum- m/g otes. In this review, we summarize the observations that marize the main features of the biosynthetic pathway of O- ly c have opened up the field and highlight characteristics of mannosyl glycans and address their biological significance in ob O-mannosylationinthedifferentdomains/kingdomsoflife. pro-andeukaryotes. /artic le Keywords:congenitalmusculardystrophy/glycosylation/ -ab mannosyltransferase/O-mannosylation ProteinO-mannosylationineukaryotes stra c OccurrenceandstructuresofO-mannosylglycans t/19 /8 Introduction O-Mannosyl glycans linked to Ser and Thr residues were first /8 1 identifiedinthelate1960sbySentandreuandNorthcote(1968) 6 Glycosylation is a highly abundant posttranslational protein /1 in bakers’ yeast. Today we know that in yeasts and fungi the 9 modificationineukaryathatalsooccursinbacteriaandarchea 8 (Abu-Qarnetal.2008;Lehleetal.2006;Yurist-Doutschetal. majorityofsecretedandcellwallproteinsaresubstantiallyO- 819 mannosylated (De Groot et al. 2005; Strahl-Bolsinger et al. 8 2008).Amongthevariousmodificationsaproteincanundergo b 1999). In all yeasts and fungi studied so far, the reducing y inalivingcell,thecovalentattachmentofcarbohydratesisthe terminal mannose residue of O-mannosyl glycans is α-linked gu mostcommonandthemostdiverse.Thirteenmonosaccharides e and eight different amino acids have been reported to be in- tmoaSnnerotorirosTehMr aanndα1m-2aMyabneα1e-x2teMnadneαd1t-oSefro/Trmhra(nGeαm1,m2-illilnaknedd st on volvedinatleast37differentcarbohydrate–proteinlinkages.A 0 Trimble1999).Thiscorestructureisfurtherprocessedaccord- 3 furthermagnitudeofcomplexityisachievedbytheformationof A ingtotheyeast/fungispecies.InSaccharomycescerevisiae,fur- p lsiintieoanr,aannddbarnaonmcheeridcocloignofisgaucrcahtiaornidse(sreovfivewareydinignlSepnigroth2,0co0m2)p.oIn- thermannoseresiduesareaddedinα1,3-linkagetoformshort ril 20 linear oligosaccharides that are up to five mannosyl residues 1 thisway,anenormousvarietyofglycanstructuresisgenerated. 9 longandcanbemodifiedbymannosylphosphate(Figure1A). Beingfarmorethanmeredecoration,carbohydratemoietiesnot Thecharacterizationofheterologouslyexpressedleech-derived onlyalterthephysico–chemicalpropertiesofaproteinbutare tryptase inhibitor revealed that S. cerevisiae is also capable alsoinvolvedinvariousfunctionalaspectsofaproteinsuchas of synthesizing O-linked Manα1-3-Manα1-2Man mannotriose cellular localization, turnover, protein quality control, and lig- and mannooligosaccharides of up to 13 mannose moieties. and interaction (for reviews, see Lehle et al. 2006; Marth and These high degrees of polymerization might be achieved by Grewal2008;TrombettaandParodi2003). theincorporationofmannosylphosphatethatcanoccuratany Based on the nature of the glycosidic bond, distinct position of the mannosyl backbone (Bergwerff et al. 1998). types of glycosylation reactions are defined. In the case of O-Glycans of the human fungal pathogen Candida albicans consistofα1,2-linkedmannosepolymersofuptofiveresidues (Herrero et al. 2002). In the methylotrophic yeast Pichia pas- 1Towhomcorrespondenceshouldbeaddressed:Tel:+49(0)6221546286; Fax:+49(0)6221545859;e-mail:[email protected] toris,themostabundantO-mannosylglycanstructuresareα1,2- ∗DedicatedtoProfessorWidmarTannerontheoccasionofhis70thbirthday. Man polymers of two, three, or rarely four mannose residues (cid:3)c TheAuthor2009.PublishedbyOxfordUniversityPress.Allrightsreserved.Forpermissions,pleasee-mail:[email protected] 816 ProteinO-mannosylation:Conservedfrombacteriatohumans 1997;Sasakietal.1998;Smalheiseretal.1998),andrecently on neuron-specific receptor protein–tyrosine phosphatase β (RPTPβ;Abbottetal.2008).DifferentfromfungalO-mannosyl glycansthatmainlyconsistofmannosylresidues,thevastma- jority of mammalian O-mannosyl glycans represent variations ofthetetrasaccharideNeuAcα2-3Galβ1-4GlcNAcβ1-2Manα1- Ser/Thr with different lengths (e.g., asialo) and fucose (α1,3- linked to GlcNAc) contents (Chiba et al. 1997; Sasaki et al. 1998; Smalheiser et al. 1998). Further, branched structures with2,6-di-substitutedmannose(GlcNAc-linkedβ1,2andβ1,6) havebeenreported(Chaietal.1999;Figure1B).Interestingly, IIH6monoclonalantibodiesthatrecognizeanO-mannosidically linked glycoepitope on α-DG also react with α-DG from ze- D o brafish (Thornhill et al. 2008), suggesting that O-mannosyl w n glycan structures are conserved in vertebrates. In addition to lo a d theconservedtetrasaccharide,O-mannosylglycanscontaining e d 3thGeahlβu1m-4aGnlncaNtuacraβl1k-2ilMlear-n1-S(/HTN(KFi-g1u)reep1iBto)pheaHveSbOe4e-n3GdelcteAcβte1d- from i(nYuseignneifitacla.n1t9a9m7)o,uanntdsraemceonntglytootnalRrPaTbPbiβtfbrroaminngeluyrcoobplaepsttoidmeas https cells(Abbottetal.2008). ://a c a d e m ic .o u BiosynthesisofO-mannosylglycans p.c o m InitiationofO-mannosylationintheendoplasmicreticulum /g ThebiosynthesisofO-mannosylglycanshasbeenuncoveredin lyc o S.cerevisiaebythepioneeringworkofTannerandco-workers. b /a Theelucidationofthebiosyntheticpathwaystartedwiththedis- rtic Fcoigre.1st.ruSccthuermeMatiacnr0e−p2r-eMseannt-aStieorn/TohfrOis-mcoamnnmoosynlignlaylclaynesa.s(tAs.)IAnnS.αc1e,r2e-vliinsikaeed, ckoavryeoryticofcedlolsli(cBhyehlrpehnoss1p9h7a0te;-Taacntinvearte1d9m69o;nToasnancecrhaarniddeBseihnreenus- le-abs mannosylphophatecanadditionallybeincorporatedatanypositionofthe 1971).BabczinskiandTanner(1973)thendemonstratedthatin tra oGlaiglβo1m-4anGnlocNseAbcaβc1k-b2oMnea.n(-BSe)rI/nThmraimscmoamlsmthoencinoraellstOru-mctuanrenosylglycans.On vitroamajorityofthemannosylresiduesderivedfromdolichyl ct/1 α-DGthiscorestructurecanbesialylated(1)orfucosylated(2).Frombrain phosphate-bound mannose (Dol-P-Man) are directly linked to 9/8 glycopeptides,branchedstructures(3)andO-mannosidicallylinkedHNK-1 protein (Sharma et al. 1974). Orlean (1990) finally confirmed /8 1 (4)epitopeshavebeendescribed. that Dol-P-Man serves as the mannosyl donor for the initial 6 /1 transferreactionintheO-mannosylationpathwayinvivo.Dol- 9 8 P-ManissynthesizedfromGDP-ManandDol-Ponthecytosolic 81 (Duman et al. 1998). Some of the α1,2-Man and α1,2-Man faceof theendoplasmic reticulum (ER)membrane (Figure2). 98 structures can be capped by a Manβ1-2Manβ31-2-disaccharide4 Inyeast,thisreactioniscatalyzedbytheGDP-Man:Dol-Pman- by g (Trimbleetal.2004;Figure1A).InthefissionyeastSchizosac- nosyltransferaseDpm1p(Haselbeck1989;Orleanetal.1988). u e charomyces pombe, linear α1,2-linked mannobiose and man- Subsequently,Dol-P-Manflip-flopsintotheERlumenthrougha st o notrioseareformed.Inaddition,uptotwogalactosylresidues putativeflippasemachinery.Thereitservesasthesolemannose n maybeaddedtotheα1,2-linkedmannosebackbonetogenerate donor for the ER resident glycosyltransferases. For structure, 03 A bturraen(cGheedmOm-illilnakneddTorliigmobslaec1ch9a9r9i;dFesigoufrteh1eAG)a.lS0−h2oMrtalinn1e−a3rsαtr1u,2c-- fthuanscetioinn,yaenasdtsreagnudlamtioamnmofalDs,owl-eP-rMefaenr taonda rDecoel-nPt-rMevainewsybny- pril 2 0 linked mannose polymers (Man1−3) have been isolated from MaedaandKinoshita(2008). 