Oligosaccharides Research Articles

The Core Oligosaccharide and Thioredoxin of Vibrio cholerae Are Necessary for Binding and Propagation of Its Typing Phage VP3

VP3 is a T7-like phage and was used as one of the typing phages in a phage-biotyping scheme that has been used for the typing of Vibrio cholerae O1 biotype El Tor. Here, we studied the receptor and other host genes of V. cholerae necessary for the lytic propagation of VP3. Six mutants resistant to VP3 infection were obtained from the random transposon insertion mutant bank of the sensitive strain N16961. The genes VC0229 and VC0231, which belong to the wav gene cluster encoding the core oligosaccharide (OS) region of lipopolysaccharide, were found to be interrupted by the transposon in five mutants, and the sixth mutant had the transposon inserted between the genes rhlB and trxA, which encode the ATP-dependent RNA helicase RhlB and thioredoxin, respectively. Gene complementation, transcription analysis, and the loss of VP3 sensitivity by the gene deletion mutants confirmed the relationship between VP3 resistance and VC0229, VC0231, and trxA mutation. The product of VP3 gene 44 (gp44) was predicted to be a tail fiber protein. gp44 could bind to the sensitive wild-type strain and the trxA mutant, but not to VC0229 and VC0231 mutants. The results showed that OS is a VP3 receptor on the surface of N16961, thioredoxin of the host strain is involved in the propagation of the phage, and gp44 is the tail fiber protein of VP3. This revealed the first step in the infection mechanism of the T7-like phage VP3 in V. cholerae.

The Fine Structure of Neisseria meningitidis Lipooligosaccharide from the M986 Strain and Three of Its Variants

Neisseria meningitidis is a cause of fatal sepsis and epidemic meningitis. A major virulence factor is cell wall lipooligosaccharide (LOS). The M986 strain has been used extensively in immunological and vaccine research. Yet, the LOS repertoire of this strain is not known. Here we have investigated the LOS structures of M986 and three of its variants OP1, OP2-, and OP2+. This strain and its variants present a series of related LOS families that are increasingly truncated in their listed order. The major structural differences are seen in the lacto-N-neotetraose alpha-chain. The gamma-chain Hep II contains two phosphoethanolamine (PEA) substitutions at C3 and C6/7. These substitutions were seen in all strains except OP2+ where the canonical core Hep II is missing. The PEA disubstitution was present in nearly stoichiometric amounts with only minor amounts of monosubstitution observed, and no glycomers devoid of PEA were seen. This was also the case in LOS with a complete lacto-N-neotetraosyl alpha-chain even though previous reports suggested that the presence of an extended alpha-chain hinders C3 PEA substitution of Hep II. Approximately 50% of gamma-chain GlcNAc was present in its 3-OAc-substituted form. Because Hep II C3 PEA substitution and gamma-chain GlcNAc OAc addition have been reported to negatively interact, the co-existence of these two modifications in these strains is unique. The LOS structures of M986 and three of its variants have been determined, which better defines these strains as tools for immunological and vaccine research.

1,5-α-d-Mannoseptanosides, Ring-Size Isomers That Are Impervious to α-Mannosidase-Catalyzed Hydrolysis

1,5-D-mannoseptanosyl di- and trisaccharide ring-size isomers of the corresponding mannopyranosyl oligosaccharides have been prepared. Remarkably, these compounds show no inhibition of the alpha-mannosidase-catalyzed hydrolysis of p-nitrophenyl-alpha-D-mannopyranoside.

Structural elucidation of the novel core oligosaccharide from LPS of Burkholderia cepacia serogroup O4

