RESUMEN
The binding of Strep 9, a mouse monoclonal antibody (mAb) of the IgG3 subclass directed against the cell-wall polysaccharide of Group A Streptococcus (GAS), has been characterized. The intact antibody and proteolytic fragments of Strep 9 bind differently to GAS: the intact mAb and F(ab)2' have greater affinity for the carbohydrate epitope than the monomeric Fab or F(ab)'. A mode of binding in which Strep 9 binds bivalently to portions of the polysaccharide on adjacent chains on GAS is proposed. A competitive ELISA protocol using a panel of carbohydrate inhibitors shows that the branched trisaccharide, beta-D-GlcpNAc-(1-->3)-[alpha-L-Rhap-(1-->2)]-alpha-L-Rhap, and an extended surface are key components of the epitope recognized by Strep 9. Microcalorimetry measurements with the mAb and two synthetic haptens, a tetrasaccharide and a hexasaccharide, show enthalpy-entropy compensation as seen in other oligosaccharide-protein interactions. Molecular modeling of the antibody variable region by homology modeling techniques indicates a groove-shaped combining site that can readily accommodate extended surfaces. Visual docking of an oligosaccharide corresponding to the cell-wall polysaccharide into the site provides a putative model for the complex, in which a heptasaccharide unit occupies the site and the GlcpNAc residues of two adjacent branched trisaccharide units occupy binding pockets within the groove-shaped binding site.
Asunto(s)
Anticuerpos Monoclonales/química , Antígenos Bacterianos/inmunología , Fragmentos de Inmunoglobulinas/química , Streptococcus pyogenes/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Antígenos Bacterianos/química , Sitios de Unión , Calorimetría , Secuencia de Carbohidratos , Epítopos , Inmunoensayo , Fragmentos de Inmunoglobulinas/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Polisacáridos Bacterianos/química , Polisacáridos Bacterianos/inmunología , Unión Proteica , TermodinámicaRESUMEN
An improved synthesis of propyl 4-O-(3,6-di-O-methyl- beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L-rhamnopyranoside, a disaccharide corresponding to the phenolic glycolipid of Mycobacterium leprae using a trichloroacetimidate as a glycosyl donor is described. The synthetic strategy is also applied to the preparation of three corresponding disaccharide analogues containing 13C-labeled methyl groups. The preparation of the trisaccharide, propyl 2-O-[4-O-(3,6-di-O-methyl-beta-D-glucopyranosyl)-2,3-di-O-methyl-alpha-L - rhamnopyranosyl]-3-O-methyl-alpha-L-rhamnopyranoside is also reported. The di- and tri-saccharides were characterized by 1H and 13C NMR spectroscopy.
Asunto(s)
Antígenos Bacterianos/química , Disacáridos/síntesis química , Glucolípidos/síntesis química , Mycobacterium leprae/química , Trisacáridos/síntesis química , Secuencia de Carbohidratos , Isótopos de Carbono , Haptenos/química , Marcaje Isotópico , Datos de Secuencia Molecular , Mycobacterium leprae/inmunología , Resonancia Magnética Nuclear BiomolecularRESUMEN
To investigate the molecular basis of antigenic mimicry by peptides, we studied a panel of closely related mAbs directed against the cell-wall polysaccharide of group A Streptococcus. These antibodies have restricted V-gene usage, indicating a shared mechanism of binding to a single epitope. Epitope mapping studies using synthetic fragments of the cell-wall polysaccharide supported this conclusion. All of the mAbs isolated crossreactive peptides from a panel of phage-displayed libraries, and competition studies indicated that many of the peptides bind at or near the carbohydrate binding site. Surprisingly, the peptides isolated by each mAb fell into distinct consensus-sequence groups that discriminated between the mAbs, and in general, the peptides bound only to the mAbs used for their isolation. Similar results were obtained with polyclonal antibodies directed against synthetic oligosaccharide fragments of the streptococcal cell-wall polysaccharide. Thus, the peptides appear to be specific for their isolating antibodies and are not recognized by the same mechanism as their carbohydrate counterparts.
Asunto(s)
Anticuerpos Monoclonales , Reacciones Cruzadas , Oligosacáridos/inmunología , Péptidos/inmunología , Polisacáridos Bacterianos/inmunología , Streptococcus pyogenes/inmunología , Secuencia de Aminoácidos , Especificidad de Anticuerpos , Secuencia de Carbohidratos , Ensayo de Inmunoadsorción Enzimática , Inmunoglobulina G , Inmunoglobulina M , Cadenas kappa de Inmunoglobulina , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/inmunología , Oligosacáridos/síntesis química , Oligosacáridos/química , Péptidos/química , Salmonella/inmunología , Shigella flexneri/inmunologíaRESUMEN
Glycosylated antitumor ether lipids (GAELs), analogs of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (1, ET-18-OCH3, edelfosine), were synthesized in good overall yields by glycosylation of 1-O-alkyl-2-O-methyl-sn-glycerol and tested for in vitro antineoplastic activity against a variety of murine and human tumor cell lines. Stereospecific glycosylation was achieved by the use of 2-O-acetyl-3,4,6-tri-O-benzylglucopyranosyl and -mannopyranosyl trichloroacetimidates as donors, with trimethylsilyl trifluoromethanesulfonate as catalyst in the presence of molecular sieves at -78 degrees C. The GAELs differ from 1 in having the sn-3-phosphocholine residue replaced by one of the following monosaccharide residues: beta- and alpha-2-deoxy-D-arabino-hexopyranosyl, alpha-D-mannopyranosyl, 2-O-methyl-beta-D-glucopyranosyl, and 2-O-methyl-alpha-D-mannopyranosyl. 1-O-Hexadecyl-2-O-methyl-3-O-(2'-deoxy-beta-D-arabino-hexopyranosyl)- sn-glycerol (2) was more effective than 1 in inhibiting the growth of MCF-7 (human breast cancer) and its adriamycin-resistant form MCF-7/adriamycin, and murine Lewis lung cancer cells. 2-Deoxy-beta-D-arabino-hexopyranoside 2 was also an effective growth inhibitor of two drug-resistant leukemic cell lines, P388/Adr and L1210/vmdr.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/toxicidad , Supervivencia Celular/efectos de los fármacos , Éteres Fosfolípidos/síntesis química , Éteres Fosfolípidos/toxicidad , Animales , Antineoplásicos/química , Neoplasias de la Mama , Línea Celular , Femenino , Glicósidos , Humanos , Indicadores y Reactivos , Leucemia L1210 , Leucemia P388 , Neoplasias Pulmonares , Espectroscopía de Resonancia Magnética , Metilación , Ratones , Estructura Molecular , Éteres Fosfolípidos/química , Células Tumorales CultivadasRESUMEN
A convergent synthesis of a hexasaccharide corresponding to the cell-wall polysaccharide of the beta-hemolytic Streptococcus Group A is described. The strategy relies on the preparation of a key linear trisaccharide unit beta-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-L-Rhap which has previously resisted our efforts. The trisaccharide functions both as a glycosyl acceptor and donor to give an elusive hexasaccharide. This fully functionalized unit can serve, in turn, as a glycosyl acceptor or donor for the synthesis of higher-order structures. Deprotection gives a hitherto unknown hexasaccharide for use as a hapten in immunochemical studies. The characterization of all compounds by high-resolution 1H and 13C NMR spectroscopy is also described.