19 glucoamylase of the filamentous fungus Aspergillus awamori Tanner and co-workers further demonstrated that biosyn- var. kawachi (Oka et al. 2005). Besides, branched O-linked thesis of O-mannosyl glycans in S. cerevisiae is initiated at oligomannoses containing galactose, glucose, and galactopen- the ER (Haselbeck and Tanner 1983). Dolichyl phosphate-D- tosehavebeendescribedfromdifferentAspergillusspeciesthat mannose:protein O-mannosyltransferases catalyze the transfer havebeensummarizedinarecentreviewbyGoto(2007). ofamannosylresiduefromDol-P-ß-D-Man(LehleandTanner Althoughhighlyabundantinfungi,proteinO-mannosylation 1978) to Ser and Thr residues of secretory proteins (Gentzsch islessfrequentinmetazoa.O-Linkedmannosehasbeenidenti- et al. 1995; Gentzsch and Tanner 1996; Strahl-Bolsinger et al. fiedamongoligosaccharidesonskincollagenoftheclamworm 1993; Strahl-Bolsinger and Tanner 1991; Figure 2). While in- (Spiro and Bhoyroo 1980). In mammals, O-mannosyl glycans vertingtheanomericconfigurationofthemannosemoietyanα- were isolated from chondroitin sulfate proteoglycans and to- D-mannosidiclinkageisformed(BauseandLehle1979).Early talglycopeptidesfrombraintissue(Finneetal.1979;Krusius experimentssuggestedthatO-mannosylationoccursexclusively et al. 1986, 1987; Yuen et al. 1997), on α-dystroglycan (α- duringthetranslocationofsecretoryproteinsintotheER(Elorza DG)originatingfromnerveandmusculartissues(Chibaetal. etal.1977).Yet,morerecentworkshowedthatthetransferof 817 MLommelandSStrahl D o w n lo a d e d fro m h ttp s ://a c a d e m ic .o u p .c o m /g ly c o b bFeigtw.2e.enBfiuonsygni,thanesimisaolfs,Oa-nmdahnunmosaynls.gDlyocla-nPs-Minaenukisarsyyonttehse.s(iAze)dSocnhethmeactiyctoresporliecsefanctaetioofnthoeftEhReOm-emmabnrnaonseyalantdioflnipp-aflthowpsayin.tTohteheinEitRiallusmtepens.inMtahnenEoRseairsefcuortnhseerrved /artic transferredtoproteinsenteringthesecretorypathwaybythemembersofthePMT-family.DiversificationoccursintheGolgiapparatuswherefurtherchain le-a elongationtakesplaceasdetailedinthetext.(B)ERlocalizationofPMTproteins.Upperpanel:co-localizationofPmt4pandtheoligosaccharyltransferasesubunit b s Ost1pintheERofbakers’yeast(realizedbyJ.Hutzler).Middlepanel:InfissionyeastOma2p(PMT2type)localizestotheperinuclearandperipheralER.The tra nucleusisvisualizedwithDAPI(realizedbyF.Hutzler).Lowerpanel:MousePOMT2andtheERmarkerproteinERp72co-localizeinculturedfibroblastcells. c t/1 9 /8 O-linkedmannosecanalsooccurafterproteinshaveenteredthe Golgiglycosyltransferasesaretype-IImembraneproteinswith /8 1 ER(Hartyetal.2001). large C-terminal catalytic domains facing the Golgi lumen. In 6 /1 Five years ago, the transfer reaction catalyzed by Dol-P- theGolgi,GDP-Manservesasthemannosyldonor. 9 8 Man:protein O-mannosyltransferases has also been demon- Sofar,theelongationofO-mannosylglycansinmammalsis 81 9 strated in mammals and insects (Ichimiya et al. 2004; Manya onlypartiallyunderstood.ThetransferofGlcNActomannose 8 b etal.2004)affirmingthattheinitialstepsofO-mannosylation in the 2-OH position is catalyzed by the protein O-mannose y g areconservedbetweenthefungalandtheanimalkingdom(Fig- N-acetylglucosaminyltransferase 1 (POMGnT1; Takahashi u e ure2).Hence,metazoanO-mannosylationisdistinctfrommost etal.2001;Zhangetal.2002),whileβ1,6-N-acetylglucosaminyl st o other O-glycosylation reactions that take place exclusively in transferase (GnT-IX) links GlcNAc to mannose in the 6-OH n 0 theGolgiapparatus(Lehleetal.2006). position in vitro (Inamori et al. 2004; Figure 2A). The 3 addition of GlcNAc in β1,2-Man linkage by PomGnT1 is Ap required before GnT-IX can add GlcNAc, strongly suggest- ril 2 ChainelongationintheGolgiapparatus ing that GnT-IX is responsible for the formation of 2,6- 01 9 Further extension of the O-linked mannose residue takes branched structures in brain O-mannosyl glycans (Inamori place in the Golgi apparatus. In S. cerevisiae, three α1,2- et al. 2004). Further enzymes directly involved in the elon- mannosyltrasferases of the KTR family (ScKre2p, ScKtr1p, gationofO-mannosesaccharidesremaintobeidentifiedamong and ScKtr3p) catalyze the stepwise addition of the first and familiesofβ1,4-galactosyltransferases,α2,3-sialyltransferases, secondα1,2-linkedmannoseinthemedialGolgicompartment α1,3-fucosyltransferases,andHNK-1-specificglucuronyltrans- (reviewedinLussieretal.(1999);Strahl-Bolsingeretal.(1999); ferasesethathavebeenfoundinmammals.Threegenes,fukutin, Figure 2A). Another member of the KTR family, ScMnn6p, fukutin-related protein, and Large, seem to be involved in is a mannosylphosphate transferase implicated in the man- the formation of O-mannosyl glycans on α-DG (Barresi and nosylphosphorylation of O-linked glycans (Jigami and Odani Campbell 2006). The corresponding proteins show substan- 1999). Further α1,3-linked mannose residues are added by tial similarities to known glycosyltransferases (Aravind and α1,3-mannosyltrasferases including ScMnn1p, ScMnt2p, and Koonin1999;Esapaetal.2002;Grewaletal.2001;Kobayashi ScMnt3p (Lussier et al. 1999; Romero et al. 1999) which re- et al. 1998); however, their specific functions remain to be side in the medial and trans-Golgi (Graham et al. 1994). All determined. 818 ProteinO-mannosylation:Conservedfrombacteriatohumans (81% similarity). In view of this, high degree of conservation tandemgeneduplicationofPMT1issupportedbythefactthat PMT1(YDL095w)andPMT5(YDL093w)arelocateddirectly next to each other on chromosome IV. Further, the report of WolfeandShields(1997)suggeststhatPMT2(YAL023c;chro- mosomeI)andPMT3(YOR321w;chromosomeXV)constitute agenepairthatderivesfromanancientduplicationoftheentire yeast genome (Wolfe and Shields 1997). Different from Sac- charomycetes, in the Schizosaccharomycete S. pombe, in the basidiomycetous yeast C. neoformans, and in all filamentous fungianalyzedsofar,onlyasinglememberofeachPMTsub- family is present (Goto 2007; Olson et al. 2007; Willer et al. 2005).Incontrasttofungi,inanimalsasinglememberofboth D o thePMT2andthePMT4subfamilyispresent,whereasPMT1 w n homologshavenotbeenfound(Figure3). lo a d e d Fig.3. PhylogenyofthethreePMTsubfamilies.Proteinsequenceswere YeastproteinO-mannosyltransferases from agnraaplyhziceadlluysiunsgintghethCelPushtyalloWde(nTdhroomnpsosoftnweatrea.l.M19em94b)earlsgoofritthhemPaMndT1d,isPpMlaTye2d, As mentioned earlier, PMTs have been extensively character- http ized in bakers’ yeast where at least six PMT family mem- s afrnodmPbMacTt4ersiuabafraemsihloywanreindebpliacctked.Ainf,gAre.efunm,riegda,tuans;dCbglu,eC,.rgeslupteacmtiivceulmy.;PDmmts, bers (Pmt1p–Pmt6p) are present (Gentzsch and Tanner 1996; ://ac D.melanogaster;Dr,Daniorerio;Gg,Gallusgallus;Hs,Homosapiens;Mm, Strahl-Bolsingeretal.1993;Strahl-BolsingerandTanner1991; ad Musmusculus;Mt,M.tuberculosis;Sc,S.cerevisiae;Sp,S.pombe. Figure 3). The members