Lipopolysaccharide (LPS) is an important virulence factor of Burkholderia cepacia, an opportunistic bacterial pathogen that causes life-threatening disease in cystic fibrosis patients and immunocompromised individuals. B. cepacia LPS comprises an O-specific polysaccharide covalently linked to a core oligosaccharide (OS) which in turn is attached to a lipid A moiety. The complete structure of the LPS core oligosaccharide from B. cepacia serotype O4 was investigated by detailed NMR and mass spectrometry (MS) methods. High- (HMW) and low-molecular-weight (LMW) OSs were obtained by deacylation, dephosphorylation, and reducing-end reduction of the LPS. Glycan and NMR analyses established that both OSs contain a common inner-core structure consisting of D-glucose, L-glycero-D-manno-heptose, D-glycero-D-manno-heptose, 3-deoxy-D-manno-octulsonic acid, and D-glycero-D-talo-2-octulosonic acid. The structure of the LMW OS differed from that of the HMW OS in that it lacks a tetra-rhamnosyl GlcNAc OS extension. These structural conclusions were confirmed by tandem MS analyses of the two OS fractions as well as an OS fraction obtained by alkaline deacylation of the LPS. The location of a phosphoethanolamine substituent in the core region was determined by ESI-MS and methylation analysis of O-deacylated LPS and core OS samples. A polyclonal antibody to B. cepacia serotype O4 core OS was cross-reactive with several other serotypes indicating common structural features.

The Klebsiella pneumoniae O2a Antigen Defines a Second Mechanism for O Antigen ATP-binding Cassette Transporters

An ATP-binding cassette (ABC) transporter-dependent mechanism is responsible for the biosynthesis of polysaccharide O antigens in the lipopolysaccharides of many Gram-negative bacteria. In the Escherichia coli O9a prototype, addition of a non-reducing terminal modification regulates chain length. The terminal residue is an export signal recognized by the nucleotide-binding domain component of the cognate ABC transporter. The Klebsiella pneumoniae O2a antigen lacks a capping residue, and the corresponding nucleotide-binding protein does not contain a separate substrate-binding domain. Unlike the O9a transporter, the O2a transporter can export heterologous O antigens. Export of the O2a antigen is obligatorily coupled to chain elongation, and the O2a transporter is essential for establishing O antigen chain length. The E. coli O9a transporter operates after chain length has been determined. Furthermore biosynthesis and export can be uncoupled. O antigen chain length is a critical element in the ability of lipopolysaccharides to confer resistance to complement-mediated killing. This study establishes that two distinctly different approaches are available for the regulation of O antigen chain length and export via an ABC transporter.

My 30 Years in the Dream of the structural Analyses of the N-linked Oligosaccharides: The Discovery of Glycoamidase A to the Development of the GALAXY Database

30 年前，私はグライコアミダーゼ A という新規酵素を発見した。この酵素はペプチド構造に影響を与えることなく，糖ペプチドから糖鎖部分を切り出すことが可能である。この発見により，私はこの酵素を用いて糖タンパク質に発現している N 型糖鎖の詳細な構造解析を自分自身の研究として行うことを決心した。現在私の開発した HPLC マップ法は N 型糖鎖の構造を解析する非常に強力な方法となっている。本稿では，グリコアミダーゼ A の発見から GALAXY データベースの開発に至るまでの N 型糖鎖の構造解析の夢に生きた私の 30 年に関して述べる。

Fracionamento dos carboidratos pelas equações do Cornell Net Carbohydrate and Protein System de três cultivares de girassol na presença ou não de irrigação

Objetivou-se quantificar as frações de carboidratos pelas equações do Cornell Net Carbohydrate and Protein System (CNCPS) de três cultivares de girassol (Helianthus annuus L.) cultivados na presença ou não de irrigação. A utilização de uma preparação fibrosa, denominada parede celular (PC), nas equações da CNCPS, em substituição à fibra em detergente neutro (FDN) não promoveu diferenças nas frações de carboidratos B1 e C, mas influenciou as frações A e B2. Como os valores da fração B1, obtidos pelo modelo CNCPS foram menores que os teores de amido e pectina determinados em laboratório, supõe-se que a pectina e outros oligossacarídeos da parede celular, solubilizados pela solução de detergente neutro (fibra solúvel), nunca fizeram parte da fração B1, e sim da fração A. Apesar de os carboidratos da fibra solúvel apresentarem elevadas taxas de degradação, não parece adequada a caracterização da fibra solúvel na fração A. Parece mais adequado que a fibra solúvel (que inclui a pectina) seja alocada a uma fração exclusivamente sua, que pode ser a fração B2, e que seja criada uma nova fração, a B3, para os carboidratos digeríveis da parede celular. Assim, a fração B1 seria composta apenas de amido. A equação da fração C, que estima os carboidratos indigeríveis da parede celular, pode ser simplificada, relacionando a fração indigerível ao teor de lignina na matéria seca, e não à FDN isenta de cinzas e proteína, como atualmente utilizado. Esta proposta tem implicações práticas, uma vez que a fração indigerível da parede celular tem sido expressa em relação à FDN, e não na MS, com base no fato de que os efeitos inibitórios da lignina ocorrem sobre os componentes fibrosos da parede celular vegetal, e não sobre o conteúdo celular.

