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 unit is -->3-alpha-L-Rhap-(1-->3)-alpha-L-Rhap-(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 the affinities of a monoclonal murine IgM for 26 fragments of, or related to, the structure of the O-polysaccharide and of the IgM Fab for the intact O-specific bacterial polysaccharide. Synthetic saccharides used were methyl glycosides to ensure an anomerically defined pyranosyl ring 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 conformation of the fragment it is a part of. Addition of an alpha-(1-->2)-linked rhamnosyl residue increases the free energy of binding significantly. We propose this rhamnopyranosyl-alpha-(1-->2)-galactopyranosyl disaccharide to be the basic determinant of the Shigella O-polysaccharide. Further extension (by linkages as in the natural antigen) of this 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 rhamnosyl-alpha-(1-->2)-galactosyl sequence but less so for the nature of the attachment to the galactosyl residue on the downstream side. Measurements 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 varies from -DELTAG of 4.7 to 5.1 kcal/mol, depending on its flanking residues.