Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae

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Broad spectrum O-linked protein glycosylation in the human pathogen Neisseria gonorrhoeae
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  Broad spectrum O-linked protein glycosylation in thehuman pathogen  Neisseria gonorrhoeae  Åshild Vik a,b , Finn Erik Aas a,b , Jan Haug Anonsen a,b , Shaun Bilsborough c , Andrea Schneider d ,Wolfgang Egge-Jacobsen a,b,1 , and Michael Koomey a,b,1 a Department of Molecular Biosciences and  b Center for Molecular Biology and Neuroscience, University of Oslo, Oslo 0316, Norway;  c Agilent TechnologiesUK Ltd., Cheshire SK8 3GR, United Kingdom; and  d Bruker Daltonik GmbH, Bremen 28359, GermanyEdited by Emil C. Gotschlich, The Rockefeller University, New York, NY, and approved January 12, 2009 (received for review September 23, 2008) Protein glycosylation is an important element of biologic systemsbecause of its significant effects on protein properties and functions.Althoughprominentwithinalldomainsoflife,O-linkedglycosylationsystemsmodifyingserineandthreonineresidueswithinbacteriaandeukaryotes differ substantially in target protein selectivity. In partic-ular, well-characterized bacterial systems have been invariably ded-icated to modification of individual proteins or related subsetsthereof.HerewecharacterizeageneralO-linkedglycosylationsystemthat targets structurally and functionally diverse groups ofmembrane-associated proteins in the Gram-negative bacterium Neisseria gonorrhoeae , the etiologic agent of the human diseasegonorrhea. The 11 glycoproteins identified here are implicated inactivities as varied as protein folding, disulfide bond formation, andsolute uptake, as well as both aerobic and anaerobic respiration.Alongwiththeircommontraffickingwithintheperiplasmiccompart-ment, the protein substrates share quasi-related domains bearingsignatures of low complexity that were demonstrated to encompasssites of glycan occupancy. Thus, as in eukaryotes, the broad scope ofthis system is dictated by the relaxed specificity of the glycan trans-ferase as well as the bulk properties and context of the protein-targeting signal rather than by a strict amino acid consensus se-quence. Together, these findings reveal previously unrecognizedcommonalities linking O-linked protein glycosylation in distantlyrelated life forms. bacteria    glycoprotein    pilin    post-translational modification    pgl T argeting of a broad array of heterogeneous protein substrates isa fundamental feature of O-linked glycosylation in eukaryotesand N-linked glycosylation systems in all domains of life (1). Thecovalentadditionofuniformglycantagsenablesdiverseproteinstobe recognized and modified by conserved core processes influenc-ing protein trafficking, folding, and turnover. Glycosylation sub-strate selection involves not only colocalization with the glycosyl-ationmachinerybutalsothepresenceandavailabilityofoccupancysites. In bacterial, archaeal, and eukaryotic N-linked systems,glycosylation sites are characterized by the N-X-S/T tripeptidesequon that is necessary but not sufficient for modification (2). Incontrast, structural determinants intrinsic to O-linked protein gly-cosylation have been more difficult to characterize (3). In highereukaryotes, the seemingly most prevalent (and arguably the mostthoroughly studied) form of O-linked glycosylation is termedmucin-type glycosylation, in which cell-surface proteins are modi-fied with N-acetylgalactosamine through the action of polypeptideN-   -acetylgalactosaminyltransferases (4). Glycosylation acceptorsites in this system are associated with domains rich in serine,threonine, and proline that are localized to coiled or turn regionsand that are often tandemly repeated (5). In the case of theeukaryotic O-linked N-acetylglucosamine modification mediatedby the O-GlcNAc transferase, few clear patterns have emergedalthough systematic peptide-based assays have begun to revealsome sequence preferences (6, 