Screening of active saccharides interacted with granulocyte colony-stimulating factor using capillary electrophoresis and their effects on leukemia cells

Objective To screen out the bioactive sulfate saccharides interacted with granulocyte colony-stimulating factor (G-CSF) and provide some scientific data on their pharmacology. Methods The interactions between G-CSF and 15 sulfate saccharides with various chain lengths and extent of sulfation were studied by capillary electrophoresis. Furthermore, the effects of these G-CSF binding saccharides on the proliferation and differentiation of leukemia cell line and NFS-60 were also investigated. Results Except for the hepatosaccharide, degradable carrageenan, Dextran sulfate (DexS) with a low molecular mass the nonsulfated sancharide and alkaline chitosan oligosaccharide, the rest of saccharides had interact with G-CSF.DexS500, λ carrageenan, ι carrageenan, κ carrageenan and low molecular weight heparin-1(LMWH-1)with anticoagulant activity could inhibit the NFS-60 growth. Compared with the absorbency of the control group (100.00±3.00) after these saccharides interacted with the NPS-60 cells, their relative absorbencies were 31.33±2.08,37.67±2.08,45.00±1.20,43.00±2.65 and 74.67±1.52, respectively. Their differences had a statistical significance(t=27.94,26.71,26.42,26.61,10.54,P<0.01). The morphological analytic results showed that the LMWH and lambda carrageenan as well as DexS with a molecular mass of 500 000 induce effectively promyclocyte to transit metamyelocyte. Flow cytometry results showed enhanced expressions of CD11b, CD18 and CD45 after treatment with the LMWH, lambda carrageenan and DexS500,respectively. Cell cycle analysis indicated that the S phase population decreased after treatment with these saccharides. Conclusions Capillary electrophoresis is an effective medicine-screening technique that is used to screen some of these saccharides which could bind to G-CSF. And it is found that the interactions are correlated with the chain length, the extent and the position of sulfation of these saccharides. Key words: Granulocyte colony-stimulating factor; Polysaccharides; Electrophoresis,capillary; Leukemia; Cell line,tumor

Generation of antibacterial oligosaccharides derived from chitin using heterologous endochitinase synthesized inEscherichia coli

To synthesize two heterologous endochitinases in Escherichia coli and demonstrate their potential for applied use in generating antibacterial chitin-derived oligosaccharides (OGS). Heterologous endochitinase genes, chiA Nima and chiA74, were expressed in E. coli. Endochitinases were secreted by the E. coli export machinery and by approximately 20 h maximal chitinolytic activity was observed. The highest chitinolytic activity was observed with ChiA Nima, which produced antibacterial OGS with activities against Enterobacter cloacae, Escherichia coli, Staphylococcus aureus and S. xylosus. It was shown that the export machinery of E. coli is well suited for the secretion of bioactive ChiA74 and ChiA Nima endochitinases, and that the latter can generate antibacterial OGS. Our study suggests that it is feasible to synthesize endochitinases ChiA Nima and ChiA74 codified by E. coli and mass-produce these enzymes in culture supernatants. As signal peptides in native ChiA Nima and ChiA74 were recognized by the protein export molecular apparatus in E. coli, these short peptides could be included as signal sequences for transport in E. coli of other proteins with applied value. This is the first report suggesting that ChiA Nima can be used to produce OGS to control food-borne pathogenic bacteria.

Glycomics analysis of serum: a potential new biomarker for ovarian cancer?

