Bivalency and epitope specificity of a high-affinity IgG3 monoclonal antibody to the Streptococcus group A carbohydrate antigen. Molecular modeling of a Fv fragment.
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.
Pitner
J B
JB
Becton Dickinson Research Center, Research Triangle Park, NC 27709, USA.
Beyer
W F
WF
Venetta
T M
TM
Nycz
C
C
Mitchell
M J
MJ
Harris
S L
SL
Mariño-Albernas
J R
JR
Auzanneau
F I
FI
Forooghian
F
F
Pinto
B M
BM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000276:immunology; D000942:Antigens, Bacterial / Q000737:chemistry / Q000276:immunology*; D001665:Binding Sites; D002151:Calorimetry; D002240:Carbohydrate Sequence; D000939:Epitopes; D007118:Immunoassay; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000276:immunology; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011135:Polysaccharides, Bacterial / Q000737:chemistry / Q000276:immunology; D011485:Protein Binding; D013297:Streptococcus pyogenes / Q000276:immunology*; D013816:Thermodynamics
D000911:Antibodies, Monoclonal; D000942:Antigens, Bacterial; D000939:Epitopes; D007128:Immunoglobulin Fragments; D011135:Polysaccharides, Bacterial; C055966:immunoglobulin Fv