7).Bacterial protein glycosylation systems have come under en-hanced scrutiny because of the increasing frequencies with whichthey are seen in pathogenic and symbiotic species, as well as theirpotential for exploitation in recombinant glycoprotein engineeringstrategies. Studies of these systems also have the potential toprovide insight into the evolution of glycan biosynthesis, glycosyl-transferases, and structural determinants of substrate recognition.Indeed, the Gram-negative species  Campylobacter jejuni  targetsmultiple proteins at asparagine residues like its eukaryotic coun-terparts and uses an oligosaccharyltransferase (OTase) srcinallyrecognized on the basis of its sequence identity with the STT3, thecatalytic subunit of eukaryotic OTases (8). An STT3-like OTasealso acts in N-linked glycosylation in the thermophilic archaeaon  Pyrococcusfuriosus (9).Incontrast,noclearrelationshipshavebeendelineated for conventional O-linked systems that predominate ineukaryotes and bacteria. Most conspicuously, bacterial O-linkedsystems are uniformly dedicated to the modification of individualproteins or sets of highly related proteins. Prime examples includethe subunits of S-layers, flagella, and Type IV pili, as well asnon-pilus adhesins, such as autotransporters and 2-partner secre-tion pathway exoproteins (10). Evidence for glycosylation of mul-tiple, structurally related protein substrates has been reported in  Bacteroides fragilis , although the nature of the glycan linkageinvolved remains unknown (11). Two surface lipoproteins of   My- cobacterium tuberculosis  have been proven to be O-glycosylated,and an implicated transferase shares some structural similarity toeukaryotic protein mannosyltransferases (12, 13). However, whether this system is representative of bona fide general proteinglycosylation has not been established.The PilE pilin protein subunit of the Type IV pilus colonizationfactor expressed by the Gram-negative human pathogen  Neisseria gonorrhoeae  (  Ngo ) undergoes O-glycosylation at a single, definedserineresidue(Ser-63)(14).Geneticanalysesincludinginterspeciescomplementation have revealed that this system (termed  pgl  for  p ilin  gl  ycosylation) is remarkably similar to the N-linked system of  C. jejuni  with regard to the use of a peptide-proximal 2,4 diacet-amido-2,4,6 trideoxyhexose (DATDH) sugar and related biosyn-thetic pathways for generating lipid-linked glycan substrates (15).These systems then diverge at the step of oligosaccharide transfer with the Ser-targeting PglO OTase acting in  Ngo  and the sequontargeted PglB OTase in  C. jejuni  (supporting information Fig. S1). Thus, in contrast to eukaryotic O-linked glycosylation systems, thegonococcalO-linkedsystemuses  enbloc transferofoligosaccharidefrom a lipid-linked donor rather than single UDP-monosaccharidedonors and downstream glycan elaboration. Results Evidence for Multiple Glycoproteins in  N. gonorrhoeae  .  Wemadetheobservation that polyclonal rabbit antiserum raised against glyco- Author contributions: Å.V., F.E.A., W.E.-J., and M.K. designed research; Å.V., F.E.A., J.H.A.,S.B.,A.S.,andW.E.-J.performedresearch;Å.V.,F.E.A.,W.E.-J.,andM.K.analyzeddata;andÅ.V. and M.K. wrote the paper.The authors declare no conflict of interest.This article is a PNAS Direct Submission.Freely available online through the PNAS open access option. 1 To whom correspondence may be addressed. E-mail: johnk@imbv.uio.no or w.m.egge- jacobsen@imbv.uio.no.This article contains supporting information online at www.pnas.org/cgi/content/full/ 0809504106/DCSupplemental. www.pnas.org  cgi  doi  10.1073  pnas.0809504106 PNAS    March 17, 2009    vol. 106    no. 11    4447–4452      M     I     C     R     O     B     I     O     L     O     G     Y  sylatedPilEprotein(inpurifiedTypeIVpili)reactedwithmultiplegonococcal proteins when examined by immunoblotting (Fig. 1).This pattern of immunoreactivity occurred independently of PilEexpression and PilE glycosylation (as seen in a PilE S63A back-ground)butwasabolishedinall  