Identification and Characterization of the Unique N-Linked Glycan Common to the Flagellins and S-layer Glycoprotein of Methanococcus voltae

The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of β-ManpNAcA6Thr-(1–4)-β-Glc-pNAc3NAcA-(1–3)-β-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway. The flagellum of Methanococcus voltae is composed of four structural flagellin proteins FlaA, FlaB1, FlaB2, and FlaB3. These proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel indicating significant post-translational modification. We describe here the structural characterization of the flagellin glycan from M. voltae using mass spectrometry to examine the proteolytic digests of the flagellin proteins in combination with NMR analysis of the purified glycan using a sensitive, cryogenically cooled probe. Nano-liquid chromatography-tandem mass spectrometry analysis of the proteolytic digests of the flagellin proteins revealed that they are post-translationally modified with a novel N-linked trisaccharide of mass 779 Da that is composed of three sugar residues with masses of 318, 258, and 203 Da, respectively. In every instance the glycan is attached to the peptide through the asparagine residue of a typical N-linked sequon. The glycan modification has been observed on 14 of the 15 sequon sites present on the four flagellin structural proteins. The novel glycan structure elucidated by NMR analysis was shown to be a trisaccharide composed of β-ManpNAcA6Thr-(1–4)-β-Glc-pNAc3NAcA-(1–3)-β-GlcpNAc linked to Asn. In addition, the same trisaccharide was identified on a tryptic peptide of the S-layer protein from this organism implicating a common N-linked glycosylation pathway. Glycosylation of prokaryotic proteins is now well accepted, and examples of N- and O-glycosylation and of attachment of glycosylphosphatidylinositol anchors can now be found in the literature (1Benz I. Schmidt M.A. Mol. Microbiol. 2002; 45: 267-276Crossref PubMed Scopus (175) Google Scholar, 2Messner P. Schaffer C. Herz W. Falk H. Kirby G.W. Progress in the Chemistry of Organic Natural Products. Springer Verlag, Wien, Germany2002: 51-124Google Scholar). However, in contrast to eukaryotic glycosylation systems, prokaryotic systems display considerable diversity in the structure of the respective glycans and the proximal monosaccharide linkage. As a consequence there is considerable interest in determining the structural and genetic basis of glycan production among these diverse prokaryotic systems. The archaeal flagellum is a unique motility structure that is distinct from the well characterized bacterial flagellum (3Thomas N.A. Bardy S.L. Jarrell K.F. FEMS Microbiol. Rev. 2001; 25: 147-174Crossref PubMed Google Scholar). In contrast to bacterial flagellar assembly where newly synthesized flagellin is incorporated at the distal tip of the filament, it is believed that mature archaeal flagellin is incorporated at the base of the filament. In recent studies, the assembly of archaeal flagellum has been shown to more closely resemble a second bacterial motility system, the type IV pilus, where the structural protein pilin is synthesized with an unusual signal peptide and a hydrophobic N terminus. In Archaea, signal peptidases have been shown to cleave a signal peptide of the preflagellin proteins to produce mature flagellin, which is then incorporated into the filament (4Bardy S.L. Jarrell K.F. Mol. Microbiol. 2003; 50: 1339-1347Crossref PubMed Scopus (102) Google Scholar). Flagellated archaeal species have one to five flagellin genes organized into a fla locus (3Thomas N.A. Bardy S.L. Jarrell K.F. FEMS Microbiol. Rev. 2001; 25: 147-174Crossref PubMed Google Scholar). The marine archaeon Methanococcus voltae has four flagellin structural genes organized in two transcriptional units: one unit contains flaA, whereas the second unit contains flaB1, flaB2, and flaB3 along with other co-transcribed accessory genes (3Thomas N.A. Bardy S.L. Jarrell K.F. FEMS Microbiol. Rev. 2001; 25: 147-174Crossref PubMed Google Scholar, 5Bardy S.L. Ng S.Y. Jarrell K.F. J. Mol. Microbiol. Biotechnol. 2004; 7: 41-51Crossref PubMed Scopus (34) Google Scholar). Early work demonstrated that FlaB1 and FlaB2 are the major species of the extended filament, whereas FlaB3 is localized proximal to the cell surface and is a major component of the curved hook region. The minor component FlaA is believed to be distributed throughout the filament (6Bardy S.L. Mori T. Komoriya K. Aizawa S. Jarrell K.F. J. Bacteriol. 