Glycosylation of Pseudomonas aeruginosa 1244 pilin: glycan substrate specificity

The structural similarity between the pilin glycan and the O-antigen of Pseudomonas aeruginosa 1244 suggested that they have a common metabolic origin. Mutants of this organism lacking functional wbpM or wbpL genes synthesized no O-antigen and produced only non-glycosylated pilin. Complementation with plasmids containing functional wbpM or wbpL genes fully restored the ability to produce both O-antigen and glycosylated pilin. Expression of a cosmid clone containing the O-antigen biosynthetic gene cluster from P. aeruginosa PA103 (LPS serotype O11) in P. aeruginosa 1244 (LPS serotype O7) resulted in the production of strain 1244 pili that contained both O7 and O11 antigens. The presence of the O11 repeating unit was confirmed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry. Expression of the O-antigen biosynthesis cluster from Escherichia coli O157:H7 in strain 1244 resulted in the production of pilin that contained both the endogenous Pseudomonas as well as the Escherichia O157 O-antigens. A role for pilO in the glycosylation of pilin in P. aeruginosa is evident as the cloned pilAO operon produced glycosylated strain 1244 pilin in eight heterologous P. aeruginosa strains. Removal of the pilO gene resulted in the production of unmodified strain 1244 pilin. These results show that the pilin glycan of P. aeruginosa 1244 is a product of the O-antigen biosynthetic pathway. In addition, the structural diversity of the O-antigens used by the 1244 pilin glycosylation apparatus indicates that the glycan substrate specificity of this reaction is extremely low.     

Flagellin glycans from two pathovars of Pseudomonas syringae contain rhamnose in D and L configurations in different ratios and modified 4-amino-4,6-dideoxyglucose

Flagellins from Pseudomonas syringae pv. glycinea race 4 and Pseudomonas syringae pv. tabaci 6605 have been found to be glycosylated. Glycosylation of flagellin is essential for bacterial virulence and is also involved in the determination of host specificity. Flagellin glycans from both pathovars were characterized, and common sites of glycosylation were identified on six serine residues (positions 143, 164, 176, 183, 193, and 201). The structure of the glycan at serine 201 (S201) of flagellin from each pathovar was determined by sugar composition analysis, mass spectrometry, and (1)H and (13)C nuclear magnetic resonance spectroscopy. These analyses showed that the S201 glycans from both pathovars were composed of a common unique trisaccharide consisting of two rhamnosyl (Rha) residues and one modified 4-amino-4,6-dideoxyglucosyl (Qui4N) residue, beta-D-Quip4N(3-hydroxy-1-oxobutyl)2Me-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap. Furthermore, mass analysis suggests that the glycans on each of the six serine residues are composed of similar trisaccharide units. Determination of the enantiomeric ratio of Rha from the flagellin proteins showed that flagellin from P. syringae pv. tabaci 6605 consisted solely of L-Rha, whereas P. syringae pv. glycinea race 4 flagellin contained both L-Rha and D-Rha at a molar ratio of about 4:1. Taking these findings together with those from our previous study, we conclude that these flagellin glycan structures may be important for the virulence and host specificity of P. syringae.     

Structural analysis of the core region of lipopolysaccharides from Proteus mirabilis serotypes O6, O48 and O57.

The structure of lipid A core region of the lipopolysaccharides (LPS) from Proteus mirabilis serotypes O6, O57 and O48 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of LPS: [see text for structure]. Incomplete substitutions are indicated by bold italic type. All sugars are present in pyranose form, alpha-Hep is the residue of L-glycero-alpha-D-manno-Hep, alpha-DD-Hep is the residue of D-glycero-alpha-D-manno-Hep, L-Ara4N is 4-amino-4-deoxy-L-arabinose, Qui4NAlaAla is the residue of 4-N-(L-alanyl-L-alanyl)-4-amino-4,6-dideoxyglucose. All sugars except L-Ara4N have D-configuration. beta-GalA* is partially present in the form of amide with 1,4-diaminobutane (putrescine)-HN(CH2)4NH2 or spermidine-HN(CH2)3NH(CH2)4NH2.     