of the PMT1 subfamily (Pmt1p, em ic Pmt5p) form heterodimeric complexes with members of the .o u PMT2subfamily(Pmt2p,Pmt3p;GirrbachandStrahl2003).In p ThePMTfamilyofproteinO-mannosyltransferases .c wild-type yeast, Pmt1p–Pmt2p and Pmt5p–Pmt3p complexes o m Dol-P-Man:proteinO-mannosyltransferases(PMTs)arethekey exist, yet the vast majority of mannosyl transfer activity is /g enzymes initiating O-mannosylation in the ER. The first PMT due to Pmt1p–Pmt2p complexes. Under certain physiological lyc o hasbeenidentifiedinbakers’yeast.Basedonitsenzymaticac- conditions, however, Pmt1p interacts also with Pmt3p, and b /a tivity, ScPmt1p was purified and the corresponding gene was Pmt5p with Pmt2p, suggesting a compensatory cooperation rtic cloned (Strahl-Bolsinger and Tanner 1991; Strahl-Bolsinger that guarantees the maintenance of O-mannosylation. Unlike le etal.1993).DuetothehomologytoScPMT1,thePMTfamily the PMT1/PMT2 subfamily members, Pmt4p acts as a homo- -ab s wasidentifiedinS.cerevisiae(Gentzschetal.1995;Gentzsch mericcomplex.Mutationalanalysesshowedthatthesamecon- tra and Tanner 1996; Lussier et al. 1995) and subsequently also served protein domains underlie both heteromeric and homo- ct/1 in other yeasts and filamentous fungi such as C. albicans meric interactions (Girrbach and Strahl 2003). Pmt1p–Pmt2p 9 /8 (Timpeletal.1998),Aspergillusfumigatus(ShawandMomany complexeshavealsobeendemonstratedinfissionyeast(Willer /8 1 2002),Trichodermareesei(Zakrzewskaetal.2003),S.pombe etal.2005)suggestingthatcomplexformationismandatoryfor 6 /1 (Willer et al. 2005), and Cryptococcus neoformans (Olson PMTfunction.TheremightbeanumberofreasonswhyPMTs 9 8 et al. 2007). In metazoa, PMTs were first identified in formhomo-orheteromericproteincomplexes.Oneisthatthese 81 9 Drosophila melanogaster (Martin-Blanco and Garcia-Bellido complexes ensure efficient O-mannosylation of a wide range 8 b 1996) and in humans (Jurado et al. 1999). Whole genome se- of target proteins. A common feature of O-glycosylated pro- y g quencesofvariousorganismsrevealedthatPMTsarenotlimited teins is that O-linked carbohydrate chains are clustered in dis- u e tofungi,butareconservedthroughouttheanimalkingdom.To tinctSer/Thr-richregions(Jentoft1990).Suchareasarethought st o date all PMT family members are classified as glycosyltrans- to adopt rod-like structures important for protein function. O- n 0 ferase family 39 (GT39) according to the carbohydrate-active Mannosylationcanoccurwhileproteinsaretranslocatedinthe 3 A enzymes(CAZy)database(http://www.cazy.org).Interestingly, lumen of the ER. Thus, the clustering of O-linked sugars re- p PMTgenesareabsentinthegenomesofCaenorhabditiselegans quireshighlyefficientsugartransfer,whichcouldbeprovided ril 2 0 andplants(e.g.,Arabidopsisthaliana;Oryzasativa). byPMTcomplexes. 1 9 Phylogenetic analyses showed that the PMT family is di- Among all PMTs, S. cerevisiae Pmtp1 is characterized vided in three subfamilies: the PMT1, PMT2, and PMT4 sub- best. Pmt1p is an integral ER membrane glycoprotein with family (Girrbach et al. 2000; Willer et al. 2002; Figure 3). In seventransmembranespanningdomains(Strahl-Bolsingerand S. cerevisiae and C. albicans, the PMT1 and PMT2 subfam- Scheinost 1999; Figure 4). Since hydropathy profiles are con- ilies are redundant (two or more members of each subfamily) servedbetweenPMTs,itishighlylikelythattheproposedtopol- whereasonlyasinglememberofthePMT4subfamilyispresent ogy also applies to all of them. An invariant arginine residue (GentzschandTanner1996;Prilletal.2005).Thesameistrue of the transmembrane domain 2 is essential for the formation for other yeasts of the class Saccharomycetes. Redundancy of and/orstabilityofPMTcomplexes(GirrbachandStrahl2003; PMT1 and PMT2 subfamily members might have evolved by Figure 4B). The Pmt1p amino terminus faces the cytoplasm gene duplication (Girrbach and Strahl 2003). In S. cerevisiae, whereasthecarboxylterminusfacesthelumenoftheER.Two the PMT1 subfamily members Pmt1p and Pmt5p feature an majorhydrophilicdomainsthatarelocatedbetweentransmem- overallaminoacidsequenceidentityof53%(72%similarity), brane spans 1 and 2 (loop 1) and transmembrane spans 5 and and the PMT2 subfamily members Pmt2p and Pmt3p of 65% 6 (loop 5), respectively, are oriented toward the ER lumen. 819 MLommelandSStrahl D o w n lo a d e d fro m h ttp s ://a c a d e m Fig.4. StructureofPMTproteins.(A)PMTsfromeukaryotesandbacteriasharesimilarhydropathyprofiles.HydropathyprofilesofScPmt1andMt-Pmtwere ic generatedaccordingtoKyteandDoolittle(1982).Loop5domainsareshadedingray.(B)TopologymodelsofScPmt1andMt-PmtaccordingtoStrahl-Bolsinger .ou andScheinost(1999).ConservedArgresidueswithintransmembranedomainoneandtwoandconservedAsp-Glumotifinloop1thatarecrucialforenzymatic p.c activityareindicatedinred(Girrbachetal.2000;VanderVenetal.2005). o m /g ly c o Site-directedmutagenesisofconservedaminoacidsandaffinity Scheinost1999).PSI-BLASTanalysesrevealedweakhomolo- b /a labeling with a synthetic acceptor peptide showed that amino gies between loop 4 and regions in other Dol-P-Man utilizing rtic acidsAsp77andGlu78(locatedinloop1;Figure4B)arecrucial enzymes (S. Strahl, unpublished data); however, functions of le forsubstratebindingandenzymeactivity(Girrbachetal.2000; thecytoplasmicloopsinPmt1premaintobeelucidated. -ab s A.SchottandS.Strahl,unpublisheddata).TheseDEx/Dex-like Due to the architecture described above, PMTs are catego- tra motifsplayakeyroleforcatalysisinmanyglycosyltransferases, rized as GT-C fold glycosyltransferases. The predicted GT-C ct/1 suggestingthatloop1representsthecatalyticdomain.Although fold is that of a large hydrophobic integral membrane protein 9 /8 deletionoftheloop5domaindoesnotaffectbindingofanar- locatedintheERortheplasmamembranehavingseveraltrans- /8 1 tificial acceptor peptide to Pmt1p (V. Girrbach and S. Strahl, membrane helices and an active site located on a loop region. 6 /1 unpublisheddata),itresultsinthecompletelossoftransferase Interestingly, most of the GT-C enzymes utilize phosphate- 9 8 activity(Girrbachetal.2000).Furthermore,exchangeofhighly activated donor sugar substrates (reviewed in Lairson et al. 8 1 9 conservedleucineresiduestoalanineinloop5causesadramatic (2008)). 8 b decreaseinenzymaticactivity(Girrbachetal.2000).Thisloop y 5domainisnotpresentinbacterialPMTs(seebelow;Figure4), AnimalproteinO-mannosyltransferases gu e suggesting that it fulfills a – yet unknown – function specific In recent years, PMTs have been identified within differ- st o to eukaryotes. Comparison of PMTs from different organisms ent animal phyla. Two PMT homologs have been character- n 0 defined highly conserved peptide motifs within the loop 5 re- ized from D. melanogaster. The genes, rotated abdomen (rt; 3 A gion(Girrbachetal.2000).Theseso-calledMIRmotifsarealso DmPOMT1) and twisted (tw; DmPOMT2), encode members p foundininositoltriphosphateandryanodinereceptors(Ponting of the PMT4- and PMT2-type enzymes, respectively (Jurado ril 2 2000).Moreover,mammalianSDF2(stromal-cell-derivedfac- etal.1999;Martin-BlancoandGarcia-Bellido1996;Willeretal. 