BINDING OF THE O-ANTIGEN OF SHIGELLA-DYSENTERIAE TYPE-1 AND TYPE-26 RELATED SYNTHETIC FRAGMENTS TO A MONOCLONAL IGM ANTIBODY

Shigella dysenteriae type 1 possesses an O-antigen whose repeating uni t is 1-->2)-alpha-D-Galp-(1-->3)-alpha-D-GlcpNAc-(1-->, where Rhap is rhamnopyranosyl, Galp is galactopyranosyl, and Glcp is glucopyranosyl. Using ligand-induced protein fluorescence change, we have measured th e affinities of a monoclonal murine IgM for 26 fragments of, or relate d to, the structure of the O-polysaccharide and of the IgM Fab for the intact O-specific bacterial polysaccharide. Synthetic saccharides use d were methyl glycosides to ensure an anomerically defined pyranosyl r ing conformation. The galactosyl residue is the only monosaccharide of the antigenic epitope that shows quantifiable binding: approximately 3.0 kcal/mol of binding free energy, depending on the structure and co nformation of the fragment it is a part of. Addition of an alpha-(1--> 2)-linked rhamnosyl residue increases the free energy of binding signi ficantly. We propose this rhamnopyranosyl-alpha-(1-->2)-galactopyranos yl disaccharide to be the basic determinant of the Shigella O-polysacc haride. Further extension (by linkages as in the natural antigen) of t his oligosaccharidic ligand toward the upstream end (in an oligo- (or poly-)saccharide, such as A-->B-->C-->D-->E-->m, where A, B, C, D, and E are sugars and m is any moiety, such as methyl, we define A as the glycosyl- or upstream terminus, and E as the glycoside- or downstream terminus) by rhamnosyl and N-acetylglucosaminyl moieties improves the binding only minimally. The antibody is quite specific for the rhamnos yl-alpha-(1-->2)-galactosyl sequence but less so for the nature of the attachment to the galactosyl residue on the downstream side. Measurem ents using IgM Fab and the intact O-specific polysaccharide show that the antibody can bind internal segments on the antigen chain. The free energy of binding of this antibody for the disaccharide determinant v aries from -DELTAG of 4.7 to 5.1 kcal/mol, depending on its flanking r esidues.