Observations on the binding of four anti-carbohydrate monoclonal antibodies to their homologous ligands.

The binding of four monoclonal immunoglobulins, two with specificity for beta(1-->6)-linked D-galactopyranans (IgA X24 and IgA J539) and two with specificity for the chain terminus of alpha(1-->6)-linked d-glucopyranans (IgA W3129 and IgA 16.4.12E), was measured with a number of their homologous oligosaccharide ligands at different temperatures. The results show a linear relationship between lnKa and 1/T, where Ka is the affinity constant and T is the absolute temperature. The unitary free energy of binding, DeltaGu, is virtually independent of T, and the DeltaSu is small when compared with DeltaGu. The enthalpy changes derived from van't Hoff plots are large and negative, indicating an exothermic binding effect, whereas the entropy changes are small and negative, indicating minor overall hydrophobic contributions. Measurements of the free energies of binding, in low and high salt buffers, of methyl beta-d-galactopyranoside and the methyl glycoside of beta(1-->6)-D-galactopyranotetraose with anti-galactan IgA X24 indicate that the monosaccharide has no hydrophobic interaction with the highest affinity subsite of IgA, whereas the tetraoside might have a modest hydrophobic interaction with the three other hapten-binding subsites of IgA. The standard entropy change of binding of the two groups (galactosyl and glucosyl) of oligosaccharides to the two respective sets (anti-galactan and anti-dextran) of antibodies shows a distinct, differing correlation with the hapten chain length within each set. This correlation agrees with the type of association previously established between the antibodies and either the interior determinants of the antigen (in the case of the anti-galactans) or the chain terminus (in the case of the anti-dextrans).

Binding of the O-antigen of Shigella dysenteriae type 1 and 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 -delta G of 4.7 to 5.1 kcal/mol, depending on its flanking residues.

Binding characteristics of IgA 16.4.12E, a monoclonal antibody with specificity for the nonreducing terminal epitope of alpha-(1----6)-dextrans. Comparisons between IgA hybridoma 16.4.12E and myeloma W3129.

IgA 16.4.12E is a murine monoclonal antibody obtained following immunization with isomaltohexose linked to keyhole limpet hemocyanin. We have studied its interaction with methyl alpha-D-glucopyranoside and its derivatives bearing deoxy or deoxyfluoro groups, and with the methyl alpha-glycosides of a series of isomalto-oligosaccharides, some bearing deoxy or deoxy-fluoro groups at selected positions. From the data it is concluded that the antibody binds optimally to 4 sequential glucopyranosyl residues and that the protein subsite possessing the major affinity binds the terminal, nonreducing glucosyl group of that antigenic epitope. All the hydroxyl groups of that terminal glucosyl group are involved in hydrogen bonding, some in a donating and some in an accepting capacity. In the last part of the paper we report the construction of a possible model of the antibody, derived from its known amino acid sequence and the known crystalline structures of two closely related antibodies. It shows a pronounced cavity in the general immunoglobulin combining area which is flanked by 2 solvent-exposed tryptophanyl residues. A model recently reported for anti-dextran IgA W3129 shows a similar cavity with one such residue. Guided by hydrogen bonds, experimentally deduced from the comparison of the affinities of variously derivatized ligands, we suggest a speculative fitting for the nonreducing terminus of the dextran antigen, in the respective cavities of both IgA 16.4.12E and W3129.

Synthesis of 2,3-epoxypropyl beta-glycosides of (1----6)-beta-D-galactopyranooligosaccharides and their binding to monoclonal anti-galactan IgA J539 and IgA X24.

Reaction of 2,3,4-tri-O-acetyl-6-O-(bromoacetyl)-alpha-D-galactopyranosyl bromide (2) with allyl 2,3,4-tri-O-acetyl-beta-D-galactopyranoside gave allyl O-[2,3,4-tri-O-acetyl-6-O-(bromoacetyl)-alpha- and -beta-D-galactopyranosyl]-(1----6)-2,3,4-tri-O-acetyl-beta-D- galactopyranoside, 4 (4%) and 5 (88%), respectively. Selective removal of the bromoacetyl group from 5 gave allyl O-(2,3,4-tri-O-acetyl-beta-D-galactopyranosyl)-(1----6)-2,3,4-tri-O-a cet yl-beta - D-galactopyranoside (6), which, after condensation with 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (1) yielded both allyl O-(2,3,4,6-tetra-O-acetyl-alpha- and -beta-D-galactopyranosyl)-(1----6)-O-(2,3,4-tri-O-acetyl-beta-D- galactopyranosyl)-(1----6)-2,3,4-tri-O-acetyl-beta-D-galactopyranoside, 7 (10%) and 8 (70%), respectively. When 6 was condensed with 2, allyl O-[2,3,4-tri-O-acetyl-6-O-(bromoacetyl)-beta-D-galactopyranosyl]-(1----6 )-O- (2,3,4-tri-O-acetyl-beta-D-galactopyranosyl)-(1----6)-2,3,4-tri-O-acetyl -beta-D - galactopyranoside (75%) was obtained. This was selectively O-de(bromoacetyl)ated to yield the nonaacetate, which was condensed with bromide 1 to give allyl O-(2,3,4,6-tetra-O-acetyl-alpha- and -beta-D-galactopyranosyl)-(1----6)-O-(2,3,4-tri-O-acetyl-beta-D- galactopyranosyl)-(1----6)-O-(2,3,4-tri-O-acetyl-beta-D-galactopyranosyl )- (1----6)-2,3,4-tri-O-acetyl-beta-D-galactopyranoside, 14 (4%) and 15 (70%). Epoxidation of the allyl group of 8 and 15 with m-chloroperoxybenzoic acid, and removal of the acetyl protecting groups with sodium methoxide, gave, respectively, 2,3-epoxypropyl O-beta-D-galactopyranosyl-(1----6)-O-beta-D-galactopyranosyl-(1----6)-be ta-D- galactopyranoside (17) and the corresponding tetrasaccharide 19. Sequential acetylation and O-debenzylation of 6-O-benzyl-D-galactose, followed by coupling of the product with bromide 1, yielded O-(2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1----6)-1,2,3,4-tetr a-O- acetyl-beta-D-galactopyranose (12). Conversion of 12 into the bromide by treatment with bromotrimethylsilane, and condensation of the product with nucleophile 6 also gave the beta-linked tetrasaccharide 15 of this series. Epoxidation of the allyl group, followed by removal of acetyl blocking groups in the latter compound, again gave 2,3-epoxypropyl O-beta-D-galactopyranosyl-(1----6)-O-beta-D-galactopyranosyl-(1----6)-O- beta-D- galactopyranosyl-(1----6)-beta-D-galactopyranoside (19).(ABSTRACT TRUNCATED AT 400 WORDS)