01 9 tor2)andSDF2-like1(Fukudaetal.2001;Hamadaetal.1996) 2002).AsdemonstratedbyRNAinterference,DmPOMT1and are soluble proteins residing in the ER that show significant DmPOMT2 act as O-mannosyltransferases in vivo (Ichimiya homologytotheMIRmotifsofthePMT-loop5domain.SDF2- et al. 2004). Coexpression of both genes is necessary to in- like1hasbeenidentifiedasacomponentofamultiproteincom- crease POMT activity in insect cells suggesting the formation plex of chaperons and folding enzymes in the ER, suggesting of DmPOMT1–DmPOMT2 complexes (Ichimiya et al. 2004). a function in protein folding (Meunier et al. 2002). A similar In situ hybridization revealed high levels of DmPOMT1 and rolemightbeenvisagedfortheloop5domainofPMTs,which DmPOMT2 transcripts at stage 14 of larvae development that yet needs to be demonstrated. Two other major domains that correspondstotheperiodofactivemuscledifferentiation(Lyalin arelocatedbetweentransmembranespans4and5(loop4)and etal.2006). transmembranespans6and7(loop6),respectively,areoriented In mammals, POMT1 (PMT4-type) and POMT2 (PMT2- towardthecytosoland,likeloop5,arealsoessentialforPmt1p type) have been identified (Jurado et al. 1999; Willer et al. activity (Girrbach et al. 2000). Loop 4 is partially hydropho- 2002). Analogous to the yeast system, complex formation be- bic,butdoesnotspantheERmembrane(Strahl-Bolsingerand tween POMT1 and POMT2 proteins has been demonstrated. 820 ProteinO-mannosylation:Conservedfrombacteriatohumans These POMT1–POMT2 complexes are crucial for mannosyl- lumen.Theimplementationofthesefindingsinabioinformat- transferase enzyme activity in vitro (Akasaka-Manya et al. ics approach identified several novel Pmt4p substrate proteins 2006), confirming that complex formation is mandatory for (Hutzleretal.2007). PMTfunctionalsoinmetazoa.Duringembryonicdevelopment In mammals the only well-characterized PMT substrate is in mice, Pomt1 is expressed in brain, muscle, and eye (Prados α-DG,amembrane-associatedproteinofthedystrophinglyco- et al. 2007; Willer et al. 2004). In adult rodents and humans, protein complex (Barresi and Campbell 2006). Several pep- POMT1 and POMT2 are expressed in all tissues investigated, tides deduced from α-DG have been identified as useful in predominantly intestis(Manyaet al.2006; Willeretal.2004, vitro substrates of POMT1–POMT2 complexes (Manya et al. 2002). Interestingly, Pomt2 of mice is transcribed from two 2007).Basedontheseinvitropeptidesubstrates,theconsensus different transcription initiation sites leading to transcripts of sequence IXPT(P/X)TXPXXXXPT(T/X)XX was proposed as different length in certain tissues. The shorter transcript (so- a sufficient trigger for O-mannosylation in mammals (Manya matic Pomt2; sPomt2) is present in all tissues examined. The et al. 2007). Yet, the consensus sequence established in vitro longer transcript (testis Pomt2; tPomt2) is highly abundant in couldnotbeverifiedinvivo.Breloyetal.(2008)usedrecombi- D o testis and encodes a testis-specific tPOMT2 protein (Willer nantfragmentsofα-DGtomonitorPOMTactivityinvivo.This w n etal.2002).Specificantibodieswereusedtodirectlyprovethat way,preferredtargetsitesofPOMT1–POMT2complexeswere lo a tPOMT2 contains a 70-aa N-terminal extension that is highly localized within a region of α-DG that contains Ser/Thr-rich de d conserved among mammals and humans. This testis-specific clustersandsingleThrresiduesthatareflankedbyoneortwo fro tPOMT2 isoform is restricted to the acrosome (Lommel et al. basicaminoacids.Furthermore,initiationofO-mannosylation m 2008),asperm-specificorganellethatisindispensableforsuc- was shown to be dependent on the presence of two structural http cessful fertilization (Abou-Haila and Tulsiani 2000). Interest- elements (located between aa 346–367 and aa 377–417 ofhu- s ingly,severallinesofevidenceindicatethattPOMT2doesnot manα-DG)upstreamofthetargetsite(Breloyetal.2008).All ://a c act as O-mannosyltransferase in the acrosome in vivo, while together, the in vivo results obtained by Hutzler et al. (2007) ad e POMT1–sPOMT2 complexes in the ER of spermatogenic and and Breloy et al. (2008) suggest that O-mannosylation signals m ic nonspermatogeniccellsareenzymaticallyactive(Lommeletal. arenotjustlinearsequencesofaprotein’sprimarystructurebut .o u 2008).Evenifitsfunctionisnotclearatthemoment,thehigh ratherrepresentstructuraldeterminantsintheflankingregions p .c degree of conservation of the testis-specific tPOMT2 isoform ofSer/Thr-richdomains. o m between mammals and human points toward a crucial role in /g acrosomematurationorevenfertilization. lyc o b BiologicalsignificanceofproteinO-mannosylationinfungi /a The isolation and characterization of pmt(cid:2) mutants clearly rticle SubstratespecificityofproteinO-mannosyltransferases demonstratedthatproteinO-mannosylationrepresentsanessen- -ab s In contrast to other types of glycosylation, signals causing tial modification in fungi. Tanner and co-workers first showed tra Opl-emxabnontohsyinlatfiuonngaolfaSnedr ainndanTihmrarlecseidlluse.sEaaprlpyeairntvoitbroe csotumd-- tchoamtbininaSt.iocnesreovfisthiareeethPeMsTimfaumltailnyemouesmkbneorsck(pomutt1opfmpta2rptmictu4l(cid:2)ar ct/19 iesinS.cerevisiaeusingsyntheticacceptorpeptidessuggested and pmt2pmt3pmt4(cid:2)) is lethal (Gentzsch and Tanner 1996). /8/8 1 that in general Thr is more frequently modified than Ser and Similarly,inS.pombethesimultaneousknockoutofthePMT1 6 /1 that this Thr/Ser mannosylation is influenced by the neigh- andPMT4subfamilymembers,butevenmoredrastically,also 9 8 boring amino acids (Sharma et al. 1991; Strahl-Bolsinger and the single knockout of the PMT2 subfamily member is lethal 81 9 Tanner 1991). However, these in vitro observations could not (Willer et al. 2005). Similar results were obtained for C. al- 8 b be verified in vivo. After the identification of the PMT family bicans (Prill et al. 2005) and C. neoformans (Olson et al. y g in S. cerevisiae, it became obvious that Pmt1p–Pmt2p com- 2007). However, the question remains why the lack of O- u e plexesandPmt4pcomplexesmannosylatedifferenttargetpro- mannosylation results in lethality. As summarized below, pro- st o teins (Ecker et al. 2003; Gentzsch and Tanner 1997). It was teinO-mannosylationaffectsnumerousimportantcellularpro- n 0 shownthatasubsetofO-mannosylatedproteinssuchasKre9p, cesses.However,itsmajorroleseemstobethemaintenanceof 3 A Cts1p, Bar1p, Pir2p, and Aga2p is exclusively mannosylated anintactcellwall,anessentialstructureinfungi. p byPmt1p–Pmt2pcomplexes,whileKex2p,Gas1p,Axl2p,and TheroleofO-mannosylglycansisbestunderstoodinS.cere- ril 2 Fus1p are O-mannosylated by Pmt4p only (Ecker et al. 2003; visiae. Analyses of viable pmt(cid:2) single and conditional lethal 01 9 Gentzsch