pgl nullmutantsexceptthatlackingPglF (encoding a putative flippase), in which the case the signalintensitieswerediminished.PglFhasbeenmodeledasamembranetranslocase for the lipid-linked oligosaccharide, and we previouslyshowed that PilE is hypoglycosylated in a  pglF  null mutant [residualglycosylation presumably being due to the presence of partiallycompensatory translocases in this background (14)]. In addition,specific immunoreactivity was absent in backgrounds expressingeither the DATDH monosaccharide (  pglA ) or the O-acetylated(O-Ac) HexHexDATDH trisaccharide (  pglE on ) glycan precursorforms. These findings suggested that the immunoreactive proteinsshared specific glycan-associated epitopes generated by the sameglycosylation pathway that acts on PilE. Identification of a Major Subset of  N. gonorrhoeae   Glycoproteins.  Toexamine these possibilities in more detail, a cell fraction enrichedfor the immunoreactive proteins was subjected to 2D gel electro-phoresis and probed by immunoblotting with antiglycan antibodies(Fig. S2). Of the more than 15 immunoreactive proteins detected, 10 candidates were identified using MS analyses of correspondingspots excised from preparative gels. However, insufficient material was available by this method to unambiguously characterize glyco-sylation status. Therefore, gonococcal strains were individuallyengineered so that each protein carried a hexahistidine (6xHis)extension at its carboxy-terminus while remaining expressed fromits endogenous locus and promoter. After affinity purification, it wasconfirmedthateachofthe10proteinsretained  pglC -dependentreactivity with glycan-specific antibodies, and in the case of Ng1328this property was also dependent on  pglA  and  pglO  (but not  pglF  )(Fig. 2  A–C ). Mass Spectrometric Confirmation of Glycosylation Status via Glyco-peptide Identification.  We next identified glycosylated peptidesderived by proteolysis from these purified proteins using liquidchromatography-electrospray ionization–tandem mass spectrome-try and collision-induced dissociation (CID). Data indicative of glycopeptides carrying the O-AcHexDATDH glycan moiety [asdiagnosticoxoniumionfragmentsappearingatmasstochargeratio(  m  /   z ) 433.2 due to CID] were obtained from 7 of the 10 proteins(Fig. 3). In the case of Ng1043, 2 non-overlapping glycopeptides were detected (Fig. 3  G  and  H  ), and the signals seen for the largerindicated the presence of 2 glycans. Signals indicating the presenceof 2 glycans were also detected on the glycopeptide found forNg1276 (Fig. 3 C ). Evidence for Glycan Linkage at Serine Residues.  Consistent with the  pgl -dependent glycosylation of Ser-63 of PilE, we noted that serineresidues were present in all 8 glycopeptides, whereas threonineresidues were represented in only 5 instances (see below). Becausethe Ng1371 glycopeptide only contained 2 serines, missense mu-tants bearing a substitution individually at each site were con-structed and examined. Whereas substitution at Ser-327 with anon–hydroxyl-bearing amino acid had no effect on glycan antibodyreactivity(datanotshown),thatatSer-337abolishedreactivity(Fig.2  B ). Directly locating the exact sites of glycosylation using conven-tional MS/MS CID techniques is difficult owing to the relativelability of glycosidic bonds. We therefore used ion trap MS withelectron transfer dissociation (ETD). As shown for the 165-DNAASGTASAPA-176 glycopeptide from Ng1328, the ETDspectrum provided the necessary peptide backbone fragmentationto assign Ser-173 as bearing the glycan (Fig. 4). Its location toSer-173 is derived by detection of fragment ions c 9  (DNAASG-TAS )  and c 11  (DNAASGTASAP )  at  m  /   z  1224.6 and 1392.7,respectively, representing the only c-ions of the c-ion series c 6  toc 11   withamassincreaseof432Da,whichcorrespondstotheglycanresidue. Nonetheless, a missense form of Ng1328 with a non–hydroxyl-bearing amino acid replacement at this residue retainedreactivitywiththeglycanantibodies(datanotshown),showingthatthis protein carries an additional acceptor site. Taken together with   w   t  p  g   l   C  p   i   l   E   S   6   3   A  w   t  p  g   l   A  p  g   l   E  o  n  p  g   l   F  p  g   l   O  p  g   l   C  p  g   l   D  pilE  : + + + - - - - - - - 25503720kDa PilE Fig. 1.  