We recently reported the use of matrix-assisted laser desorption ionization (MALDI) Fourier transformation mass spectrometry (FTMS) techniques to identify unique glycan markers in ovarian cancer cell lines which may be biomarkers for diagnosis of ovarian cancer. Glycan markers and CA125 levels are compared in a series of ovarian cancer patients and normal control subjects. Oligosaccharides (OS) were cleaved from the serum glycoproteins and isolated using solid phase extraction. MALDI-FTMS was then used to identify unique mass spectrometry (MS) peaks. Sensitivity, specificity, and the area under the receiver operating characteristic (ROC) curve were calculated to measure the test performance of glycan markers. Sixteen unique OS MS signals were identified in ovarian cancer patient sera. Their additive mass/charge intensities were used to determine their presence or absence. The ovarian cancer patients varied in their disease status, with initial cancer stages ranging from IC to IV. Forty-four of 48 patients had detectable OS signals, with CA125 values between 2 and 17,044. Four patients had undetectable signals and their CA125 ranged between 7 and 10. Twenty-three of 24 control subjects had no detectable glycan markers, with CA125 levels between 10 and 64. Sensitivity and specificity values were determined to be 91.6% and 95.8%, respectively. The area under the ROC curve for all 72 samples was 0.954 (95% CI: 0.896, 1.0) using the glycomics assay, which was superior to CA125 in discriminating between cases and controls. This preliminary study suggests that glycomics profiling may be useful for the detection of ovarian cancer.

Characterization of intact lipopolysaccharides from the Haemophilus influenzae strain RM 118 using electrophoresis‐assisted open‐tubular liquid chromatography‐mass spectrometry

We have applied an electrophoresis-assisted open-tubular LC-MS method for analyzing intact lipopolysaccharides (LPSs) from Haemophilus influenzae strain RM118 (Rd). We were able to obtain structural information on both core oligosaccharides (OSs) and the lipid A moiety including the sialylation, glycylation, and the distribution of fatty acid residues on the disaccharide backbone of lipid A. The fragmentation patterns of sodiated and protonated LPS molecules were investigated for determining the location of sialic acid. It was found that the tandem mass spectra of sodiated ions provided unambiguous evidence of both sialylated lactose and sialylated lacto-N-neotetraose. In contrast, the fragment ions of protonated ions only offered the evidence for the existence of sialylated lacto-N-neotetraose. The lipid A of Gram-negative bacteria, as the principal endotoxic component of LPS, plays a major role in the pathogenesis of bacterial infections. We have previously characterized lipid A species after mild acid hydrolysis of LPS during which lipid A precipitates. In this study, intact LPS was directly introduced to a tandem mass spectrometer. In-source dissociation strategy was employed, followed by multiple-stage MS/MS on the ions originating from the lipid part to obtain structural information. This is the first time that the structure of lipid A of H. influenzae was characterized by MS/MS on intact LPS molecules without any prior chemical modifications. In the same way information on the OS can be obtained by MS/MS by focusing on ions originating from core OS.

Structural elucidation of lipopolysaccharide core oligosaccharides from lic1 and lic1/lic2 mutants of Haemophilus influenzae type b strain Eagan

The structures of lipopolysaccharides (LPSs) of lic1 and lic1/lic2 mutants from Haemophilus influenzae type b strain Eagan (RM153) were investigated using methylation analysis, electrospray ionization - mass spectrometry, and nuclear magnetic resonance spectroscopy on O-deacylated, O- and N-deacylated core oligosaccharide (OS); and deacylated, dephosphorylated, and terminally reduced samples. The backbone OS derived from the major LPS glycoforms were determined to consist of the inner-core triheptosyl unit, L-alpha-D-Hepp-(1-2)-L-alpha-D-Hepp-(1-3)-L-alpha-D-Hepp-(1-, common to all H. influenzae strains investigated to date that is linked to the lipid A region of the molecule via a Kdo residue to which beta-D-Glcp and beta-D-Galp residues are attached in 1,4 and 1,2 linkages to the proximal (HepI) and distal (HepIII) heptose residues, respectively. It was found that the lic1 mutant predominately elaborates the Hex4 LPS glycoforms previously identified in the parent strain where a beta-D-Glcp-(1-4)-alpha-D-Glcp unit is linked in a 1,3 linkage to the central heptose (HepII) of the triheptosyl moiety. The lic1 locus consists of 4 genes (lic1A to lic1D) in a single transcriptional unit that directs phase variable expression of phosphocholine. The lic1A gene is phased off in the RM153 isolate of strain Eagan. LPS from the double mutant, lic1/lic2 had a similar structure to that of lic1 mutant except that there was no chain extension from the central heptose in the inner core (HepII). The lic2 locus consists of 4 genes (lic2A to lic2D). Our structural data were consistent with the proposed function of lic2C, providing the first definitive evidence for its role as the glycosyltransferase required for chain initiation from HepII. The presence of an O-acetyl group at O-3 of the distal heptose (HepIII) was elucidated by 1H NMR on the mild acid liberated core OS samples.