and Tanner 1997; Proszynski et al. 2004; Sanders pmt(cid:2) double mutants showed that O-mannosylation is crucial etal.1999).AthirdgroupofproteinsincludingMid2p,theWSC for the stability, localization and/or function of plasma mem- familymembers,andCcw5pisglycosylatedbybothcomplexes. brane proteins and thus affects various cellular processes such However,differentdomainswithintheseproteinsaremannosy- asmatingandmaintenanceofcellpolarity(Lommeletal.2004; lated by the different complexes (Ecker et al. 2003; Lommel Proszynskietal.2004;Sandersetal.1999).Forexample,hap- et al. 2004). Recently, first signals determining Pmt4p- loidpmt4(cid:2)mutantsexhibitunipolar,ratherthanaxialbudding dependentO-mannosylationinS.cerevisiaeinvivowereiden- that is characteristic for haploid cells. This change in cell po- tified. Hutzler et al. (2007) proved that Pmt4p mannosylates larity is caused by aberrant O-mannosylation of the landmark exclusively membrane-bound proteins whereas Pmt1p/Pmt2p proteinAxl2p,whichisrequiredfortheaxialbuddingpattern. complexesactonbothsolubleandmembraneproteins.Analy- IncellslackingPmt4p,Axl2pisincompletelyO-mannosylated, sisofspecificmodelproteinsdemonstratedthatthenatureofthe unstable,andmislocalized(Sandersetal.1999). membrane anchoring sequence is not relevant for recognition, AnotherfunctionofO-mannosylationinS.cerevisiae isthe aslongasitisflankedbyaSer/Thr-richdomainfacingtheER solubilizationofmisfoldedproteinsintheER.Whenmisfolded 821 MLommelandSStrahl proteins accumulate in the ER upon stress conditions, the un- O-mannosylation leads to incorrect proteolytic processing of foldedproteinresponseistriggeredtoremovetheseproteinsby these proteins, which in turn results in impaired activation of the ER-associated degradation (ERAD; Ellgaard and Helenius CWI and finally causes cell death in the absence of osmotic 2003). Upon induction of ER stress, transcription of PMT1 stabilization(Lommeletal.2004).However, allCWImutants (PMT1 and PMT5) and PMT2 (PMT2 and PMT3) subfamily areviablewhenosmoticallystabilized(Levin2005).Thus,the members is upregulated (Travers et al. 2000). Several mutant loss of CWI sensor function does not completely explain why secretoryproteinsthatareunabletofoldproperlyintheERare pmt2pmt4(cid:2)doublemutantscanberescuedbyosmoticstabiliz- O-mannosylated by Pmt1p and/or Pmt2p whereas their wild- ers while the additional deletion of PMT1 in pmt1pmt2pmt4(cid:2) type counterparts are not (Coughlan et al. 2004; Harty et al. mutants causes cell death even under osmotically stabilizing 2001; Hirayama et al. 2008; Nakatsukasa et al. 2004; Vashist conditions(GentzschandTanner1996).BesidestheCWIsen- et al. 2001). In pmt1pmt2(cid:2) mutant strains, these mutant pro- sors, many other cell wall mannoproteins are affected in the teinstendtoaggregateindicatingthatthemodificationbyPmt1p absence of O-mannosylation (summarized in Lengeler et al. and Pmt2p prevents aggregation that might in turn be cyto- 2008).Further,itwassuggestedthatO-mannosylationprecedes D o toxic(Nakatsukasaetal.2004).Therefore,itwassuggestedthat and potentially controls the N-glycosylation of glycoproteins w n O-mannosylation might function as a fail-safe mechanism for (Ecker et al. 2003). Thus, diminished O-mannosylation might lo a d ERADbysolubilizingmisfoldedproteinsandtherebyreducing change general glycosylation patterns of cell wall mannopro- e d theloadforERchaperones(Nakatsukasaetal.2004).Veryre- teinstherebyfurtheraffectingproteinmaturationandfunction. fro cently,itwasshownthatPmt4p-dependentO-mannosylationof Consideringthesevariousaspects,itseemslikelythatoncethe m heterologously expressed human β-amyloid precursor protein O-mannosylating capacity falls below a critical level, folding, http counteracts aggregation of the protein in the ER (Murakami- maturation,stability,and/orfunctionoftoomanyglycoproteins s Sekimata et al. 2009). Thus, O-mannosylation might play a isperturbed and cells areno longer able tocompensate forall ://a c generalroleinthesolubilizationofproteinsintheER. resultingdefects. ad Probably, the most important function of protein O- Phenotypes of pmt(cid:2) mutants in other yeasts and filamen- em mannosylationinfungiisitsroleinformation/maintenanceofa tousfungiarehighlysimilartothoseobservedinS.cerevisiae ic.o stablecellwall.Analysesofviablepmt(cid:2)mutantsfromdifferent pmt(cid:2) strains. In addition, it has been demonstrated that O- up .c yeastsandfilamentousfungirevealedthatmostofthesemutants mannosylation of surface mannoproteins of fungal pathogens o m are susceptible toward cell wall disturbing agents and display contributes significantly to virulence, as recently summarized /g aberrant cell wall morphologies (Gentzsch and Tanner 1996; byGoto(2007)andLengeleretal.(2008). lyc o Goto2007;Olsonetal.2007;Timpeletal.1998,2000;Willer b et al. 2005). For example, several S. cerevisiae pmt(cid:2) mutants /artic show increased sensitivity toward Calcofluor white and form BiologicalsignificanceofproteinO-mannosylationin le large cell clumps during vegetative growth. These phenotypes animalsandhumans -ab s rbeetlsyae,lmo.rb1gl9ae9n9itzh)a.otsSieoinmo,ifloaryrlemya,asitSn.mtepnuoatmannbcteesoowfmiatah1ri(cid:2)dgeidf(ePccMetslTliw1naotlhrlteh(Loaolsorsgbeuemreg)- Tzooadiasteα,-DthGe.bIetstc-ostmudpireisdesOt-wmoangnloosbyullaatreddopmroatienisn sienpamraettead- tract/19 andoma4(cid:2)(PMT4orthologue)mutantsarerounded,irregular by a Ser/Thr-rich mucin-like region that is substantially O- /8/8 mannosylated (Chiba et al. 1997; Sasaki et al. 1998). α-DG 1 shaped, and form aggregates. Ultrastructural analysis revealed 6 isanessentialcomponentofthedystrophin–glycoproteincom- /1 dramaticchangesincellwallarchitectureanddefectiveseptum 9 formation(Willeretal.2005).Again,themolecularbasisofcell plex (DGC) in skeletal muscle (Barresi and Campbell, 2006). 881 walldefectsinpmt(cid:2)mutantsisbestknowninS.cerevisiae,as Deficiencies within DGC result in inherited forms of muscu- 98 lar dystrophy (Blake et al. 2002). Remarkably, in flies, mice, b summarizedbelow. y and humans, most of the defects associated with impaired O- g In bakers’ yeast, many cell wall proteins are highly N- u glycosylated, O-mannosylated, or both. These mannoproteins mα-aDnGnoasnydlaatiroensicmanilabrelyexopblsaeirnveeddbiynddiymstirnoigshlyecdafnumncutitoanntasl.ityof est o are structurally and/or functionally important cell wall com- n 0 ponents, which determine cell wall permeability, hydrophilic 3 properties, and accessibility of cell wall glucan, and are also IntactO-mannosylationisessentialforviability Ap involved in cell–cell recognition processes (for reviews, see offliesandmice ril 2 Klis et al. 2006; Lesage and Bussey 2006). They are crucial In D. melanogaster, recessive mutations in the rt/DmPOMT1 01 9 for cell wall biogenesis and/or remodeling in order to main- gene (PMT4-type) lead to poorly viable flies with defects in