Conformation of the O-specific polysaccharide of Shigella dysenteriae type 1: molecular modeling shows a helical structure with efficient exposure of the antigenic determinant alpha-L-Rhap-(1-->2)-alpha-D-Galp.

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.

Crystal structure of an anti-carbohydrate antibody directed against Vibrio cholerae O1 in complex with antigen: molecular basis for serotype specificity.

The crystal structure of the murine Fab S-20-4 from a protective anti-cholera Ab specific for the lipopolysaccharide Ag of the Ogawa serotype has been determined in its unliganded form and in complex with synthetic fragments of the Ogawa O-specific polysaccharide (O-SP). The upstream terminal O-SP monosaccharide is shown to be the primary antigenic determinant. Additional perosamine residues protrude outwards from the Ab surface and contribute only marginally to the binding affinity and specificity. A complementary water-excluding hydrophobic interface and five Ab-Ag hydrogen bonds are crucial for carbohydrate recognition. The structure reported here explains the serotype specificity of anti-Ogawa Abs and provides a rational basis toward the development of a synthetic carbohydrate-based anti-cholera vaccine.

Preparation of novel conjugates involving immunomodulating peptidoglycan monomer.

Peptidoglycan monomer, the disaccharide pentapeptide beta-D-Glcp-N-Ac-(1-->4)-D-Murp-N-Ac-L-Ala-D-mesoA2pm- (epsilonN H2)-D-Ala-D-Ala (PGM) is an immunomodulator. PGM and/or its derivative N-tert-butyloxycarbonyl-L-tyrosyl peptidoglycan monomer (Boc-Tyr-PGM) were coupled to two polysaccharides: the glucuronoxylomannan (GXM) from Cryptococcus neoformans, type B, solubilized by ultrasonic irradiation (MW 12-400 kDa) and to the dextran FP 70 (MW 70 kDa). Both polysaccharides were activated by CNBr. Initially, unprotected PGM was coupled via its amino group to GXM. The reactions yielded 42%-52% of the conjugate, containing only 0.18%-0.31% of PGM. In another approach Boc-Tyr-PGM (having its amino group blocked) was reacted via its free carboxyl group. Both CNBr-activated polysaccharides were first coupled to adipic acid dihydrazide (ADH) and then subsequently coupled to Boc-Tyr-PGM. The dextran conjugate (approximately 80% yield ) contained 6.3% of Boc-Tyr-PGM. The isolation of GXM conjugate required several modifications and it was obtained in lower yield (approximately 30%) but contained 13.7% of Boc-Tyr-PGM. Both conjugates were water soluble and apyrogenic and suitable for further testing of biological activity.

Synthesis of tri- and tetrasaccharide fragments of the Shigella dysenteriae type 1 O-antigen deoxygenated and fluorinated at position 3 of the methyl alpha-D-galactopyranoside terminus.

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.

Binding of modified fragments of the Shigella dysenteriae type 1 O-specific polysaccharide to monoclonal IgM 3707 E9 and docking of the immunodeterminant to its modeled Fv.

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.

Synthetic C-oligosaccharides mimic their natural, analogous immunodeterminants in binding to three monoclonal immunoglobulins.

The binding of three monoclonal antigalactan immunoglobulins, IgAs X24, J539 and X44 to their natural haptens methyl beta-D-galactopyranosyl-(1-->6)-beta-D-galactopyranoside, and the corresponding tri- and tetrasaccharides, was compared to the binding of these immunoglobulins with the comparable C-linked oligosaccharide analogues 1-3. The near identity of affinities of the two sets of oligosaccharides indicated the absence of any hydrogen bond involvement by the intersaccharidic oxygen atoms in the natural immunodeterminants.

On the antigenic determinants of the lipopolysaccharides of Vibrio cholerae O:1, serotypes Ogawa and Inaba.

Monoclonal, murine IgG1s S-20-4, A-20-6, and IgA 2D6, directed against Vibrio cholerae O:1 Ogawa-lipopolysaccharide exhibited the same fine specificities and similar affinities for the synthetic methyl alpha-glycosides of the (oligo)saccharide fragments mimicking the Ogawa O-polysaccharide (O-PS). They did not react with the corresponding synthetic fragments of Inaba O-PS. IgG1s S-20-4 and A-20-6 have absolute affinity constants for synthetic Ogawa mono- to hexasaccharides of from approximately 10(5) to approximately 10(6) M-1. For IgG1s S-20-4, A-20-6, and IgA 2D6, the nonreducing terminal residue of Ogawa O-PS is the dominant determinant, accounting for approximately 90% of the maximal binding energy shown by these antibodies. Binding studies of derivatives of the Ogawa monosaccharide and IgGs S-20-4 and A-20-6 revealed that the C-2 O-methyl group fits into a somewhat flexible antibody cavity and that hydrogen bonds involving the oxygen and, respectively, the OH at the 2- and 3-position of the sugar moiety as well as the 2'-position in the amide side chain are required. Monoclonal IgA ZAC-3 and IgG3 I-24-2 are specific for V. cholerae O:1 serotypes Ogawa/Inaba-LPS.1 The former did not show binding with members of either series of the synthetic ligands related to the O-antigens of the Ogawa or Inaba serotypes, in agreement with its reported specificity for the lipid/core region (1). Inhibition studies revealed that the binding of purified IgG3 I-24-2 to Ogawa-LPS might be mediated by a region in the junction of the OPS to the lipid-core region of the LPS. cDNA cloning and analysis of the anti-Ogawa antibodies S-20-4, A-20-6, and 2D6 revealed a very high degree of homology among the heavy chains. Among the light chains, no such homology between S-20-4 and A-20-6 on the one hand, and 2D6 on the other hand, exists. For the anti-Inaba/Ogawa antibodies I-24-2 and ZAC-3, their heavy chains are completely different, with some homology among the light chains.