2002; 184: 5223-5233Crossref PubMed Scopus (59) Google Scholar). Glycosylation of archaeal flagellin using glycoprotein-specific stains such as thymol sulfuric acid or periodic acid-Schiff is commonly reported (7Faguy D.M. Bayley D.P. Kostyukova A.S. Thomas N.A. Jarrell K.F. J. Bacteriol. 1996; 178: 902-905Crossref PubMed Google Scholar, 8Kalmokoff M.L. Jarrell K.F. Koval S.F. J. Bacteriol. 1988; 170: 1752-1758Crossref PubMed Google Scholar), although a detailed structural analysis of the post-translational modification present is limited to a single organism, Halobacterium salinarum (formerly Halobacterium halobium) (9Ventosa A. Oren A. Int. J. Syst. Bacteriol. 1996; 46: 347-348Crossref Scopus (52) Google Scholar). The flagellin of this organism has been shown to be glycosylated with N-linked sulfated oligosaccharides composed of glucose in 1,4 linkage to hexuronic acids (10Sumper M. Biochim. Biophys. Acta. 1987; 906: 69-79Crossref PubMed Scopus (79) Google Scholar, 11Wieland F. Paul G. Sumper M. J. Biol. Chem. 1985; 260: 15180-15185Abstract Full Text PDF PubMed Google Scholar). This post-translational modification is also found on purified S-layer protein from this organism. This is in marked contrast to recent studies of bacterial flagellin glycosylation in Campylobacter, Helicobacter, Pseudomonas, and Listeria where each unique glycan was shown to be O-linked to the respective flagellin proteins (12Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 13Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (222) Google Scholar, 14Schirm M. Arora S.K. Verma A. Vinogradov E. Thibault P. Ramphal R. Logan S.M. J. Bacteriol. 2004; 186: 2523-2531Crossref PubMed Scopus (100) Google Scholar, 15Schirm M. Kalmokoff M. Aubry A. Thibault P. Sandoz M. Logan S.M. J. Bacteriol. 2004; 186: 6721-6727Crossref PubMed Scopus (93) Google Scholar). Although the M. voltae flagellins did not react with glycoprotein-specific stains, these proteins possess a total of 15 potential N-linked sequons (NX(S/T)) and show a mass shift on an SDS-polyacrylamide gel suggesting significant post-translational modification. In this study we provide a detailed structural characterization of the unique glycan found on all of the M. voltae flagellin proteins and demonstrate that the S-layer protein is also post-translationally modified with the same novel trisaccharide. Microbial Strains and Growth Conditions—M. voltae cells were grown in Balch medium III under a head space gas mixture of 80% H2, 20% CO2 at 37 °C as described previously (8Kalmokoff M.L. Jarrell K.F. Koval S.F. J. Bacteriol. 1988; 170: 1752-1758Crossref PubMed Google Scholar). Flagellin and S-layer Purification—Crude flagellar filament preparations were isolated by shearing followed by banding in a KBr gradient (8Kalmokoff M.L. Jarrell K.F. Koval S.F. J. Bacteriol. 1988; 170: 1752-1758Crossref PubMed Google Scholar). For isolation of flagella containing some attached basal structure (whole intact flagella), cells were extracted with the non-ionic detergent OP-10 without prior flagella shearing as described by Bardy et al. (6Bardy S.L. Mori T. Komoriya K. Aizawa S. Jarrell K.F. J. Bacteriol. 2002; 184: 5223-5233Crossref PubMed Scopus (59) Google Scholar). S-layer protein was released from M. voltae membranes by heat treatment at 60°C for 1 h in 50 mm HEPES buffer as described previously (16Koval S.F. Jarrell K.F. J. Bacteriol. 1987; 169: 1298-1306Crossref PubMed Scopus (36) Google Scholar). Preflagellin Peptidase Reaction—A non-glycosylated form of FlaB2 was produced in Escherichia coli as described previously (17Correia J.D. Jarrell K.F. J. Bacteriol. 2000; 182: 855-858Crossref PubMed Scopus (32) Google Scholar). This form has the signal peptide of FlaB2 still attached. To remove this, E. coli membranes containing FlaB2 were used as substrate in an in vitro preflagellin peptidase assay using M. voltae membranes as a source of the preflagellin peptidase. Both the unprocessed and processed forms of FlaB2 are detected in a Western blot using anti-flagellin antisera (17Correia J.D. Jarrell K.F. J. Bacteriol. 