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1.
Proc Natl Acad Sci U S A ; 105(29): 9976-81, 2008 Jul 22.
Article in English | MEDLINE | ID: mdl-18621718

ABSTRACT

The anti-LPS IgG mAb F22-4, raised against Shigella flexneri serotype 2a bacteria, protects against homologous, but not heterologous, challenge in an experimental animal model. We report the crystal structures of complexes formed between Fab F22-4 and two synthetic oligosaccharides, a decasaccharide and a pentadecasaccharide that were previously shown to be both immunogenic and antigenic mimics of the S. flexneri serotype 2a O-antigen. F22-4 binds to an epitope contained within two consecutive 2a serotype pentasaccharide repeat units (RU). Six sugar residues from a contiguous nine-residue segment make direct contacts with the antibody, including the nonreducing rhamnose and both branching glucosyl residues from the two RUs. The glucosyl residue, whose position of attachment to the tetrasaccharide backbone of the RU defines the serotype 2a O-antigen, is critical for recognition by F22-4. Although the complete decasaccharide is visible in the electron density maps, the last four pentadecasaccharide residues from the reducing end, which do not contact the antibody, could not be traced. Although considerable mobility in the free oligosaccharides can thus be expected, the conformational similarity between the individual RUs, both within and between the two complexes, suggests that short-range transient ordering to a helical conformation might occur in solution. Although the observed epitope includes the terminal nonreducing residue, binding to internal epitopes within the polysaccharide chain is not precluded. Our results have implications for vaccine development because they suggest that a minimum of two RUs of synthetic serotype 2a oligosaccharide is required for optimal mimicry of O-Ag epitopes.


Subject(s)
Antibodies, Bacterial/chemistry , Antibodies, Monoclonal/chemistry , O Antigens/chemistry , Shigella flexneri/chemistry , Shigella flexneri/immunology , Animals , Antigen-Antibody Complex/chemistry , Bacterial Vaccines/chemistry , Binding Sites, Antibody , Carbohydrate Conformation , Carbohydrate Sequence , Crystallography, X-Ray , Dysentery, Bacillary/immunology , Dysentery, Bacillary/prevention & control , Glycosylation , Models, Molecular , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/immunology , Protein Conformation , Serotyping , Shigella flexneri/classification , Shigella flexneri/pathogenicity
2.
Glycobiology ; 11(11): 945-55, 2001 Nov.
Article in English | MEDLINE | ID: mdl-11744629

ABSTRACT

The O-specific polysaccharide of Shigella dysenteriae type 1, which has the repeating tetrasaccharide unit -->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcNAcp-(1--> (A-B-C-D), is a major virulence factor, and it is believed that antibodies against this polysaccharide confer protection to the host. The conformational properties of fragments of this O-antigen were explored using systematic search with a modified HSEA method (GLYCAN) and with molecular mechanics MM3(96). The results show that the alpha-D-Gal-(1-->3)-alpha-D-GlcNAc linkage adopts two favored conformations, phi/psi approximately equal to -40 degrees /-30 degrees (I) and approximately 15 degrees /30 degrees (II), whereas the other glycosidic linkages only have a single favored phi/psi conformational range. MM3 indicates that the trisaccharide B-C-D and tetrasaccharides containing the B-C-D moiety exist as two different conformers, distinguished by the conformations I and II of the C-D linkage. For the pentasaccharide A-B-C-D-A' and longer fragments, the calculations show preference for the C-D conformation II. These results can explain previously reported nuclear magnetic resonance data. The pentasaccharide in its favored conformation II is sharply bent, with the galactose residue exposed at the vertex. This hairpin conformation of the pentasaccharide was successfully docked with the binding site of a monoclonal IgM antibody (E3707 E9) that had been homology modeled from known crystal structures. For fragments made of repetitive tetrasaccharide units, the hairpin conformation leads to a left-handed helical structure with the galactose residues protruding radially at the helix surface. This arrangement results in a pronounced exposure of the galactose and also the adjacent rhamnose in each repeating unit, which is consistent with the known role of the as alpha-L-Rhap-(1-->2)-alpha-D-Galp moiety as a major antigenic epitope of this O-specific polysaccharide.


Subject(s)
O Antigens/chemistry , Shigella dysenteriae/chemistry , Shigella dysenteriae/immunology , Antibodies, Monoclonal/chemistry , Binding Sites, Antibody , Carbohydrate Conformation , Carbohydrate Sequence , Epitopes/chemistry , In Vitro Techniques , Models, Molecular , Molecular Sequence Data , Thermodynamics
3.
Carbohydr Res ; 311(3): 121-33, 1998 Sep.
Article in English | MEDLINE | ID: mdl-9825517

ABSTRACT

The blockwise synthesis of methyl alpha tri- and tetrasaccharide analogs of the biochemical repeating unit of the Shigella dysenteriae type 1 O-polysaccharide is described. Modifications include deoxygenation and deoxyfluorination at position 3 of the galactopyranoside residue. Methyl 4,6-O-benzylidene-3-deoxy-alpha-D-xylo-hexopyranoside (8) and methyl 4,6-O-benzylidene-3-deoxy-3-fluoro-alpha-D-galactopyranoside (9) were condensed with (2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl)-(1-->3) -2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride to give, after deprotection, the target trisaccharide methyl alpha-L-rhamnopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyranoside and the corresponding fluorinated oligosaccharide. For the tetrasaccharide synthesis, the glycosyl acceptors 8 and 9 were condensed with the temporarily protected (2,4-di-O-benzoyl-3-O-chloroacetyl-alpha-L- rhamnopyranosyl)-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride. Removal of the chloroacetyl group was followed by condensation of the resulting selectively deblocked trisaccharides with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride. Reduction and deprotection then gave the free methyl 2-acetamido-2-deoxy- alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl- (1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyra noside and the fluorinated analog.