Recovery of monoclonal antibody from its complex with ligand.

The separation of antibody from its excess and bound ligand is important following affinity chromatography or the use of methods requiring large amounts of antibody, such as microcalorimetry. Using radioactively labeled ligands we show that these separations can be effected by using commercially available, short polyacrylamide size-exclusion columns. By using both low (K alpha = 5 x 10(2) M-1) and medium high-affinity (K alpha = 0.6 x 10(6) M-1) ligands in the presence of antibody, it is shown that the latter system requires more dilute loading concentrations than the former system does in order to achieve acceptable separation. Since the degree of association between a protein and a ligand is solely governed by the affinity constant for the binding equilibrium, these results are applicable to any system represented by this range of binding constants.

Of four murine, anti-Shigella dysenteriae type 1 O-polysaccharide antibodies, three employ V-genes that differ extensively from those of the fourth.

Three murine, monoclonal antibodies, IgM 5286 F2, IgM 5297 C1, and IgG 5338 H4 were generated against Shigella dysenteriae type 1 O-specific polysaccharide (O-SP)-conjugate. They are specific for the O-SP, which is a poly-[alpha-L-rhamnopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-al pha-D-galactopyranosyl-(1-->3)-2-deoxy-2-amino-N-acetyl-alpha-D-glucopyr anosyl]. The VH and VL genes of these antibodies were cloned and their sequences determined. They showed 93% homology, but were quite different to the primary sequence of IgM 3707 E9, of the same O-SP-specificity, previously reported. The fine-specificities of both IgG 5338 H4 and IgM 3707 E9 were for the same disaccharide moiety in the O-SP, while IgMs 5286 F2 and 5297 C1 showed fine-specificity for the entire repeating unit of the O-SP. Therefore, divergent sequences can confer upon antibodies similar-, or even identical-carbohydrate-epitope fine-specificity. In addition, close primary sequence-homology does not preclude differences in antibody fine-specificity.

Synthesis of the methyl alpha-glycosides of some isomalto-oligosaccharides specifically deoxygenated at position C-3.

Methyl alpha-isomaltoside and methyl alpha-isomaltotrioside specifically deoxygenated at position C-3 of various glucopyranosyl units were synthesized by condensation of either 1,6-di-O-acetyl-2,4-di-O-benzyl-3-deoxy-alpha,beta-D-ribo-hexopyranos e (7) or 1,6-di-O-acetyl-2,3,4-tri-O-benzyl-alpha,beta-D-glucopyranose [mediated by silver perchlorate and tin(IV) chloride] with suitably blocked derivatives of methyl alpha-D-glucopyranoside, its 3-deoxy analog (6), or methyl 3'-deoxy alpha-isomaltoside (10), respectively.

Observations on the binding of four anti-carbohydrate monoclonal antibodies to their homologous ligands.

The binding of four monoclonal immunoglobulins, two with specificity for beta(1-->6)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(1-->6)-linked d-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnKa and 1/T, where Ka is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaGu, is virtually independent of T, and the DeltaSu is small when compared with DeltaGu. The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-d-galactopyranoside and the methyl glycoside of beta(1-->6)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans).

Synthesis of the methyl alpha-glycosides of some isomalto-oligosaccharides specifically deoxygenated at position C-4.

Methyl alpha-isomaltoside and methyl alpha-isomaltotrioside specifically deoxygenated at position C-4 of various glucopyranosyl units were synthesized by condensation of either 1,6-di-O-acetyl-2,3-di-O-benzyl-4-deoxy-alpha,beta-D-xylo-hexopyranos e (7) or 1,6-di-O-acetyl-2,3,4-tri-O-benzyl-alpha,beta-D-glucopyranose (10) [mediated by silver perchlorate and tin(IV) chloride] with suitably blocked derivatives of methyl alpha-D-glucopyranoside, its 4-deoxy analog 6, or methyl 4'-deoxy alpha-isomaltoside (13), respectively.

A murine antibody to Shigella dysenteriae type 1 employs V-genes that contain a rearranged codon for the lambda light chain.

The cDNA coding for a hybridoma anti Shigella dysenteriae type 1 antibody (3707 E9) has been cloned, and sequenced. Binding patterns with fragments of the bacterial polysaccharide antigen had already been studied in detail. The VH sequence utilizes the VH441 gene, first identified amongst beta-(1,6)galactan-binding antibodies, while the VL is closely related to the V lambda 1 gene. We found that the VL3707 E9 gene employed a VL-J combinatorial joining leading to a rare Trp-->Leu substitution at position L96.

Synthesis of methyl glycosides of some alpha-isomalto oligosaccharides specifically deoxygenated at position C-2.

Methyl alpha-isomaltoside and methyl alpha-isomaltotrioside analogues specifically deoxygenated at position C-2 of various glucopyranosyl units were synthesized by condensation of either 6-O-acetyl-3-O-benzoyl-4-O-benzyl-1-O-tert-butyldimethylsilyl-2-deoxy- beta-D-arabino-hexopyranose (trimethylsilyl triflate mediated) or 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride (mediated by silver carbonate and silver triflate) with suitably blocked derivatives of methyl alpha-D-glucopyranoside, its 2-deoxy analogue, or methyl 2'-deoxy-alpha-isomaltoside.

Synthesis of specifically monofluorinated ligands related to the O-polysaccharide of Shigella dysenteriae type 1.

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.