Pharmacological Strategies for Inhibition of Thrombin Activity

For decades, the options for therapeutic anticoagulation were limited to unfractionated heparin (UFH) and vitamin K antagonists (VKA), and their well-known limitations had to be accepted. With the introduction of the various LMWHs, the short-term anticoagulation could be much improved. The heparins delivered the proof of concept that FXa and thrombin represent suitable targets for therapeutic anticoagulation. Consequently, the search for new anticoagulants focus on inhibitors of thrombin or FXa. Apart from the VKA, the anticoagulants presently available or in an advanced stage of development can thus be divided in two classes: One are the glyco-anticoagulants with the natural sulfated glycosaminoglycans (GAGs) (UFH, LMWHs, and danaparoid) and the synthetic oligosaccharides (OS) (fondaparinux, idraparinux, and SR123781A). The other class are the xenobiotic anticoagulants, i.e. proteins and synthetic chemical entities. Die glyco-anticoagulants act partially (GAGs) or exclusively (oligosaccharides) by catalysing antithrombin, whereas the xenobiotic anticoagulants are direct inhibitors of either thrombin or FXa. At present, three direct thrombin inhibitors (DTI) (lepirudin, argatroban, and bivalirudin) are clinically used for limited indications, whereas there is still no direct FXa inhibitor available. The DTI ximelagatran represented the first oral anticoagulant since the introduction of VKA, but was withdrawn due to safety concerns. Among numerous drug candidates in the clinical development, two orally active anticoagulants dabigatran etexilate, a DTI, and rivaroxaban, the direct FXa inhibitor, are in the most advanced stage of development and may allow a paradigm change in anticoagulation in the foreseeable future. This review describes the pharmacological profile of all these anticoagulants.

Oligosaccharide polyester and triterpenoid saponins from the roots of Polygala japonica

An oligosaccharide polyester, 1- O-( E)- p-coumaroyl-(3- O-benzoyl)-β- d-fructofuranosyl-(2 → 1)-[6- O-( E)-feruloyl-β- d-glucopyranosyl-(1 → 2)]-[6- O-acetyl-β- d-glucopyranosyl-(1 → 3)-(4- O-acetyl)-β- d-glucopyranosyl-(1 → 3)]-4- O-[4- O-α- l-rhamnopyranosyl-( E)- p-coumaroyl]-α- d-glucopyranoside (polygalajaponicose I), and four triterpenoid saponins, 3β, 23, 27-trihydroxy-29- O-β- d-glucopyranosyl-(1 → 2)-β- d-glucopyranosyl-olean-12-en-28-oic acid (polygalasaponin XLVII), 3- O-β- d-glucopyranosyl presenegenin 28- O-α- l-rhamnopyranosyl-(1 → 2)-β- d-fucopyranosyl ester (polygalasaponin XLVIII), 3- O-β- d-glucopyranosyl presenegenin 28- O-β- d-galactopyranosyl-(1 → 5)-β- d-apiofuranosyl-(1 → 4)-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-β- d-glucopyranosyl ester (polygalasaponin XLIX) and 2β, 27-dihydroxy-3- O-β- d-glucopyranosyl 11-oxo-olean-12-en-23, 28-dioic acid 28- O-β- d-galactopyranosyl-(1 → 5)-β- d-apiofuranosyl-(1 → 4)-β- d-xylopyranosyl-(1 → 4)-α- l-rhamnopyranosyl-(1 → 2)-β- d-fucopyranosyl ester (polygalasaponin L), in addition to five known compounds have been isolated from the roots of Polygala japonica.