The structure of the carbohydrate backbone of the core-lipid A region of the lipopolysaccharide from Proteus mirabilis serotype O28

The following structure of the lipid A-core region of the lipopolysaccharide (LPS) from Proteus mirabilis serotype O28 was determined using NMR, MS, and chemical analysis of the core oligosaccharide, obtained by mild acid hydrolysis of LPS, and of the products of alkaline deacylation of the LPS: carbohydrate sequence [see text] where S = beta-GalALys (amide of beta-D-galactopyranosyluronic acid with the alpha-amino group of L-lysine) or beta-GalALys4PEtN are present in the ratio of approximately 1:1. beta-GalA and Ara4N (indicated by bold italics) are present in non stoichometric amount. All sugars are present in the pyranose form and all except L-Ara4N have the D configuration.     

Pseudomonas aeruginosa 1244 pilin glycosylation: glycan substrate recognition

The pilin of Pseudomonas aeruginosa 1244 is glycosylated with an oligosaccharide that is structurally identical to the O-antigen repeating unit of this organism. Concordantly, the metabolic source of the pilin glycan is the O-antigen biosynthetic pathway. The present study was conducted to investigate glycan substrate recognition in the 1244 pilin glycosylation reaction. Comparative structural analysis of O subunits that had been previously shown to be compatible with the 1244 glycosylation machinery revealed similarities among sugars at the presumed reducing termini of these oligosaccharides. We therefore hypothesized that the glycosylation substrate was within the sugar at the reducing end of the glycan precursor. Since much is known of PA103 O-antigen genetics and because the sugars at the reducing termini of the O7 (strain 1244) and O11 (strain PA103) are identical (beta-N-acetyl fucosamine), we utilized PA103 and strains that express lipopolysaccharide (LPS) with a truncated O-antigen subunit to test our hypothesis. LPS from a strain mutated in the wbjE gene produced an incomplete O subunit, consisting only of the monosaccharide at the reducing end (beta-d-N-acetyl fucosamine), indicating that this moiety contained substrate recognition elements for WaaL. Expression of pilAO(1244) in PA103 wbjE::aacC1, followed by Western blotting of extracts of these cells, indicated that pilin produced has been modified by the addition of material consistent with a single N-acetyl fucosamine. This was confirmed by analyzing endopeptidase-treated pilin by mass spectrometry. These data suggest that the pilin glycosylation substrate recognition features lie within the reducing-end moiety of the O repeat and that structures of the remaining sugars are irrelevant.     

Structural analysis of the lipopolysaccharide core of a rough, cystic fibrosis isolate of Pseudomonas aeruginosa.

Lipopolysaccharide (LPS) expressed by isolates of Pseudomonas aeruginosa from cystic fibrosis patients lacks the O-polysaccharide chain but the degree to which the rest of the molecule changes has not been determined. We analyzed, for the first time, the core structure of an LPS from a rough, cystic fibrosis isolate of P. aeruginosa. The products of mild acid hydrolysis and strong alkaline degradation of the LPS were studied by ESI MS, MALDI MS, and NMR spectroscopy. The following structure was determined for the highest-phosphorylated core-lipid A backbone oligosaccharide isolated after alkaline deacylation of the LPS: [structure: see text] where Kdo and Hep are 3-deoxy-D-manno-octulosonic acid and L-glycero-D-manno-heptose, respectively; all sugars are in the pyranose form and have the D configuration unless stated otherwise. The outer core region occurs as two isomeric glycoforms differing in the position of rhamnose (Rha). The inner core region carries four phosphorylation sites at two Hep residues, HepI being predominantly bisphosphorylated and HepII monophosphorylated. In the intact LPS, both Hep residues carry monophosphate and diphosphate groups in nonstoichiometric quantities, GalN is N-acylated by an L-alanyl group, HepII is 7-O-carbamoylated, and the outer core region is nonstoichiometrically O-acetylated at four sites. Therefore, the switch to the LPS-rough phenotype in cystic fibrosis isolates of P. aeruginosa is not accompanied by losses of core monosaccharide, phosphate or acyl components. The exact positions of the O-acetyl groups and the role of the previously undescribed O-acetylation in the LPS core of P. aeruginosa remain to be determined.     