2000; 182: 855-858Crossref PubMed Scopus (32) Google Scholar). Enzymatic Digestion of Flagellin and S-layer Protein—Flagellin or S-layer protein extract (50–200 μg) was digested with either trypsin (Promega, Madison, WI) or Glu-C (Roche Applied Science) at a ratio of 40:1 (protein:enzyme, v/v) in 100 mm ammonium bicarbonate, pH 8.5, at 37 °C overnight. Double digestion was achieved by overnight incubation of the tryptic digest with Glu-C at the same ratio (40:1) at 37 °C. Nano-liquid Chromatography-Electrospray Tandem Mass Spectrometry Analysis—Protein digests were by used mass total using a mass to a The digests μg) were on a using the gradient acid in in The mass was to on and were for the of unusual of of the flagellin tryptic digests was using an with a The digests μg) were on a using the gradient acid at The was at to the were every at The were to on a in of and by using a acid was used as the and containing potential were by of each were into a and using the were by of which the or of a glycan modification. analysis the of to the as they the mass This was achieved by the from to were then by as described This was used to the of glycan linkage and to of the glycan residue mass of the glycan in was achieved by using a of the peptide as mass were then for each glycan the of the unusual glycan of the of the glycan was on a of the J. Mol. Biol. 1996; Google Scholar). were in 100 of ammonium and to at for The were then to in of using and by in the described Flagellin from was used as a This protein is modified with O-linked glycans (12Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). of flagellin protein containing an of glycan were digested with in mm pH flagellin to incubation for h at 37 °C a second of was and the digestion was to for The digest was in to a and to a in the described previously S.L. Ng S.Y. Jarrell K.F. J. Mol. Microbiol. Biotechnol. 2004; 7: 41-51Crossref PubMed Scopus (34) Google Scholar). The from each was in to and by as described were then by NMR of were at °C on at and The purified glycan was and in either for where were The was The pH in for was and and and were for the of the glycan and were as described previously J.R. E. J. Chem. 2002; Scopus Google Scholar, Brisson J.R. Kelly J. Jarrell F. E. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). were then used to in single and for structure and were with to the of an at and respectively. were using with and were processed with the Mass of M. voltae masses of the M. voltae flagellins as by Western with antisera FlaA, FlaB1, or FlaB2, or and The FlaB2 also with FlaB1, and the two proteins have masses of and This is the masses from the FlaA, FlaB1, FlaB2, and FlaB2 is in E. a protein of is detected by FlaB2 to the FlaB2 protein with the signal peptide of acids still attached. This signal peptide is not present on the mature flagellin protein isolated from intact flagella of M. This signal peptide can be from the E. FlaB2 in an in vitro preflagellin peptidase assay as shown in (17Correia J.D. Jarrell K.F. J. Bacteriol. 2000; 182: 855-858Crossref PubMed Scopus (32) Google Scholar). This processed FlaB2 produced in E. coli has an mass of which a in mass from the mature FlaB2 flagellin protein isolated from the of This is of significant post-translational modification of the protein in M. to the M. voltae flagellins with stains such as the periodic acid were D.M. Jarrell K.F. J. Kalmokoff M.L. J. Microbiol. PubMed Scopus Google Scholar). to the intact mass of the respective flagellins by mass spectrometry and have of Flagellin the tryptic digest of the flagellin proteins was by of the be to flagellin tryptic However, a of the were from modified with to be a glycan composed of three residues with masses of 318, 258, and 203 Da, the that the glycan is linked to the the residue is a and that the is the This was by the of the at and the of the at The at and the at that the residue is the trisaccharide. Although it was to the peptide from the mass of the the of the peptide to In these was on the peptide to provide this For analysis of the at observed in the of this peptide as from FlaB2 flagellin was to the four flagellar proteins to as sites of glycosylation as and were on tryptic on Glu-C and on digests of the flagellin This well for FlaB1 and FlaB2, the proteins in the To the of the proteins the same were on intact flagellin preparations that a has been demonstrated previously that the FlaB3 protein is in these preparations (6Bardy S.L. Mori T. Komoriya K. Aizawa S. Jarrell K.F. J. Bacteriol. 