MOLECULAR RECOGNITION OF A SALMONELLA TRISACCHARIDE EPITOPE BY MONOCLONAL-ANTIBODY SE155-4

The binding site of monoclonal antibody Se155-4, which has been the ob ject of successful crystallographic and antibody-engineering studies, is shown by solid-phase immunoassays to be complementary to a branched trisaccharide, alpha-D-Galp(1-->2) [alpha-D-Abep(1-->3)]-alpha-D-Manp (l, rather than to the tetrasaccharide repeating unit -D-Abep(1-->3)]- alpha-D-Manp(1-->4)alpha-L-Rhap(1- of the bacterial antigen. Specifici ty for the 3,6-dideoxy-D-xylo-hexose (3,6-dideoxy-D-galactose) epitope present in Salmonella paratyphi BO-antigens was ensured by screening hybridoma experiments with glycoconjugates derived from synthetic olig osaccharides. Detailed epitope mapping of the molecular recognition by modified and monodeoxy oligosaccharide derivatives, showed that compl ementary surfaces and three antibody-saccharide hydrogen bonds are ess ential for full binding activity. Both hydroxyl groups of the 3,6-dide oxy-D-galactose residue were obligatory for binding and consistent wit h the directional nature of their involvement in carbohydrate-protein hydrogen bonds; related tetrasaccharides built from the isomeric 3,6-d ideoxyhexoses, 3,6-dideoxy-D-glucose, paratose, and 3,6-dideoxy-D-mann ose, tyvelose were not bound by the antibody. Titration microcalorimet ry measurements were consistent with the hydrogen-bonding map inferred from the crystal structure and suggest that the displacement of water molecules from the binding site accounts for the favorable entropy th at accompanies binding of the native trisaccharide determinant. The pr otein Sequences determined for the antibody V-L and V-H domains reveal somatic mutation of the V-L germ line gene, implying that this antibo dy-binding site results from a mature antibody response.