RESUMEN
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.
Asunto(s)
Antígenos O/química , Shigella dysenteriae/química , Shigella dysenteriae/inmunología , Anticuerpos Monoclonales/química , Sitios de Unión de Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Epítopos/química , Técnicas In Vitro , Modelos Moleculares , Datos de Secuencia Molecular , TermodinámicaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Antígenos Bacterianos/inmunología , Lipopolisacáridos/inmunología , Vibrio cholerae/inmunología , Animales , Anticuerpos Antibacterianos/inmunología , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Reacciones Antígeno-Anticuerpo/inmunología , Secuencia de Carbohidratos , Cristalografía por Rayos X , Fragmentos Fab de Inmunoglobulinas/química , Fragmentos Fab de Inmunoglobulinas/inmunología , Lipopolisacáridos/química , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Serotipificación , Relación Estructura-ActividadRESUMEN
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.
Asunto(s)
Adyuvantes Inmunológicos/química , Peptidoglicano/química , Conformación de Carbohidratos , Secuencia de Carbohidratos , Cryptococcus neoformans/química , Datos de Secuencia Molecular , Polisacáridos/químicaRESUMEN
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.
Asunto(s)
Metilgalactósidos/química , Antígenos O/química , Shigella dysenteriae/química , Secuencia de Carbohidratos , Flúor , Datos de Secuencia Molecular , Oligosacáridos/química , Oxidación-ReducciónRESUMEN
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.
Asunto(s)
Reacciones Antígeno-Anticuerpo , Epítopos Inmunodominantes/inmunología , Fragmentos de Inmunoglobulinas/metabolismo , Inmunoglobulina M/inmunología , Antígenos O/inmunología , Shigella dysenteriae/inmunología , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Secuencia de Carbohidratos , Modelos Moleculares , Datos de Secuencia Molecular , Método de Montecarlo , Unión ProteicaRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/metabolismo , Inmunoglobulina A/metabolismo , Imitación Molecular , Oligosacáridos/metabolismo , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos , Secuencia de Carbohidratos , Haptenos/inmunología , Enlace de Hidrógeno , Inmunoglobulina A/inmunología , Datos de Secuencia Molecular , Oligosacáridos/química , Oligosacáridos/inmunologíaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Especificidad de Anticuerpos , Antígenos O/inmunología , Vibrio cholerae/inmunología , Secuencia de Aminoácidos , Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/genética , Unión Competitiva , Clonación Molecular , Epítopos , Inmunoglobulina G/genética , Inmunoglobulina G/inmunología , Ligandos , Datos de Secuencia Molecular , Monosacáridos/inmunología , Oligosacáridos/inmunología , Especificidad de la Especie , Vibrio cholerae/clasificaciónAsunto(s)
Anticuerpos/química , Afinidad de Anticuerpos , Anticuerpos/inmunología , Fluorescencia , LigandosRESUMEN
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.
Asunto(s)
Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Monoclonales/metabolismo , Cromatografía Liquida/métodos , Afinidad de Anticuerpos , Secuencia de Carbohidratos , Cromatografía Liquida/instrumentación , Galactanos/inmunología , Inmunoglobulina A/aislamiento & purificación , Inmunoglobulina A/metabolismo , Ligandos , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/genética , Genes de Inmunoglobulinas/inmunología , Región Variable de Inmunoglobulina/genética , Antígenos O/inmunología , Polisacáridos Bacterianos/inmunología , Homología de Secuencia de Aminoácido , Shigella dysenteriae/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/química , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/genética , Unión Competitiva/inmunología , Secuencia de Carbohidratos , Cadenas Pesadas de Inmunoglobulina/química , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas Ligeras de Inmunoglobulina/química , Cadenas Ligeras de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/química , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia MolecularRESUMEN
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.
Asunto(s)
Disacáridos/síntesis química , Trisacáridos/síntesis química , Secuencia de Carbohidratos , Datos de Secuencia Molecular , Oxígeno/químicaRESUMEN
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).
Asunto(s)
Afinidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Carbohidratos/inmunología , Anticuerpos Monoclonales/inmunología , Carbohidratos/química , Ligandos , Solubilidad , Relación Estructura-Actividad , Temperatura , Termodinámica , Agua/químicaRESUMEN
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.
Asunto(s)
Disacáridos/síntesis química , Metilglicósidos/síntesis química , Trisacáridos/síntesis química , Secuencia de Carbohidratos , Desoxiazúcares/síntesis química , Desoxiazúcares/química , Disacáridos/química , Espectroscopía de Resonancia Magnética , Metilglicósidos/química , Datos de Secuencia Molecular , Trisacáridos/químicaRESUMEN
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.
Asunto(s)
Anticuerpos Antibacterianos/genética , Anticuerpos Monoclonales/genética , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Shigella dysenteriae/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Codón , Cartilla de ADN/química , Hibridomas , Ratones , Datos de Secuencia Molecular , Mutación PuntualAsunto(s)
Desoxiazúcares/química , Glicósidos/química , Oligosacáridos/química , Arabinosa/metabolismo , Conformación de Carbohidratos , Secuencia de Carbohidratos , Desoxiazúcares/metabolismo , Disacáridos/química , Disacáridos/metabolismo , Isomaltosa/química , Isomaltosa/metabolismo , Metilglicósidos/química , Metilglicósidos/metabolismo , Datos de Secuencia Molecular , Monosacáridos/química , Monosacáridos/metabolismo , Trisacáridos/química , Trisacáridos/metabolismoRESUMEN
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.
Asunto(s)
Isomaltosa/química , Metilglicósidos/síntesis química , Oligosacáridos/síntesis química , Secuencia de Carbohidratos , Desoxiglucosa , Espectroscopía de Resonancia Magnética , Metilglicósidos/química , Datos de Secuencia Molecular , Oligosacáridos/químicaAsunto(s)
Fluoruros , Polisacáridos Bacterianos/química , Shigella dysenteriae/química , Animales , Anticuerpos Monoclonales , Sitios de Unión de Anticuerpos , Conformación de Carbohidratos , Secuencia de Carbohidratos , Inmunoglobulina M , Indicadores y Reactivos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Ratones/inmunología , Datos de Secuencia Molecular , Estructura Molecular , Antígenos O , Rotación Óptica , Polisacáridos Bacterianos/inmunología , Shigella dysenteriae/inmunologíaRESUMEN
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.