2002; 184: 5223-5233Crossref PubMed Scopus (59) Google Scholar). was for FlaB1 FlaB2 and FlaB3 for FlaA were also one These are with the that FlaA is the of the four flagellin of the flagellar filament (6Bardy S.L. Mori T. Komoriya K. Aizawa S. Jarrell K.F. J. Bacteriol. 2002; 184: 5223-5233Crossref PubMed Scopus (59) Google Scholar). In addition, FlaA be to digestion as a consequence of the flagellar filament. of the acid that the glycan well be N-linked to asparagine residues in the eukaryotic sequon In that 14 of the 15 N-linked sequons the The that was the N-linked sequon of the FlaA flagellin However, modified or were detected from this of To the of glycan attachment we on the of the the monosaccharide The that were to and peptide the mass These two were not present in the as shown in In addition, the modified of the on the is the 203 at which the asparagine This of the monosaccharide is N-linked to the asparagine residue of the N-linked sequon to on either of the present in this peptide were To this we of Although the the O-linked glycans from the modification of the M. voltae flagellin was observed not This that the glycan linkage on these flagellin proteins is not This through an residue was for an S-layer peptide of the mass were on the glycan in the in to for the The masses of peptide were used as in a to that described previously (12Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). The masses and the are in I. The masses were Da of the masses of the sugar residues as identified by The mass and for the N-linked sugar that this is a whereas that for the sugar that this is a hexuronic masses of the glycan and in a The and glycan were by The for the was The at of from the of an The of a from this the at and the of a second of acid forms the at and the of a acid This the in indicating that this is a hexuronic This was by NMR The of in also the monosaccharide although not to a The was by the of three from the and is of the of at three As with the of a and of acid that this contains one and one S-layer the analysis of from the intact flagella we identified from other M. voltae in the S-layer are two potential sites of N-linked glycosylation the of this and a tryptic peptide of mass to an 779 Da was found the analysis of flagellin To this protein was also glycosylated we digested a S-layer protein and characterized a of by We were to of the S-layer protein and we were also to show that tryptic peptide which contains one of the N-linked is with the glycan NMR of the Flagellin and NMR were used to the structure of the novel N-linked flagellin which is in The the of three at and respectively. to well of these and are to the glycan and glycan J.R. G. J. T. NMR of Germany2002: Google Scholar, Brisson NMR in and Scholar). and through of the three in the glycan are in The and to and to with the from the at residue and are with of glucose residues in the The of and from to from the and the to and to observed in the of the at are with that of in the The for at in residues and the of a at of the N-linked flagellin with a of of the of residues and to the the glycan to the with a of of the of residues and to the glycan and the and to the glycan structure in The of the glycan in revealed the of four linked to the glycan of the observed in and an at of each the is linked to of residue observed each and a of at that these are of the to was also through the of in and which from the of through to the and the The of the was of of at of all three residues and of residue of the trisaccharide revealed the of a with that of the acid with to the glycan In the were observed the and of with the and at and of residue that this acid is N-linked to residue through an to the of purified to the of not be is also that the of glycan for analysis by NMR with to was achieved through the of a in on the at the of of from the of in of the trisaccharide The shift for the at in single residue a as the proximal N-linked In addition, this shift the linkage of the glycan as N-linked and not of the for residue a with of K. A. PubMed Scopus Google a shift of for of the of residue with of M. T. S. S. T. J. 2003; PubMed Scopus Google Scholar, A. N.A. J. Chem. 2000; Scopus Google was with a at although there were some with the for the and of et al. M. T. S. S. T. J. 2003; PubMed Scopus Google Scholar). The for the to of residue were in with of the monosaccharide S. A. K. H. J. 1987; PubMed Scopus Google Scholar). was a