Evidence for multiple  Ngo  glycoproteins revealed via detection ofglycan-associatedepitopes.Immunoblottingofwhole-celllysateswithrabbitserum (termed antiglycan) raised against purified PilE bearing the O-AcHexDATDH glycan reacts with multiple proteins in a  pgl   pathway–dependentfashion(seeFig.S1foranoverviewofthe  pgl  pathway).Notethatthe lack of immunoreactivity observed in the  pglA and  pglE  on  backgrounds isnotassociatedwithalackofglycosylationbutratherthepresenceofmonosac-charide DATDH and trisaccharide O-AcHexHexDATDH glycans, respectively. Ng1717 Ng1548Ng2139Ng1371 Ng1494Ng1276Ng1237Ng1328Ng0372   w   t  p  g   l   C     w     t   w   t  w   t  p  g   l   C  p  g   l   A  p  g   l   O  p  g   l   F  p  g   l   C  p  g   l   C     p    g     l     C   w   t     w     t    w     t    w     t    p    g     l     C    p    g     l     C     S     3     3     7     G IB:anti-glycanIB:anti-His   w   t  p  g   l   C Ng1043Ng0994Ng1225   w   t  w   t  w   t  p  g   l   C  p  g   l   C  p  g   l   C IB:anti-glycanIB:anti-His AB C D Fig. 2.  Affinity purification confirms the identities of candidate Ngo glycoproteins. (  A–D ) C-terminally 6xHis-tagged candidate proteins were affinity purifiedfromwild-type( wt  )and  pgl  backgroundsandtestedforglycan-associatedantigenicitybyimmunoblotting.( B )ForNg1371,amutantformoftheproteincarryingaglycinesubstitutionatSer-337wasalsopurifiedandtested,whereasforNg1717( C  ),theNg1548paralogue(Fig.S3)wasexaminedinparallel.( D )Ng1225wasidentified as a potential glycoprotein on the basis of its lipoprotein processing site and proximal ASP-rich LCR (Table S1). 4448    www.pnas.org  cgi  doi  10.1073  pnas.0809504106 Vik et al.  Ng1328 1228.8433.2b 10 846.41032.5*1260.6+MS2(2+, 733.2)0.00.51.01.52.05x10Intens.200 400 600 800 1000 1200 1400 1600m/z A Ng1371 1433.2y 7 683.3y 24 1116.1++1484.6++*y 28 1300.1+++MS2(3+, 1134.2)01235x10Intens.250 500 750 1000 1250 1500 1750 m/zy 22 1031.9++ B Ng1276 2433.2654.11083.51389.2++*+MS/MS (4+, 911.6)0.00.51.01.52.04x10Intens.200 400 600 800 1000 1200 1400 1600m/z391.2 C Ng1237 1433.11316.1++*+MS/MS (3+, 1022.1)0.00.20.40.60.81.05x10Intens.250 500 750 1000 1250 1500 m/z1418.1++ D Ng2139 1433.1609.4y 9 830.41370.71642.7*+MS/MS (3+, 1038.9)0.00.51.01.52.05x10Intens.250 500 750 1000 1250 1500 m/z E Ng1717 1229.1433.1722.41215.5*1419.4+MS/MS (2+, 824.6)0.00.20.40.60.85x10Intens.200 400 600 800 1000 1200 1400 m/z F Ng1043 1228.8433.2517.0++609.4804.5*1014.4+MS/MS (2+, 619.5)012344x10Intens.200 400 600 800 1000 m/z211.8 G Ng1043 2433.2707.31056.4++1678.8*2111.0+MS/MS (2+, 1272.7) 0.00.51.01.52.05x10Intens.500 1000 1500 2000 m/z H Fig. 3.  Identification of glycopeptides derived from endoproteolytic cleavage of affinity-purified proteins using tandem MS. CID experiments of theglycopeptide-related molecular ions yield mainly cleavage at glycosidic bonds, revealing characteristic oxonium ions at m  /   z  433 and m  /   z  229 (corresponding toO-AcHexDATDHandDATDHglycans,respectively).Diamondsdenotethe m  /   z  oftheprecursorionfragmentedinthespectrum.Superscriptnumberingappendedto ORF designations indicates peptides bearing 1 ( 1 ) or 2 ( 2 ) glycans. The asterisk marks the unmodified peptide after loss of the glycan moiety resulting fromCID. (  A ) MS/MS spectrum of [M  2H]  2  at  m  /   z   733.2 derived from Ng1328 by AspN cleavage. ( B ) MS/MS spectrum of [M  3H]  3  at  m  /   z   1134.2 derived fromNg1371 by tryptic cleavage. ( C  ) MS/MS spectrum of [M  4H]  4  at  m  /   z   911.6 derived from Ng1276 by tryptic cleavage. ( D ) MS/MS spectrum of [M  4H]  4  at m  /   z  1022.1derivedfromNg1237byAspNcleavage.( E  )MS/MSspectrumof[M  3H] 3  at m  /   z  1038.9derivedfromNg2139bytrypticcleavage.( F  )MS/MSspectrumof[M  2H] 2  at m  /   z  824.6derivedfromNg1717byproteinaseKcleavage.