Glycosylation of Pseudomonas aeruginosa strain Pa5196 type IV pilins with mycobacterium-like alpha-1,5-linked d-Araf oligosaccharides

Pseudomonas aeruginosa is a gram-negative bacterium that uses polar type IV pili for adherence to various materials and for rapid colonization of surfaces via twitching motility. Within the P. aeruginosa species, five distinct alleles encoding variants of the structural subunit PilA varying in amino acid sequence, length, and presence of posttranslational modifications have been identified. In this work, a combination of mass spectrometry and nuclear magnetic resonance spectroscopy was used to identify a novel glycan modification on the pilins of the group IV strain Pa5196. Group IV pilins continued to be modified in a lipopolysaccharide (wbpM) mutant of Pa5196, showing that, unlike group I strains, the pilins of group IV are not modified with the O-antigen unit of the background strain. Instead, the pilin glycan was determined to be an unusual homo-oligomer of alpha-1,5-linked d-arabinofuranose (d-Araf). This sugar is uncommon in prokaryotes, occurring mainly in the cell wall arabinogalactan and lipoarabinomannan (LAM) polymers of mycobacteria, including Mycobacterium tuberculosis and Mycobacterium leprae. Antibodies raised against M. tuberculosis LAM specifically identified the glycosylated pilins from Pa5196, confirming that the glycan is antigenically, as well as chemically, identical to those of Mycobacterium. P. aeruginosa Pa5196, a rapidly growing strain of low virulence that expresses large amounts of glycosylated type IV pilins on its surface, represents a genetically tractable model system for elucidation of alternate pathways for biosynthesis of d-Araf and its polymerization into mycobacterium-like alpha-1,5-linked oligosaccharides.     

Structural determination of the O-antigenic polysaccharide from the Shiga toxin-producing Escherichia coli O171

The structure of the O-antigenic part of the lipopolysaccharide (LPS) obtained from the verotoxin-producing Escherichia coli O171 has been determined. (1)H and (13)C NMR spectroscopy techniques in combination with component analysis were used to elucidate the O-antigen structure of O-deacylated LPS. Subsequent NMR analysis of the native LPS revealed acetylation at O-7/O-9 of the sialic acid residue. The sequence of sugars was determined by inter-residue correlations in (1)H,(1)H-NOESY and (1)H,(13)C-heteronuclear multiple-bond correlation spectra. The O-antigen is composed of pentasaccharide repeating units with one equivalent of O-acetyl groups distributed over two positions: -->4)-alpha-Neu5Ac7,9Ac-(2-->6)-beta-D-Galp-(1-->6)-beta-DGlcp-->(1-->3)-beta-D-Galp-(1-->3)-beta-D-GalpNAc-(1--> Based on biosynthetic considerations, this should also be the biological repeating unit.     

Structural analysis of oligosaccharides from lipopolysaccharide (LPS) of Escherichia coli K12 strain W3100 reveals a link between inner and outer core LPS biosynthesis