tainstabilityofthisessentialstructureinyeastandfungalcells. muscledevelopmentthatresultina60–90◦twistoftheabdomen Inaddition,anumberofhighlyO-mannosylatedplasmamem- relative to the body axis (Martin-Blanco and Garcia-Bellido branereceptors/sensorsofdifferentMAPKpathwayshavebeen 1996).AsimilarphenotypeisobservedupontheRNAiknock- identified which serve as key functions in the sensing of en- down of DmPOMT1 (Ichimiya et al. 2004). Likewise, reces- vironmental changes and different stress conditions. Among sivemutantallelesandRNAiknockdownofthetw/DmPOMT2 thosearethesensorsofthecellwallintegritypathway(CWI)– gene(PMT2-type)inducethesametwistedabdomenphenotype the WSC family members and Mid2p – that control cell wall (Ichimiyaetal.2004;Lyalinetal.2006).Alreadyduringlarval integrity during vegetative growth, periods of environmental development,mutationsineitherDmPOMT1orDmPOMT2re- stress, and pheromone-induced morphogenesis (Levin 2005). sultindefectsinmuscleattachmentandchangesinmusclecon- Analyses of conditionally lethal pmt2pmt4(cid:2) mutants revealed traction(Hainesetal.2007).Remarkably,thesephenotypesare thatO-mannosylglycansareindispensableforstabilityandpro- evenmorepronounceduponcombinationofeitherDmPOMT1 cessingoftheCWIsensorsWsc1p,Wsc2p,andMid2p.Reduced or DmPOMT2 mutations with a dystroglycan mutant allele, 822 ProteinO-mannosylation:Conservedfrombacteriatohumans suggesting that PMTs are required for the proper function of de Bernabe and coworkers, who identified POMT1 mutations dystroglycaninflies(Hainesetal.2007). in6outof30WWSpatients(Beltran-ValerodeBernabeetal. In mice, during embryogenesis, the mPomt1 and mPomt2 2002).Inaddition,mutationsinPOMT1havebeenreportedthat genes are prominently expressed in the neural tube, the devel- resultinMEB(Godfreyetal.2007),aswellasmilderformsof oping eye, and the mesenchyme (Lommel et al. 2008; Willer CMDsuchascongenitalmusculardystrophywithmentalretar- et al. 2004). Interestingly, these expression patterns in mice dation (CMD/MR; van Reeuwijk et al. 2006) and LGMD-2K correlate with the sites of main tissue alterations observed in (Balcietal.2005).MutationsinPOMT2havebeendetectedin patients with mutations in hPOMT1 or hPOMT2 (see below). WWS patients, a milder MEB-like patient, and CMD/MR pa- The disruption of murine Pomt1 results in embryonic lethality tients(Mercurietal.2006;vanReeuwijketal.2005;Yanagisawa aroundembryonicday7.5–10.5whichisduetostructuraland etal.2007).Further,aPOMT2mutationhasbeendescribedin functionalperturbationsofthefirstbasalmembrane(Reichert’s apatientwithanLGMD-2Nphenotype,amildclinicalformof membrane)separatingtheembryofromthesurroundingmater- CMDwithnoneurologicalinvolvement(Biancherietal.2007). nal tissue (Willer et al. 2004). It was shown that in pomt1−/− A recent study with a large cohort of dystroglycanopathy pa- D o embryos,O-mannosylationofα-DGisabolishedindicatingthat tientsidentifiedmutationsineitherPOMT1orPOMT2inabout w n α-DG-mannosylationisessentialfortheproperformationofthe 20 % of the cases, finally establishing POMTs as causatives lo a d Reichert’smembrane. Accordingly, asimilarphenotype isob- forCMDassociatedwithclinicalheterogeneity(Godfreyetal. e d sInercvoendtirnasdtytsotrpoogmlytc1a−n/-−demfiicciee,nPtmOiMceG(nWTinlluiallm-msountaenttaml.i1c9e9a7r)e. 2007). from vshiaobwlesbduytstsrhoopwhicsecvhearnegdeesvseulochpmasensmtalaldlerfoeucntsd.eSdkfiebleetraslamnudstchlee PathologiesarisingfromPOMTmutations https infiltrationoffatcells(Liuetal.2006).Thecerebelluminbrains As emphasized above, phenotypes observed in POMT mu- ://a c ofmutantmiceissmallerandthecerebralcortexisthinner.Se- tants in animals and humans correlate with α-DG deficien- ad e vereperturbationsofthebrainarchitectureareobservedsuchas cies. Within the DGC, α-DG is noncovalently associated with m laminationdefectsofthecortexandbreachesorevenabsenceof β-dystroglycan,amembraneproteinofthesarcolemmathatin- ic.o u thebasalmembranethatseparatestheneuronaltissuefromthe teractsdirectlywithsubsarcolemmaproteinssuchasdystrophin. p menningen(Huetal.2007;Liuetal.2006).Additionally,eye α-DG interacts with components of the extracellular matrix .co abnormalities are described in pomGnT−/− mice that include (ECM)suchaslaminin,providingaphysicallinkbetweenthe m/g reduced size of the optic nerve and retinal abnormities (Liu subsarcolemmacytoskeletonandbasementmembranes(Barresi lyc o etal.2006). andCampbell2006).ItisbelievedthatDGCconfersstructural b /a stabilitytothesarcolemmaduringcontraction,althoughprecise rtic mechanismsarenotyetunderstood(Petrofetal.1993).Inher- le ImpairedproteinO-mannosylationcausescongenital ited deficiencies in most of the proteins within DGC result in -ab musculardystrophies CMD(Blakeetal.2002),probablyduetomembranemicrole- stra Inhumans,impairedO-mannosylationisassociatedwithaclin- sionsandcompromisedmusclemembranefunctionthatmight ct/1 icallyandgeneticallyheterogeneousgroupofautosomalreces- beinducedbymechanicalstressfrommusclecontraction(Blake 9 sive muscular dystrophies associated with variable brain and etal.2002;DeconinckandDan2007).Hypoglycosylatedα-DG /8/8 1 ocularabnormalities(Yoshidaetal.2001;Muntonietal.2004). loses the ability to interact with its ECM-ligands in muscle 6 /1 Thesecongenitalmusculardystrophies(CMD)arealsoreferred (e.g.,laminin,agrin,andperlecan)andinbrain(e.g.,neurexin; 9 8 toassecondaryα-dystroglycanopathiessinceacommonpatho- Michele et al. 2002; Michele and Campbell 2003; Muntoni 81 logical feature is the hypoglycosylation of α-DG. Phenotypic et al. 2004). Thus, loss of α-DG O-mannosyl glycans reduces 98 b severityofdystroglycanopathypatientsishighlyvariable.The the association between DGC and ECM, therefore leading to y g mostseveredisorderisWalker–Warburgsyndrome(WWS)fol- musculardystrophy.Asidefromskeletalmuscle,dystroglycan u e lowedbymuscle–eye–braindisease(MEB)andFukuyamacon- isfoundinseveralothertissueswereitisabundantlyexpressed st o genitalmusculardystrophy(FCMD;reviewedinMuntonietal. onthebasalsideofmostepithelia(Durbeejetal.1998;Durbeej n 0 2004). These diseases are characterized by CMD associated etal.1995).Thus,ageneralroleintheassemblyandorganiza- 3 A with severe brain malformations (cobblestone lissencephaly) tionofbasalmembraneswasproposed(BarresiandCampbell p and abnormalities of the eye. Due to multiple malformations, 2006).Duringnormalbraindevelopment,thetopofthecerebral ril 2 0 WWSpatientsoftendiewithinthefirstyearoflife.Incontrast, cortexisdefinedbyabasementmembrane–theglialimitans– 1 9 the mildest disorders may not present until adulthood such as attheendoftheradialgliafibersthatguideneuronsontheirway limb-girdle muscular dystrophy (LGMD) where neither brain tothecortex. Thisprevents neurons frommigratingoutof the noreyesareaffected(Biancherietal.2007).Betweentheseex- brain into the subarachnoid space. Cobblestone lissencephaly tremes,intermediatephenotypeswithCMDbutmildmentalre- inducedbydiminishedO-mannosylationofα-DGisassociated