( G )MS/MSspectraof[M  2H] 2  at m  /   z  619.5derivedfromNg1043bytrypticcleavage.( H ) MS/MS spectra of [M  2H]  2  at  m  /   z   1272.7 derived from Ng1043 by tryptic cleavage. Vik et al. PNAS    March 17, 2009    vol. 106    no. 11    4449      M     I     C     R     O     B     I     O     L     O     G     Y  the observations that serine is the only hydroxyl-bearing residuein Ng1043 and Ng2139 glycopeptides, these data strongly suggestthat  Ngo general protein glycosylation is primarily attributable tolinkage at serine residues. However, we cannot formally rule outthat threonine or other hydroxyl-bearing residues might also betargeted. Glycan Occupancy Sites Are Associated with Low-Complexity Regions. Examinationoftheglycopeptidesrevealedaremarkablyconservedset of features, including an overabundance of alanine, serine, andproline residues (Fig. 5  A ). At the same time, the relative positionsof the glycopeptides within the proteins were quite diverse, beingfound in the N-terminus in 4 cases, in the middle in 2 others, andattheC-terminusinanother.Toconfirmthebiasedcompositionof the glycopeptides, glycoproteins were examined using the SEGalgorithm to identify low-complexity regions (LCRs) (16). All 8glycopeptideswerepredictedtobeencompassedwithinLCRs(Fig.5  A ), and although specific glycopeptides have yet to be identifiedfrom the 4 remaining substrates, each of these glycoproteins carriesan alanine, serine, and proline (ASP)-rich LCR near their matureN-termini similar to those seen in the other glycoproteins (Fig. 5  B andTableS1).Togainfurtherevidencefortheassociationbetween glycosylation and these ASP-rich LCR elements, the Ng1548 para-logue of Ng1717, which lacks the corresponding ASP-rich LCRdomain (Fig. S3), was affinity tagged, purified, and found to lack reactivity with glycan antibodies (Fig. 2 C ). Finally, an  in silico approachpredicatedonthepresenceoftheASP-richLCRtogether with periplasmic targeting identified a number of candidate glyco-proteins, and among these we were able to successfully affinity tagand purify Ng1225 (Fig. 5  B  and Table S1). Glycan antibodies reacted with affinity-purified Ng1225, and this recognition wasabolished when the protein was derived from a  pglC  background(Fig. 2  D ). General Characteristics and Features of  N. gonorrhoeae   Glycopro-teins.  Ngo  glycoproteins share a number of intriguing featuresincludingtheirmembranetetheringandtranslocationtoorthroughthe periplasm (Table 1). Trafficking to this compartment is likely aprerequisite for glycosylation because the PglO OTase is predictedtoactintheperiplasm(15,17).Potentialfunctionalconnectionscanalso be discerned among them. For example, Ng1276 is a copper-containing nitrite reductase essential for growth under oxygen-limiting conditions that has been proposed to acquire electrons viaNg0994, a lipid-linked azurin (18). Ng1371 is a multiheme, c-typecytochrome of the cytochrome cbb3 oxidase complex (the solepredicted terminal respiratory oxidase in  Ngo ) and is believed totransfer electrons to the catalytic subunit (19), whereas Ng1328 isan integral-membrane, diheme c-type cytochrome that potentiallychannels electrons to both Ng1276 and Ng1371 (20). On the basisof their relative signal intensities in immunoblotting, Ng1276, Ng1328, and Ng1371 represent the most abundant glycoproteins afterPilE(Fig.S4).Ng1717isanisoformofthedisulfideoxidoreductase DsbA, whose reoxidation is linked via DsbB and quinones to theterminal oxidase of electron transport (21), which in this case is thecytochrome cbb3 oxidase. Finally, Ng1237 is a lipoprotein memberof the Sco protein family whose constituents are implicated incopper binding and cytochrome oxidase biogenesis (22). Thepresence of a thioredoxin fold in these proteins suggests that theymay have thiol/disulfide-based oxidoreductase activity and metalion homeostasis functions, and an Ng1237 null mutant was shown D N A A S G T A S A P A 533  AcHexDATDH 1225 1393 792 634705  960 DNAASGTASAPA ETD MS/MS(+2, 733.2)228.9433.21422.7012344x10Intens..200 400 600 800 1000 1200 1400 1600 1800 2000 m/z846.5c 6 533.3c 11 1392.72 x2 xc 8 705.41032.5*c 9 1224.61742.7.c 7 634.4733.2++1464.7 c11c6 c-ionsz-ions Fig. 4.  