Lipopolysaccharide (LPS) from Escherichia coli K12 W3100 is known to contain several glycoforms, and the basic structure has been investigated previously by methylation analyses (Holst, O. (1999) in Endotoxin in Health and Disease (Brade, H., Opal, S. M., Vogel, S. N., and Morrison, D., eds) pp. 115-154; Marcel Dekker, Inc., New York). In order to reveal dependences of gene activity and LPS structure, we have now determined the composition of de-O-acylated LPS by electrospray ionization-Fourier transform ion cyclotron-mass spectrometry (ESI-FT-MS) and identified 11 different LPS molecules. We have isolated the major glycoforms after de-O- and de-N-acylation and obtained four oligosaccharides that differed in their carbohydrate structure and phosphate substitution. The main oligosaccharide accounted for approximately 70% of the total and had a molecular mass of 2516 Da according to ESI-FT-MS. The dodecasaccharide structure (glycoform I) as determined by NMR was consistent with MS and compositional analysis. One minor oligosaccharide (5%) of the same carbohydrate structure did not contain the 4'-phosphate of the lipid A. Two oligosaccharides contained the same phosphate substitution but differed in their carbohydrate structure, one (5%) which contained an additional beta-D-GlcN in 1-->7 linkage on a terminal heptose residue (glycoform II) which was N-acetylated in LPS. A minor amount of a molecule lacking the terminal L-alpha-D-Hep in the outer core but otherwise identical to the major oligosaccharide (glycoform III) could only be identified by ESI-FT-MS of the de-O-acylated LPS. The other oligosaccharide (20%) contained an alpha-Kdo-(2-->4)-[alpha-l-Rha-(1-->5)]-alpha-Kdo-(2-->4)-alpha-Kdo branched tetrasaccharide connected to the lipid A (glycoform IV). This novel inner core structure was accompanied by a truncation of the outer core in which the terminal disaccharide L-alpha-D-Hep-(1-->6)-alpha-D-Glc was missing. The latter structure was identified for the first time in LPS and revealed that changes in the inner core structure may be accompanied by structural changes in the outer core.     

Structural analysis of the lipopolysaccharide from Chlamydia trachomatis serotype L2.

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 x 10(4) cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594-10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.     

Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50.

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro-inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg-gingipains of P. gingivalis are post-translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O-polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF-COSY, TOCSY, NOESY, ROESY, 1H-13C HSQC and 1H-31P HXTOCSY) and GC-MS data. These data allowed us to conclude that the O-polysaccharide is built up of the tetrasaccharide repeating sequence: -->6)-alpha-D-Glcp-(1-->4)-alpha-L-Rhap-(1-->3)-beta-D-GalNAc-(1-->3)-alpha-D-Galp-(1--> and carries a monophosphoethanolamine residue at position C-2 of the alpha-rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O-polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.     

Pilin glycosylation in Neisseria meningitidis occurs by a similar pathway to wzy-dependent O-antigen biosynthesis in Escherichia coli

Pili (type IV fimbriae) of Neisseria meningitidis are glycosylated by the addition of O-linked sugars. Recent work has shown that PglF, a protein with homology to O-antigen 'flippases', is required for the biosynthesis of the pilin-linked glycan and suggests pilin glycosylation occurs in a manner analogous to the wzy-dependent addition of O-antigen to the core-LPS. O-Antigen ligases are crucial in this pathway for the transfer of undecraprenol-linked sugars to the LPS-core in Gram-negative bacteria. An O-antigen ligase homologue, pglL, was identified in N. meningitidis. PglL mutants showed no change in LPS phenotypes but did show loss of pilin glycosylation, confirming PglL is essential for pilin O-linked glycosylation in N. meningitidis.     

A novel type of carbohydrate-protein linkage region in the tyrosine-bound S-layer glycan of Thermoanaerobacterium thermosaccharolyticum D120-70.