Functional Characterization of MigA and WapR: Putative Rhamnosyltransferases Involved in Outer Core Oligosaccharide Biosynthesis ofPseudomonas aeruginosa

Pseudomonas aeruginosa lipopolysaccharide (LPS) contains two glycoforms of core oligosaccharide (OS); one form is capped with O antigen through an alpha-1,3-linked L-rhamnose (L-Rha), while the other is uncapped and contains an alpha-1,6-linked L-Rha. Two genes in strain PAO1, wapR (PA5000) and migA (PA0705), encode putative glycosyltransferases associated with core biosynthesis. We propose that WapR and MigA are the rhamnosyltransferases responsible for the two linkages of L-Rha to the core. Knockout mutants with mutations in both genes were generated. The wapR mutant produced LPS lacking O antigen, and addition of wapR in trans complemented this defect. The migA mutant produced LPS with a truncated outer core and showed no reactivity to outer core-specific monoclonal antibody (MAb) 5C101. Complementation of this mutant with migA restored reactivity of the LPS to MAb 5C101. Interestingly, LPS from the complemented migA strain was not reactive to MAb 18-19 (specific for the core-plus-one O repeat). This was due to overexpression of MigA in the complemented strain that caused an increase in the proportion of the uncapped core OS, thereby decreasing the amount of the core-plus-one O repeat, indicating that MigA has a regulatory role. The structures of LPS from both mutants were elucidated using nuclear magnetic resonance spectroscopy and mass spectrometry. The capped core of the wapR mutant was found to be truncated and lacked alpha-1,3-L-Rha. In contrast, uncapped core OS from the migA mutant lacked alpha-1,6-L-Rha. These results provide evidence that WapR is the alpha-1,3-rhamnosyltransferase, while MigA is the alpha-1,6-rhamnosyltransferase.

Pharmakologie der Heparine und der direkten Antikoagulanzien

For decades, the options for therapeutic anticoagulation were limited to unfractionated heparin (UFH) and vitamin K antagonists (VKA), and their well-known limitations had to be accepted. With the introduction of the various LMWHs, the short- and medium-term anticoagulation could be much improved, but an alternative to VKA is still missing The heparins provided the proof of concept that FXa and thrombin represent suitable targets for therapeutic anticoagulation. Consequently, the search for new anticoagulants focuses on inhibitors of thrombin (DTI) or FXa (DXI). Apart from the VKA, the anticoagulants presently available or in an advanced stage of development can thus be divided in two classes: One are the glyco-anticoagulants with the natural sulfated glycosaminoglycans (GAGs) (UFH, LMWHs, and danaparoid) and the synthetic oligosaccharides (OS) (fondaparinux, idraparinux, and SR123781A). The other class are the xenobiotic anticoagulants, i.e. proteins and synthetic chemical entities. Die glyco-anticoagulants act partially (GAGs) or exclusively (oligosaccharides) by catalysing antithrombin, whereas the xenobiotic anticoagulants are direct inhibitors of either thrombin or FXa. At present, three parenteral DTI (lepirudin, argatroban, and bivalirudin) and since March 2008 one oral DTI (dabigatran etexilate) are clinically used for limited indications. In September 2008 rivaroxaban has been approved as the first oral DXI. This review describes the development of the anticoagualants as well as the pharmacological profile of the clinically used anticoagualants.

Recognition of Heptoses and the Inner Core of Bacterial Lipopolysaccharides by Surfactant Protein D