tardationandmicrocephalyhavebeendescribed(vanReeuwijk withgapsintheglialimitans,aswellasthefailureofneurons etal.2006). toorganizethemselveswithinthecortexregionandtheirmigra- To date, mutations in six known or putative glycosyl- tion into the subarachnoid space (Ross 2002). Similar defects transferase genes have been identified to cause dystrogly- areinducedintheconditionalknockoutofmurinedystroglycan canopathies with overlapping phenotypes. Among these are ingliacellsandbrainneuronsthatresultinbrainmalformations hPOMT1 (OMIM 607423), hPOMT2 (OMIM 607439), and resemblingthosefoundinWWSandMEBpatients(Mooreetal. hPOMGnT1 (OMIM 606822) that are directly involved in the 2002).Thereareindicationsthatdystroglycanisalsoinvolved synthesis of O-mannosyl glycans linked to α-DG. Association in the organization of synapses and of the basal membrane of of POMTs with WWS was first established by Bertran-Valero theretina(Moukhlesetal.2000;Satoetal.2008).Thus,onthe 823 MLommelandSStrahl whole, brain and ocular malformations of dystroglycanopathy nous polyprenol phosphate containing nine repeated isoprenyl patients might be explained by diminished function of α-DG units in membrane preparations of S. coelicolor. Further, mu- duetoreducedO-mannosylation. tation in ppm1 abolished the transfer of mannose to acceptor In addition, emerging evidence suggests that changes in O- peptides/protein. mannosylglycancompositionaremodulatingnotonlylaminin- Abioinformaticapproachledtotheidentificationofthefirst but also integrin-dependent adhesion and migration of human bacterial protein O-mannosyltransferase (Rv1002c/pmt) from neuronal cells (Abbott et al. 2006). In this respect, it was re- M.tuberculosis.TheMt-Pmtpproteindisplays22–24%homol- centlysuggestedthatthedevelopmentallyregulatedandneuron- ogytothePMTsofS.cerevisiae(VanderVenetal.2005).Over- specificreceptorprotein-tyrosinephosphatasebeta(RPTPβ)is expressionofMt-pmtinM.smegmatisledtoincreasedPmtac- carryingtheO-mannosyl-linkedHNK-1modification.Changes tivityofmembranefractionsinacell-freepeptideglycosylation in O-mannosylation might thus regulate RPTPβ signalling assay(VanderVenetal.2005).Pmtproteinsthatinitiatebiosyn- therebyinfluencingcell–cellandcell–matrixinteractionsinthe thesisofbacterialO-mannosylglycanshavealsobeencharacter- developingnervoussystem(Abbottetal.2008). izedfromCorynebacteriumglutamicumandfromS.coelicolor D o (Mahneetal.2006;Wehmeieretal.2009).Disruptionofboth, w n Cg-pmtandSc-pmt,completelyabolishesO-mannosylationof lo a d specific glycoproteins in vivo (Mahne et al. 2006; Wehmeier e ProteinO-mannosylationinprokaryotes d etal.2009).Todate,awholeplethoraofbacterialPmthomologs fro Prokaryotes,includingbacteriaandarchaea,areabletoglycosy- has been identified and categorized as glycosyltransferases of m lateproteins(Abu-Qarnetal.2008).Morethan70glycoproteins theGT39family(http://www.cazy.org).Despitetheirmoderate http havebeenreported(Upretietal.2003).Amongthese,somegly- homology to fungal Pmt proteins, the hydropathy profiles of s coproteinscontainingO-mannosylglycanshavebeenreported bacterialandeukaryoticPmtsaresubstantiallysimilar(Mahne ://a c including β1,4-glucanase Cex of Cellumonas fimi, and Strep- etal.2006;VanderVenetal.2005;Figure4).Yet,MIRmotifsof ad e tomyces lividans (Ong et al. 1994) and the phosphate binding thelargehydrophilicloop5domainareabsentinbacterialPmts. m ic proteinPstSofStreptomycescoelicolor(Wehmeieretal.2009). Interestingly, specific arginine residues in the first and second .o u Further,O-mannosylglycanscontainingrhamnose,2-O-methyl transmembrane domain are conserved between bacterial and p .c mannose, glucose, and glucuronic acid, attached at consensus eukaryotic PMTs (Figure 4). In S. cerevisiae, these conserved o m sites corresponding to Asp-Ser or Asp-Thr have been isolated residuesarecrucialfortheformationofPMTcomplexes,sug- /g fromproteaseP40andtheendoglycosidasesEndoF2andEndo gestingthatbacterialPmtsmightalsoactascomplexes.Inad- lyc o F3oftheGram-negativebacteriumFlavobacteriummenigosp- dition,anAsp-Glumotifpresentintheputativecatalyticloop1 b /a ticum(Plummeretal.1995).Thealanin-andproline-richanti- domainiscrucialformannosyltransferaseactivityforboth,yeast rtic gen(Apa)andsuperoxidedismutase(SodC)glycoproteinsofthe andmycobacterialPmts(Girrbachetal.2000;VanderVenetal. le Gram-positive bacterium Mycobacterium tuberculosis (Dobos 2005). Conservation of the basal mechanisms of O-mannosyl -ab s etal.1996;SartainandBelisle2008)andthesecretedantigen transferbetweenpro-andeukaryotesisfurthersupportedbythe tra MPB83 of Mycobacterium bovis (Michell et al. 2003) are O- findingthatinmycobacteriaproteinO-mannosylationdepends ct/1 mannosylatedatThrresiduesinThr/Pro-richsequences.Char- onproteintranslocationviathesecretorypathway(VanderVen 9 acterizationofApaandSodCrevealedshortlinearα1,2-linked etal.2005). /8/8 1 oligomannosechainsofuptothreemannosylresidues(Dobos Although enzymes catalyzing the initial steps of bacterial 6 /1 etal.1996;SartainandBelisle2008),whereasMBP83contains O-mannosylation have been identified, it is not clear to date 9 8 mannoseandα1,3-linkedmannobiose(Michelletal.2003).The howO-linkedmannoseisfurtherextended.However,bacterial 81 structuralsimilaritiesofmycobacterialandfungalO-mannosyl mannosyltransferases of the GT-C superfamily that add α1,2 98 b glycansalreadysuggestaconservedbiosyntheticpathway. branchestothemannancoreoflipomannan/lipoarabinomannan y g have been characterized (Kaur et al. 2006). Whether these or u e similar enzymes are also involved in the biosynthesis of O- st o mannosylglycansremainstobeelucidatedinthefuture. n BiosynthesisofbacterialO-mannosylglycans 0 3 A The biosynthetic pathway of O-mannosyl glycans in bacte- p ria was initially characterized using a cell-free peptide glyco- BiologicalsignificanceofproteinO-mannosylationin ril 20 sylation assay. Incubation of a membrane fraction from My- 1 prokaryotes 9 cobacteriumsmegmatiswithGDP-[14C]mannoseandsynthetic peptides derived from the Apa protein, led to the synthe- Despite the progress in the eukaryotic field, little is known sis of polyprenol monophosphate [14C]mannose and then to about the functional role of protein O-mannosylation in bac- [14C]mannose-peptide(Cooperetal.2002). teria.pmtmutantsofC.glutamicumareviableanddonotshow Consequently, polyprenyl monophosphomannose synthases any obvious phenotype (Mahne et al. 2006). Insights into the (Ppm1) with significant homology to the S. cerevisiae Dol-P- biological significance of protein O-mannosylation could be MansynthaseDpm1phavebeenidentifiedinmycobacteriaand obtained fromthe function ofthe respective glycoproteins. M. corynebacteria. Their function in polyprenol monophosphate tuberculosiscausespersistentinfectionbyevadingthedefense mannose (PPM) synthesis has been demonstrated (Baulard mechanisms of the infected host (Ragas et