The surface-layer (S-layer) protein of Thermoanaerobacterium thermosaccharolyticum D120-70 contains glycosidically linked glycan chains with the repeating unit structure -->4)[alpha-D-Galp-(1-->2)]-alpha-L-Rhap-(1-->3)[beta-D-Glcp-(1--> 6)] -beta-D-Manp-(1-->4)-alpha-L-Rhap-(1-->3)-alpha-D-Glcp-(1--> . After proteolytic degradation of the S-layer glycoprotein, three glycopeptide pools were isolated, which were analyzed for their carbohydrate and amino-acid compositions. In all three pools, tyrosine was identified as the amino-acid constituent, and the carbohydrate compositions corresponded to the above structure. Native polysaccharide PAGE showed the specific heterogeneity of each pool. For examination of the carbohydrate-protein linkage region, the S-layer glycan chain was partially hydrolyzed with trifluoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion-edited difference experiment, showed the O-glycosidic linkage region beta-D-glucopyranose-->O-tyrosine. No evidence was found of additional sugars originating from a putative core region between the glycan repeating units and the S-layer polypeptide. For the determination of chain-length variability in the S-layer glycan, the different glycopeptide pools were investigated by matrix-assisted laser desorption ionization-time of flight mass spectrometry, revealing that the degree of polymerization of the S-layer glycan repeats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no core structure is present and thus supports the data from the NMR spectroscopy analyses. This is the first observation of a bacterial S-layer glycan without a core region connecting the carbohydrate moiety with the polypeptide portion.     

Positions of disulfide bonds and N-glycosylation site in juvenile hormone binding protein

The juvenile hormone binding protein (JHBP) from Galleria mellonella hemolymph is a glycoprotein composed of 225 amino acid residues. It contains four Cys residues forming two disulfide bridges. In this study, the topography of the disulfide bonds as well as the site of glycan attachment in the JHBP molecule from G. mellonella was determined, using electrospray mass spectrometry. The MS analysis was performed on tryptic digests of JHBP. Our results show that the disulfide bridges link Cys10 and Cys17, and Cys151 and Cys195. Of the two potential N-glycosylation sites in JHBP, Asn4, and Asn94, only Asn94 is glycosylated. This site of glycosylation is also found in the fully biologically active recombinant JHBP expressed in the yeast Pichia pastoris.     

Isolation and structural characterization of an R-form lipopolysaccharide from Yersinia enterocolitica serotype O:8.

The lipopolysaccharide (LPS) of strain 8081-c-R2, a spontaneous R-mutant of Yersinia enterocolitica serotype O:8, was isolated using extraction with phenol/chloroform/light petroleum. Its compositional analysis indicated the presence of D-GlcN, D-Glc, L-glycero-D-manno- and D-glycero-D-manno-heptose, 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) and phosphate. From deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide, three oligosaccharides (1-3) were isolated using high-performance anion-exchange chromatography, the structures of which were determined by compositional analysis and one- and two-dimensional NMR spectroscopy as [carbohydrate structure see text] in which all sugars are pyranoses, and R and R' represent beta-D-Glc (in 1 and 2) and beta-D-GlcN (in 1 only), respectively. D-alpha-D-Hep is D-glycero-alpha-D-manno-heptose, L-alpha-D-Hep is L-glycero-alpha-D-manno-heptose, Kdo is 3-deoxy-D-manno-oct-2-ulosonic acid, and P is phosphate. The liberated lipid A was analyzed by compositional analyses and MALDI-TOF MS. Its beta-D-GlcN4P-(1-->6)-alpha-D-GlcN-1-->P backbone is mainly tetra-acylated with two amide- and one ester-linked (at O3 of the reducing GlcN) (R)-3-hydroxytetradecanoic acid residues, and one tetradecanoic acid that is attached to the 3-OH group of the amide-linked (R)-3-hydroxytetradecanoic acid of the nonreducing GlcN. Additionally, small amounts of tri- and hexa-acylated lipid A species occur.     

The structure of the core region of the lipopolysaccharide from Klebsiella pneumoniae O3. 3-deoxy-alpha-D-manno-octulosonic acid (alpha-Kdo) residue in the outer part of the core, a common structural element of Klebsiella pneumoniae O1, O2, O3, O4, O5, O8, and O12 lipopolysaccharides.