Lipopolysaccharides (LPS) of Gram-negative bacteria are important mediators of bacterial virulence that can elicit potent endotoxic effects. Surfactant protein D (SP-D) shows specific interactions with LPS, both in vitro and in vivo. These interactions involve binding of the carbohydrate recognition domain (CRD) to LPS oligosaccharides (OS); however, little is known about the mechanisms of LPS recognition. Recombinant neck+CRDs (NCRDs) provide an opportunity to directly correlate binding interactions with a crystallographic analysis of the binding mechanism. In these studies, we examined the interactions of wild-type and mutant trimeric NCRDs with rough LPS (R-LPS). Although rat NCRDs bound more efficiently than human NCRDs to Escherichia coli J-5 LPS, both proteins exhibited efficient binding to solid-phase Rd2-LPS and to Rd2-LPS aggregates presented in the solution phase. Involvement of residues flanking calcium at the sugar binding site was demonstrated by reciprocal exchange of lysine and arginine at position 343 of rat and human CRDs. The lectin activity of hNCRDs was inhibited by specific heptoses, including l-glycero-alpha-d-manno-heptose (l,d-heptose), but not by 3-deoxy-alpha-d-manno-oct-2-ulosonic acid (Kdo). Crystallographic analysis of the hNCRD demonstrated a novel binding orientation for l,d-heptose, involving the hydroxyl groups of the side chain. Similar binding was observed for a synthetic alpha1-->3-linked heptose disaccharide corresponding to heptoses I and II of the inner core region in many LPS. 7-O-Carbamoyl-l,d-heptose and d-glycero-alpha-d-manno-heptose were bound via ring hydroxyl groups. Interactions with the side chain of inner core heptoses provide a potential mechanism for the recognition of diverse types of LPS by SP-D.

Identification of a novel sugar 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-d-glycero-d-galacto-non-2-ulosonic acid present in the lipooligosaccharide of Vibrio parahaemolyticus O3:K6

A novel sugar, 5,7-diacetamido-8-amino-3,5,7,8,9-pentadeoxy-D-glycero-D-galacto-non-2-ulosonic acid (NonlA), has been identified as a component of the oligosaccharide (OS) isolated from the lipooligosaccharide (LOS) of the emerging strain of Vibrio parahaemolyticus O3:K6 associated with a global pandemic. In the present study we report the identification and characterization of this novel sugar present in the OS of V. parahaemolyticus O3:K6, using chemical analysis, NMR spectroscopy and mass spectrometry.

Role of different moieties from the lipooligosaccharide molecule in biological activities of the Moraxella catarrhalis outer membrane

Lipooligosaccharide (LOS), a major component of the outer membrane of Moraxella catarrhalis, consists of two major moieties: a lipid A and a core oligosaccharide (OS). The core OS can be dissected into a linker and three OS chains. To gain an insight into the biological activities of the LOS molecules of M. catarrhalis, we used a random transposon mutagenesis approach with an LOS specific monoclonal antibody to construct a serotype A O35Elgt3 LOS mutant. MALDI-TOF-MS of de-O-acylated LOS from the mutant and glycosyl composition, linkage, and NMR analysis of its OS indicated that the LOS contained a truncated core OS and consisted of a Glc-Kdo(2) (linker)-lipid A structure. Phenotypic analysis revealed that the mutant was similar to the wild-type strain in its growth rate, toxicity and susceptibility to hydrophobic reagents. However, the mutant was sensitive to bactericidal activity of normal human serum and had a reduced adherence to human epithelial cells. These data, combined with our previous data obtained from mutants which contained only lipid A or lacked LOS, suggest that the complete OS chain moiety of the LOS is important for serum resistance and adherence to epithelial cells, whereas the linker moiety is critical for maintenance of the outer membrane integrity and stability to preserve normal cell growth. Both the lipid A and linker moieties contribute to the LOS toxicity.

Epitope Specificities of the Group Y and W-135 Polysaccharides ofNeisseria meningitidis

Previous studies have identified the length dependency of several polysaccharide (PS) protective epitopes. We have investigated whether meningococcal polysaccharides Y and W-135 possess such epitopes. Oligosaccharides (OSs) consisting of one or more disaccharide repeating units (RU) were derived from the capsular PSs of group Y and W-135 meningococci (GYMP and GWMP, respectively) by mild acid hydrolysis. The relative affinities of anticapsular antibodies binding to derivative OSs of different chain lengths were measured in inhibition enzyme-linked immunosorbent assays. As OS size increased from two to three RU, there was a notable increase in binding inhibition of rabbit anti-group Y antiserum. This pattern of antibody binding inhibition was also observed for rabbit antiserum to group W-135, though the inhibition increase was much more pronounced. In the cases of both OS species, the concentration of inhibiting antigen required to achieve 50% inhibition of rabbit immunoglobulin binding increased progressively as the inhibiting disaccharide chain length increased from 1 RU through greater than 50 RU. These data suggest that antibodies directed against both of these meningococcal PSs recognize conformational epitopes only fully expressed in higher-molecular-weight forms of these antigens.