of for the to a at with The and for and of residue were to found in E. PubMed Scopus Google Scholar). the linkage of the glycan was to be This linkage was through the of in and to and to in the the N-linked glycan structure was to be The of the not be to the of However, in structural studies of glycans containing these the has been and of the N-linked flagellin trisaccharide from M. in a demonstrate that the flagellin structural proteins FlaA, and and the S-layer protein of M. voltae are modified with a novel N-linked trisaccharide. These of glycosylation for these proteins in Methanococcus (3Thomas N.A. Bardy S.L. Jarrell K.F. FEMS Microbiol. Rev. 2001; 25: 147-174Crossref PubMed Google Scholar). Although S-layer protein glycosylation has been for a of archaeal species 2Messner P. Schaffer C. Herz W. Falk H. Kirby G.W. Progress in the Chemistry of Organic Natural Products. Springer Verlag, Wien, Germany2002: 51-124Google and J. 2003; PubMed Scopus Google Scholar), prior to this detailed structural characterization of flagellin glycosylation was for a single The flagellin and S-layer proteins of H. salinarum were shown to be with sulfated oligosaccharides F. Paul G. Sumper M. J. Biol. Chem. 1985; 260: 15180-15185Abstract Full Text PDF PubMed Google Scholar, F. W. G. Sumper M. PubMed Scopus Google Scholar). In structural characterization of flagellar glycosylation for a of bacterial species has demonstrated that the flagellar glycan is linked to the protein through to in all In addition, the studies have revealed significant diversity in the of the glycans C. and flagellins are glycosylated with acid and (12Thibault P. Logan S.M. Kelly J.F. Brisson J.R. Ewing C.P. Trust T.J. Guerry P. J. Biol. Chem. 2001; 276: 34862-34870Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar, 13Schirm M. Soo E.C. Aubry A.J. Austin J. Thibault P. Logan S.M. Mol. Microbiol. 2003; 48: 1579-1592Crossref PubMed Scopus (222) Google Scholar), Listeria flagellin is glycosylated with M. Kalmokoff M. Aubry A. Thibault P. Sandoz M. Logan S.M. J. Bacteriol. 2004; 186: 6721-6727Crossref PubMed Scopus (93) Google Scholar), and flagellin is glycosylated with a glycan M. Arora S.K. Verma A. Vinogradov E. Thibault P. Ramphal R. Logan S.M. J. Bacteriol. 2004; 186: 2523-2531Crossref PubMed Scopus (100) Google Scholar). it that although is more common to bacterial flagellar glycosylation systems, be the of for glycosylation of archaeal Although the of this is it is to that the archaeal flagellum is distinct in and of assembly from bacterial The of N-linked glycans on the flagellins of M. voltae and on H. salinarum has for the assembly of archaeal The of N-linked glycosylation the assembly of the glycan on a on the of the followed by a of the glycan to the of the to the This not be for bacterial flagellins that are from the through the flagella structure at the distal tip of the filament Rev. Microbiol. 2003; PubMed Scopus Google Scholar). 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PubMed Scopus Google Scholar), and to the potential of bacterial or from these for as described C. P. 2004; PubMed Scopus (79) Google Scholar). characterization of a of other prokaryotic a M. 2003; PubMed Scopus Google A. Biochim. Biophys. Acta. PubMed Scopus Google and J. Biol. Chem. Full Text PDF PubMed Google has that a asparagine linkage also be although structural for each protein is still characterization of is a and in where limited of protein can be from In the we have the structural of a unique trisaccharide using a combination of mass and NMR was used to and linkage sites from each flagellin We demonstrated that all one of the 15 N-linked sequons from the four flagellin structural proteins were glycosylated with trisaccharide. The that on the FlaA protein was characterized to of the protein in the flagellar preparations and also be In addition, mass of each monosaccharide and structural NMR analysis of of purified using to the detailed linkage and for each The in this analysis to the potential to detailed structural from of glycan from other where is a is on the of respective N- and O-linked glycans found on the of flagellins and S-layer proteins to from and or the basis of The structural characterization of the unique glycan found in these the of glycan and to a of the and of each in with We E. for and T. for with with

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