The structure of lipid A-core region of the lipopolysaccharide (LPS) from Klebsiella pneumoniae serotype O3 was determined using NMR, MS and chemical analysis of the oligosaccharides, obtained by mild acid hydrolysis, alkaline deacylation, and deamination of the LPS: [carbohydrate structure see text] where P is H or alpha-Hep; J is H or beta-GalA; R is H or P (in the deacylated oligosaccharides).Screening of the LPS from K. pneumoniae O1, O2, O4, O5, O8, and O12 using deamination showed that they also contain alpha-Hep-(1-->4)-alpha-Kdo-(2-->6)-GlcN and alpha-Kdo-(2-->6)-GlcN fragments.     

Novel globoside-like oligosaccharide expression patterns in nontypeable Haemophilus influenzae lipopolysaccharide

We report the novel pattern of lipopolysaccharide (LPS) expressed by two disease-associated nontypeable Haemophilus influenzae strains, 1268 and 1200. The strains express the common structural motifs of H. influenzae; globotetraose [beta-d-GalpNAc-(1-->3)-alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and its truncated versions globoside [alpha-d-Galp-(1-->4)-beta-d-Galp-(1-->4)-beta-d-Glcp] and lactose [beta-d-Galp-(1-->4)-beta-d-Glcp] linked to the terminal heptose (HepIII) and the corresponding structures with an alpha-d-Glcp as the reducing sugar linked to the middle heptose (HepII) in the same LPS molecule. Previously these motifs had been found linked only to either the proximal heptose (HepI) or HepIII of the triheptosyl inner-core moiety l-alpha-d-Hepp-(1-->2)-[PEtn-->6]-l-alpha-d-Hepp-(1-->3)-l-alpha-d-Hepp-(1-->5)-[PPEtn-->4]-alpha-Kdo-(2-->6)-lipid A. This novel finding was obtained by structural studies of LPS using NMR techniques and ESI-MS on O-deacylated LPS and core oligosaccharide material, as well as electrospray ionization-multiple-step tandem mass spectrometry on permethylated dephosphorylated oligosaccharide material. A lpsA mutant of strain 1268 expressed LPS of reduced complexity that facilitated unambiguous structural determination. Using capillary electrophoresis-ESI-MS/MS we identified sialylated glycoforms that included sialyllactose as an extension from HepII, this is a further novel finding for H. influenzae LPS. In addition, each LPS was found to carry phosphocholine and O-linked glycine. Nontypeable H. influenzae strain 1200 expressed identical LPS structures to 1268 with the difference that strain 1200 LPS had acetates substituting HepIII, whereas strain 1268 LPS has glycine at the same position.     

Diversity in prokaryotic glycosylation: an archaeal-derived N-linked glycan contains legionaminic acid

VP4, the major structural protein of the haloarchaeal pleomorphic virus, HRPV‐1, is glycosylated. To define the glycan structure attached to this protein, oligosaccharides released by β‐elimination were analysed by mass spectrometry and nuclear magnetic resonance spectroscopy. Such analyses showed that the major VP4‐derived glycan is a pentasaccharide comprising glucose, glucuronic acid, mannose, sulphated glucuronic acid and a terminal 5‐N‐formyl‐legionaminic acid residue. This is the first observation of legionaminic acid, a sialic acid‐like sugar, in an archaeal‐derived glycan structure. The importance of this residue for viral infection was demonstrated upon incubation with N‐acetylneuraminic acid, a similar monosaccharide. Such treatment reduced progeny virus production by half 4 h post infection. LC‐ESI/MS analysis confirmed the presence of pentasaccharide precursors on two different VP4‐derived peptides bearing the N‐glycosylation signal, NTT. The same sites modified by the native host, Halorubrum sp. strain PV6, were also recognized by the Haloferax volcanii N‐glycosylation apparatus, as determined by LC‐ESI/MS of heterologously expressed VP4. Here, however, the N‐linked pentasaccharide was the same as shown to decorate the S‐layer glycoprotein in this species. Hence, N‐glycosylation of the haloarchaeal viral protein, VP4, is host‐specific. These results thus present additional examples of archaeal N‐glycosylation diversity and show the ability of Archaea to modify heterologously expressed proteins.     

