DELINEATION OF THE EPITOPE RECOGNIZED BY AN ANTIBODY SPECIFIC FOR N-GLYCOLYLNEURAMINIC ACID-CONTAINING GANGLIOSIDES

P3 is a mouse monoclonal antibody (mAb) that binds to several NeuGc-co ntaining gangliosides, It also reacts with antigens expressed in human breast tumors (Vazquez ef al, (1995) Hybridoma, 14, 551-556), In this work, the binding specificity of P3 has been characterized in more de tail using a panel of glycolipids that included several disialylated g angliosides and several chemical derivatives of NeuGc-GM3. The carboxy l group and the nitrogen function of sialic acid were found to play im portant roles in the antibody binding, whereas the glycerol tail appea rs to be nonrelevant. Molecular modeling was used to analyze the bindi ng data, including the finding that P3 selectively recognizes the inte rnal NeuGc in GD3, For this purpose, conformational studies of GD3 wer e performed using molecular dynamics. It was concluded that sialic aci d binds the P3 antibody through its upper face (the one on which the c arboxyl group is exposed) and the C4-C5 side of the sugar ring, wherea s none or very little contact between the galactose residue and the pr otein is evident, Conformational analysis of GD3 revealed that, despit e the large flexibility of the NeuGc alpha NeuGc linkage, the P3 bindi ng epitope on the external sialic acid is not well exposed for any of the possible conformations this linkage can adopt, whereas the interna l sialic acid presents the epitope in a proper way for several of thes e conformations. As a final result, a coherent picture of the epitope that fits the wide binding data was obtained.