Identification of Ser-173 of Ng1328 as a glycan acceptor site using ETD. Fragmentation of the doubly charged peptide at  m  /   z   733.2 shows the site ofO-AcHexDATDH modification by detection of fragment ions c 9  and c 11  at  m  /   z   1224.6 and 1392.7, respectively. These represent the only c-ions with a massincrease of 432 Da, which corresponds to the glycan moiety. Ng1371low complexity regionNg1328cyt ccyt cNg2139lipoprot 9Ng1717dsbANg1237sco1-senCcyt ccyt ccyt cNg1276Cu-oxidase 3Cu-oxidaseglycopeptide 358-LSDTAYAGSGAASAPAASAPAASAPAASASEK-389*316-AEPAPAAEPAPSAPAEAAQAA SEAK-34025-DNSAAQAASSSASAPAAENAAKPQTRGT-52165-DNAASGTA S APA-17627-TSVPADSAPAASAA-4025-DSAPAASAAAPSADNGAAK-43 Ng1043 85-EAV SEAAK-9238-DTAASAAESAASAVEEAK-55* lipoprotein proc.transmembraneNg0372 SBP-bac-3 Ng1225 FKBP_NFKBP_C Ng1494 SBP-bac-1 Ng0994 copper binding Pfam domain A B Fig. 5.  Domain organization and structural architecture of  Ngo  glycopro-teins. (  A ) Glycoproteins for which glycopeptides have been identified byMS/MS. Regions encompassed by glycopeptides are indicated by yellow rect-angles,andspecificresiduesarenumberedaccordingtothoseofunprocessedpolypeptides. Serines identified as acceptor sites (via ETD, substitution muta-tion,orbyvirtueofbeingthesolehydroxyl-bearingresidue)arelabeledinred.Asterisks denote glycopeptides bearing multiple glycan moieties. ( B ) Glyco-proteins for which glycopeptides have yet to be identified. Sequences of theLCRs shown for these proteins are found in Table S1. Further details and specific Pfam family identifiers are found in Table 1. 4450    www.pnas.org  cgi  doi  10.1073  pnas.0809504106 Vik et al.  tobehypersensitivetooxidativestress(23).Together,thesefindingsestablish a link between protein glycosylation, electron transportsystems, and redox components. The remaining glycoproteinsinclude a peptidyl prolyl-isomerase (Ng1225), 2 solute-bindingproteins associated with ABC transport systems (Ng0372 andNg1494), and 2 lipoproteins of unknown function (Ng1043 andNg2139). We further note that with the exceptions of PilE as wellas Ng1225 and Ng2139 [for which evidence for surface exposureexists (24, 25)], the glycoproteins are predicted to function in theperiplasmic compartment, although their precise sites of localiza-tion await confirmation. Discussion In this study, we discovered and characterized a general O-linkedglycosylation system in bacteria. Additionally, our findings docu-ment an instance in which bacterial proteins carrying O-linkedglycans are destined to carry out their roles within the cell ratherthan at the cell surface or beyond. Although we remain ignorant asto what properties might be imparted or modified by glycosylation,these findings imply that  Ngo  O-linked protein glycosylation acts inboth intracellular and extracellular capacities and contributes at asystem level to the biology of the organism. It seems unlikely,however, that  Ngo  will be the only bacterial species with a complex repertoire of O-linked glycoproteins. The closely related species  Neisseria meningitidis  expresses a very similar pilin glycosylationsystem,andimmunoblottingwiththeglycan-recognizingantibodiesused here reveals analogous patterns of reactive protein forms insomemeningococcalbackgrounds(B.Børud,unpublisheddata).Inaddition, LCRs similar in composition and localization to thoseseen in  Ngo  glycoproteins are discernible among orthologouscounterparts in some proteobacterial species. It is also perhapsrelevant to note that the ASP-rich LCRs defined here are remi-niscent of those sites of glycan occupancy documented in  M.tuberculosis  lipoglycoproteins (12, 13). Although further studies are required to delineate the specifictargeting signals associated with the ASP-rich LCRs, it seems clearthat there is no consensus amino acid sequence or