al. 2007). In this etal.2003;Cooperetal.2002;Gibsonetal.2003;Gurchaetal. context, mannose-specific C-type lectins have emerged to be 2002).Recently,directinvolvementofS.coelicolorPpm1inthe the preferential receptor used by mycobacteria to enter their protein O-mannosylation pathway could be shown (Wehmeier target cells (Nguyen and Pieters 2005). The M. tuberculosis etal.2009).Ppm1catalyzedthetransferofmannosetoendoge- cell wall-associated adhesion protein Apa has recently been 824 ProteinO-mannosylation:Conservedfrombacteriatohumans demonstratedtobindthesemannose-specificlectinswhichsug- Conflictofintereststatement gestsaroleoftheattachedO-mannosylglycansincolonization Nonedeclared. andinvasionofthehostcellbythepathogen.Ontheotherhand, Apaisoneofthemajorimmunodominantantigenssecretedby M.tuberculosis.ExperimentsusingnonmannosylatedApapro- Abbreviations teins demonstrated that its capacity to stimulate T-lymphocyte responses is highly dependent on the glycosylation. Thus, O- CMD,congenitalmusculardystrophy;CWI,cellwallintegrity mannosyl glycans contribute to the antigenicity of Apa (Horn pathway; α-DG, α-dystroglycan; Dol-P, dolichol-phosphate; etal.1999;Romainetal.1999).InS.coelicolor,mutationsin HNK-1epitope,humannaturalkiller-1epitope;LGMD,limb- pmtandppm1resultinincreasedresistancetothebacteriophage girdle muscular dystrophy, MEB, muscle-eye-brain disease; (cid:3)C31(CowlishawandSmith2001).Thisphenotypeisalsoob- WWS,Walker–Warburgsyndrome. served in ppm1 mutants (Cowlishaw and Smith 2001). These observationsleadtothesuggestionthatthereceptorfor(cid:3)C31 D o isayetundefinedO-mannosylatedcellsurfaceglycoproteinof References w n S.coelicolor(CowlishawandSmith2001).Insummary,these AbbottKL,MatthewsRT,PierceM.2008.Receptortyrosinephosphatasebeta loa d initialstudiessuggestthatO-mannosylglycansofspecificsecre- (RPTPbeta)activityandsignalingareattenuatedbyglycosylationandsub- e d toryproteinscontributetovirulenceofpathogenicmycobacteria AbbsoetqtuKeLnt,TcerolluspuerfKac,LeegealIe,cPtiienr-c1ebMin.d2i0n0g6..JIBntieoglrCinh-edmep.e2n8d3e:n3t3n0e2u6r–o3b3l0as3t5o.ma fro andmightfulfillotheryetunidentifiedimportantfunctions. m celladhesionandmigrationonlamininisregulatedbyexpressionlevelsof h twoenzymesintheO-mannosyl-linkedglycosylationpathway,PomGnT1 ttp Aboaun-dHGainlaT-AV,bT.uElxspiaCnielDlRRe.s2.030102.:M28a3m7–m2a8l5ia0n. spermacrosome:Formation, s://a c Concludingremarks contents,andfunction.ArchBiochemBiophys.379:173–182. a d Abu-QarnM,EichlerJ,SharonN.2008.NotjustforEukaryaanymore:Protein e m During the last 35 years, the biosynthetic pathway of O- glycosylationinBacteriaandArchaea.CurrOpinStructBiol.18:544–550. ic mannosyl glycans was established in bakers’ yeast. The iden- Akasaka-ManyaK,ManyaH,NakajimaA,KawakitaM,EndoT.2006.Physical .o u tdieficciapthioenrinogftPhMebTisolaongdicpamlrtomleuotfanthtsisweassseantmiaaljporrotseteinpmtoowdaifird- Arav2ani.ndJdBfuLino,clKtCioohnoeanmlin.a2sEs8oV1c:i11a9t9i39o39n9.o–Tf1h9he3u4mf5ua.knuptirnotperionteOin-mfaanmniolysy-pltrreadniscfteerdaseensz1ymaneds p.com cationinfungi.Unexpectedly,O-mannosylglycansandPMTs modifyingcell-surfacemolecules.CurrBiol.9:R836–R837. /gly werealsoidentifiedinanimalsandhumansinthelate1990s,and BabczinskiP,TannerW.1973.Involvementofdolicholmonophosphateinthe c o theirassociationwithneuromusculardiseaseshasfocusedtheat- formationofspecificmannosyl-linkagesinyeastglycoproteins.Biochem b/a tTeondtiaoyn, tohfedbifafseircenptrrinesceipalrecshoafreOas-monanpnroosteyilnatOio-nmiannneouskyalraytoiotens. BalcBGii,oBpH,heUyhsyraRnUeis,kCWGo,imnDkmilneurcne.Jr54Pet:,1Ga1l1r.o92s–s0101C52,.4W.AinllearuTto,sTomaliaml rBec,eHssailvileoglilmubG,gKirdallee rticle-a are quite well understood. However, 3D structures of PMTs, muscular dystrophy (LGMD2) with mild mental retardation is allelic to bs their association with other ER components, and signals that Walker–Warburgsyndrome(WWS)causedbyamutationinthePOMT1 tra define O-mannosylation still remain to be explored. Certainly, BarrgeesnieR.,NCeaumropmbeulslcKulPD2i0s0o6r.dD.1y5s:t2ro7g1l–y2c7a5n.:Frombiosynthesistopathogenesis ct/19 bakers’yeastwillagainbeanimportantmodelorganismtosolve ofhumandisease.JCellSci.119:199–207. /8 theseissues.Furthermore,therecentidentificationofPMTsin BaulardAR,GurchaSS,Engohang-NdongJ,GouffiK,LochtC,BesraGS. /81 bacteriahasproventhatO-mannosylationisconservedbetween 2003.Invivointeractionbetweenthepolyprenolphosphatemannosesyn- 6/1 pro-andeukaryotes.ThefutureanalysisofbacterialPmtswill thase Ppm1 and the integral membrane protein Ppm2 from Mycobac- 98 teriumsmegmatisrevealedbyabacterialtwo-hybridsystem.JBiolChem. 8 helptoresolvebasicprinciplesofO-mannosylationandtoex- 1 278:2242–2248. 9 plain how the PMT family has evolved to satisfy the different Bause E, Lehle L. 1979. Enzymatic N-glycosylation and O-glycosylation of 8 b demandsintheERofeukaryotes. synthetic peptide acceptors by dolichol-linked sugar derivatives in yeast. y g EurJBiochem.101:531–540. ue BehcroensestNraHn.sf1e9r7i0n.lDivoelri.cPhroolcmNoantolpAhcoasdphSactieUgSluAc.o6s6e::1A53n–i1n5te9r.mediateinglu- st on Beltran-ValerodeBernabeD,CurrierS,SteinbrecherA,Celli,vanBeusekom 0 Funding 3 E,VanDerZwaagB,KayseriliH,MerliniL,ChitayatD,DobynsWBetal. A 2002.MutationsintheO-mannosyltransferasegenePOMT1giverisetothe p WpeeanthUannkioTnhfeorDceountsticnhueinFgorssucphpuonrgt.sgemeinschaftandtheEuro- sGeevneeret.n7e1u:1ro0n3a3l–m10ig4r3a.tiondisorderWalker–Warburgsyndrome.AmJHum ril 201 Bergwerff AA, Stark W, Fendrich G, Knecht R, Blommers MJ, Maerki W, 9 Kragten EA, van Oostrum J. 1998. Identification of Man alpha1-3Man alpha1-2ManandMan-linkedphosphateonO-mannosylatedrecombinant Acknowledgements leech-derivedtryptaseinhibitorproducedbySaccharomycescerevisiae,de- terminationofthesolutionconformationofthemannosylatedpolypeptide. Wethankallthepresentandformermembersofthelaboratories EurJBiochem.253:560–575. ofW.TannerandourswhohelpedtothrowlightonproteinO- Biancheri R, Falace A, Tessa A, Pedemonte M, Scapolan S, Cassandrini D, Aiello C, Rossi A, Broda P, Zara F et al. 2007. POMT2 gene mutation mannosylationinyeastsandinmammals.WearegratefultoF. in limb-girdle muscular dystrophy with inflammatory changes. Biochem HutzlerandJ.HutzlerforlocalizationofyeastPMTsshownin BiophysResCommun.363:1033–1037. Figure2B.ThankstoM.Bu¨ttnerforthecriticalreadingofthe BlakeDJ,WeirA,NeweySE,DaviesKE.2002.Functionandgeneticsofdys- manuscript. S. Strahl is especially thankful to Mark Lehrman trophinanddystrophin-relatedproteinsinmuscle.PhysiolRev.82:291–329. BreloyI,SchwientekT,GriesB,RazawiH,MachtM,AlbersC,HanischFG. forhisimmensepatienceandencouragementwhilethisreview 2008.InitiationofmammalianO-mannosylationinvivoisindependentofa wasinprogress.S.StrahlismemberofCellNetworks–Cluster consensussequenceandcontrolledbypeptideregionswithinandupstream ofExcellence(EXC81). ofthealpha-dystroglycanmucindomain.JBiolChem.283:18832–18840. 825