Subject(s)
Methylgalactosides/chemistry , O Antigens/chemistry , Shigella dysenteriae/chemistry , Carbohydrate Sequence , Fluorine , Molecular Sequence Data , Oligosaccharides/chemistry , Oxidation-Reduction
4.
Carbohydr Res ; 309(3): 219-26, 1998 Jul.
Article in English | MEDLINE | ID: mdl-9742688

ABSTRACT

The O-specific polysaccharide (O-SP) of Shigella dysenteriae type 1 has been shown by others to have the structure-->3)-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(1-->2)-alp ha-D- Galp-(1-->3)-alpha-D-GlcpNAc-(1-->. We have shown in the past that IgM 3707 E9, an anti S. dysenteriae type 1 O-SP monoclonal antibody, binds specifically to the -alpha-L-Rhap-(1-->2)-alpha-D-Galp-determinant of the polysaccharide. In this report we show that determinant to have hydrogen bonds, necessary for binding to the antibody, involving positions 3, 4 and 6 of the galactopyranosyl residue. The hydroxyl groups of the rhamnopyranosyl moiety of the immunodeterminant appear not to partake in hydrogen-bond interactions with the antibody. A model is presented of the Fv of IgM 3707 E9 based on our previously established cDNA-sequence and two known, highly homologous immunoglobulin crystal structures. The methyl glycoside of the immunodeterminant alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranose is docked to the combining area of the Fv.


Subject(s)
Antigen-Antibody Reactions , Immunodominant Epitopes/immunology , Immunoglobulin Fragments/metabolism , Immunoglobulin M/immunology , O Antigens/immunology , Shigella dysenteriae/immunology , Amino Acid Sequence , Antibodies, Monoclonal , Carbohydrate Sequence , Models, Molecular , Molecular Sequence Data , Monte Carlo Method , Protein Binding
5.
Carbohydr Res ; 274: 209-22, 1995 Sep 08.
Article in English | MEDLINE | ID: mdl-7585707

ABSTRACT

The synthesis of methyl O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranosides specifically deoxygenated at position 2 (31), or 4 (21) of the rhamnopyranosyl residue was accomplished using methyl 3,4,6-tri-O-benzoyl-alpha-D-galactopyranoside (18) as the glycosyl acceptor. Phenyl thionocarbonate activation of the penta-O-benzoylated disaccharide precursor followed by Barton reduction and Zemplén transesterification gave 31, while 21 was obtained via condensation of the deoxygenated monosaccharide donor with 18, and subsequent debenzoylation of the product.


Subject(s)
Disaccharides/chemical synthesis , O Antigens/chemistry , Shigella dysenteriae/chemistry , Carbohydrate Sequence , Disaccharides/chemistry , Galactosides/chemical synthesis , Galactosides/chemistry , Magnetic Resonance Spectroscopy , Molecular Sequence Data , O Antigens/immunology , Rhamnose/chemistry
7.
Carbohydr Res ; 259(1): 21-34, 1994 Jun 02.
Article in English | MEDLINE | ID: mdl-7518745

ABSTRACT

The synthesis is reported of galactopyranose nucleophiles monofluorinated at positions 3, 4, or 6 and protected by 4,6-O-benzylidene, 3,6-di-O-benzyl, or 3,4-O-isopropylidene groups, respectively. The condensation of these nucleophiles with 2,3,4-tri-O-benzoyl-alpha-L-rhamnosyl bromide gave, after deprotection, the disaccharide analogues of methyl O-alpha-L-rhamnopyranosyl-(1-->2)-alpha-D-galactopyranoside, monofluorinated at position 3, 4, or 6 of the galactoside residue.


Subject(s)
Disaccharides/chemical synthesis , Fluorides , Polysaccharides, Bacterial/chemistry , Shigella dysenteriae/chemistry , Carbohydrate Conformation , Carbohydrate Sequence , Disaccharides/chemistry , Fluorine , Galactose , Indicators and Reagents , Ligands , Magnetic Resonance Spectroscopy , Molecular Sequence Data , O Antigens , Optical Rotation , Rhamnose , Shigella dysenteriae/immunology
8.
Carbohydr Res ; 251: 213-32, 1994 Jan 03.
Article in English | MEDLINE | ID: mdl-7511986

ABSTRACT

The title disaccharides were synthesized by condensation of 2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl bromide with suitably protected, deoxygenated derivatives of methyl alpha-D-galactopyranoside. Deoxygenation was achieved via activation of a protected methyl alpha-D-gluco- or galacto-pyranoside with N,N'-thiocarbonyldiimidazole followed by treatment with tributyltin hydride and azobisisobutyronitrile. At position 3, the deoxygenation was more successful when performed with the tri-O-benzoylated precursor, rather than the tri-O-benzylated one. The corresponding nucleophile was obtained by benzylidenation of the methyl 3-deoxy-alpha-D-xylo-hexopyranoside. The preparation of the glycosyl acceptor deoxygenated at position 4 could be pursued starting from derivatives having either the D-galacto or the D-gluco configuration. The pathway involving the former was found superior.


Subject(s)
Disaccharides/chemical synthesis , Epitopes/chemistry , Fucose/analogs & derivatives , Antibodies, Bacterial/immunology , Antigen-Antibody Reactions , Antigens, Bacterial/immunology , Carbohydrate Sequence , Lipopolysaccharides/immunology , Molecular Sequence Data , Shigella dysenteriae/immunology
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