obvious sequon. A tetrapeptide serine–alanine–proline–alanine motif is notably over-represented within both the defined glycopeptides (in 6 of 8 instancesand tandemly triplicated in Ng1276; Fig. 5  A ) and the LCRs of thoseproteinsforwhichglycopeptideshaveyettobeidentified(in2of4cases;Table S1). However, it is neither sufficient (as seen in the nonglycosy-latedNg1371mutantexpressingasubstitutionatSer-337)nornecessary(given that it is absent in PilE and Ng1043). Rather, the LCRs arethemselvesevocativeofthestructuralfeaturesassociatedwithacceptorsites in mucin-type glycoproteins. Commonalities include the biasedoverrepresentation of alanine, serine, and proline residues, the struc-tural contexts of acceptor sites confined to interdomain regions, theclustering of multiple acceptor sites, and an association with tandemrepeatelements(26).Relativesurfaceexposureandaccessibilityoftheacceptorsitesareotherlikelyshareddeterminantsoftheseelements.Itis important to note in this context that although PilE (the mostabundant glycoprotein) lacks any discernible LCR, its single acceptorsite maps on the protein surface as defined by x-ray diffraction (27).Taken together with findings suggesting that PglO exhibits relaxedspecificity for lipid-linked oligosaccharide donors (17, 28), the promis-cuity of PglO with regard to protein substrates demonstrated heresignificantly augments the potential of this system for exploitation innovel glycoprotein engineering strategies.From a larger perspective, our findings reveal remarkable sim-ilarities in both the global contexts and structural signals dictatingglycan acceptor sites of bacterial and eukaryotic O-linked glyco-sylation systems. They also extend the ways in which the  Ngo O-linked system emulates the  C. jejuni  N-linked systems to includethetargetingofmultipleperiplasmicsubstrates.Likeinthe C.jejuni system,thesefindingsraisetheobviousquestionsastowhatbiologicsignificance global protein glycosylation may have and what forcesand processes have shaped glycoproteome content. Materials and Methods BacterialStrains,Media,andCultureConditions. ThebacterialstrainsusedinthisstudyaredescribedinTableS2andweregrownonconventionalGCmediumasdescribed previously (15). Carboxyterminal 6xHis-tagged alleles as well as mis-senseallelesofNg1328andNg1371weregeneratedusingPCR-basedsplicingbyoverlapextensionreactions(29).PurifiedPCRproductswereusedtogeneticallytransform Ngo strains (30), and correct transformants were screened for by PCRusing diagnostic primer sets. Protein glycosylation null mutations were intro- Table 1. Features and properties of  Ngo  glycoproteins ORFProteinnamePfamdomain(s)Predicted mass(kDa) FunctionTarget site associated withtandem repeat elementsMembraneassociationPredictedlocalization— PilE PF07963PF00114Type IV pilus subunit    TransmembranedomainPeriplasm/ cell surfaceNg0372 — PF00497 29 ABC transporter, periplasmicbinding protein (amino acid)ND Lipoprotein PeriplasmNg0994 Laz PF00127 19 Lipid-modified azurin Cu bindingresistance to oxidative stressND Lipoprotein PeriplasmNg1043 — — 11 —    Lipoprotein PeriplasmNg1225 Mip PF00254PF0134629 Peptidyl-prolyl isomerase ND Lipoprotein Periplasm/ cell surfaceNg1237 Sco PF02630 23 Cu homeostasis, resistanceto oxidative stress   Lipoprotein PeriplasmNg1276 AniA PF00394PF0773241 Cu containing nitrite reductase    Lipoprotein PeriplasmNg1328 CycB PF00034 29 Cytochrome c5    TransmembranedomainPeriplasmNg1371 CcoP PF00034 48 Cytochrome cbb3 oxidase subunit    TransmembranedomainPeriplasmNg1494 PotF PF01547 41 ABC transporter, periplasmicbinding protein (polyamine)ND Lipoprotein PeriplasmNg1717 DsbA PF01323 25 Thiol-disulfide isomerase    Lipoprotein PeriplasmNg2139 Gna1946 PF03180 31 ABC transporter, periplasmicbinding protein (metal)   Lipoprotein Periplasm/ cell surface ND, acceptor sites not yet defined. Vik et al. PNAS    March 17, 2009    vol. 106    no. 11    4451      M     I     C     R     O     B     I     O     L     O     G     Y
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