Structural characterization of the O-antigenic polysaccharide of the lipopolysaccharide from Rhizobium etli strain CE3. A unique O-acetylated glycan of discrete size, containing 3-O-methyl-6-deoxy-L-talose and 2,3,4-tri-O-,methyl-l fucose

The O-antigenic polysaccharide of the Rhizobium etli CE3 lipopolysaccharide (LPS) was structurally characterized using chemical degradations (Smith degradation and beta-elimination of uronosyl residues) in combination with alkylation analysis, electrospray, and matrix-assisted laser desorption ionization-time of flight mass spectrometry, tandem mass spectrometry, and (1)H COSY and TOCSY nuclear magnetic resonance spectroscopy analyses of the native polysaccharide and the derived oligosaccharides. The polysaccharide was found to be a unique, relatively low molecular weight glycan having a fairly discrete size, with surprisingly little variation in the number of repeating units (degree of polymerization = 5). The polysaccharide is O-acetylated and contains a variety of O-methylated glycosyl residues, rendering the native glycan somewhat hydrophobic. The molecular mass of the major de-O-acetylated species, including the reducing end 3-deoxy-d-manno-2-octulosonic acid (Kdo) residue, is 3330 Da. The polysaccharide is comprised of a trisaccharide repeating unit having the structure -->4)-alpha-d-GlcpA-(1-->4)-[alpha-3-O-Me-6-deoxy-Talp-(1--> 3)]-alpha -l-Fucp-(1-->. The nonreducing end of the glycan is terminated with the capping sequence alpha-2,3, 4-tri-O-Me-Fucp-(1-->4)-alpha-d-GlcpA-(1-->, and the reducing end of the molecule consists of the non-repeating sequence -->3)-alpha-l-Fucp-(1-->3)-beta-d-Manp-(1-->3)-beta-QuiNA cp-(1-->4)-a lpha-Kdop-(2-->, where QuiNAc is N-acetylquinovosamine (2-N-acetamido-2,6-dideoxyglucose). The reducing end Kdo residue links the O-chain polysaccharide to the core region oligosaccharide, resulting in a unique location for a Kdo residue in LPS, removed four residues distally from the lipid A moiety. Structural heterogeneity in the O-chain arises mainly from the O-acetyl and O-methyl substitution. Methylation analysis using trideuteriomethyl iodide indicates that a portion of the 2,3,4-tri-O-methylfucosyl capping residues, typically 15%, are replaced with 2-O-methyl- and/or 2,3-di-O-methylfucosyl residues. In addition, approximately 25% of the 3,4-linked branching fucosyl residues and 10% of the 3-linked fucosyl residues are 2-O-methylated. A majority of the glucuronosyl residues are methyl-esterified at C-6. These unique structural features may be significant in the infection process.     

The structure of the carbohydrate backbone of the rough type lipopolysaccharides from Proteus penneri strains 12, 13, 37 and 44

The following structure of the lipid A-core backbone of the rough type lipopolysaccharides (LPS) from Proteus penneri strains 12, 13, 37, and 44 was determined using NMR and mass spectroscopy and chemical analysis of the oligosaccharides obtained by mild-acid hydrolysis, alkaline O,N-deacylation, O-deacylation with hydrazine, and deamination of the LPSs:where K=H, R=PEtN, R(1)=alpha-Hep-(1-->2)-alpha-DDHep, and R(2)=alpha-GalN (strains 12 and 13) or beta-GlcNAc-(1-->4)-alpha-GlcN (strains 37 and 44). LPS from each strain contained several structural variants. LPS from strain 12 contained a variant with R(1)=alpha-DDHep, whereas LPS from strains 13, 37, and 44 contained structures with K=amide of beta-GalA with putrescine or spermidine. The phosphate group at O-1 of the alpha-GlcN residue in the lipid part was partially substituted with Ara4N.