Evidence for the Extended Helical Nature of Polysaccharide Epitopes. The 2.8 .ANG. Resolution Structure and Thermodynamics of Ligand Binding of an Antigen Binding Fragment Specific for .alpha.-(2.fwdarw.8)-Poly(sialic acid)

Abstract

The antigen binding fragment from an IgG2a kappa murine monoclonal antibody with specificity for alpha-(2-->8)-linked sialic acid polymers has been prepared and crystallized in the absence of hapten. Crystals were grown by vapor diffusion equilibrium with 16-18% polyethylene glycol 4000 solutions. The structure was solved by molecular replacement methods and refined to a conventional R factor of 0.164 for data to 2.8 A. The binding site is observed to display a shape and distribution of charges that is complementary to that of the predicted conformation of the oligosaccharide epitope. A thermodynamic description of ligand binding has been compiled for oligosaccharides ranging in length from 9 to 41 residues, and the data for the largest ligand has been used in a novel way to estimate the size of the antigen binding site. A model of antigen binding is presented that satisfies this thermodynamic data, as well as a previously reported requirement of conformational specificity of the oligosaccharide. X-ray crystallographic and thermodynamic evidence are consistent with a binding site that accommodates at least eight sialic acid residues.

High affinity binding of long-chain polysialic acid to antibody, and modulation by divalent cations and polyamines

“…5, 7 and 16 at 100 M; data not shown), although there were no significant differences in the inhibitory effects of various oligomers tested. The results for penta-and hepta-mer are in accordance with the independence of binding affinities on the chain length observed for short oligomers (Evans et al, 1995). A more pronounced inhibition with the longer 16-mer could have been masked by the increase in the observed response due to the binding of the oligomer itself to the antibody (Table 2).…”

“…2) indicated concentration-dependent bivalent binding (MacKenzie et al, 1996). This behavior of mAb735 was previously observed with a 41-mer PSA (Evans et al, 1995) and is similar to the binding of heparan sulfate to lipoprotein lipase (Lookene et al, 1996). Although, we have shown previously, that only one antibody combining site is needed for binding of mAb735 to polysialic acids (Häyrinen et al, 1989), both sites of the antibody can be saturated using higher ligand concentrations.…”

“…Also, variations in the experimental conditions (temperature, pH, ionic strength and buffer composition) make the comparison of results difficult, since not much is known about their effect on the binding. Although several models for the PSA epitopes have been constructed based on the results obtained by NMR spectroscopy, potential energy calculations, ligand docking studies and X-ray crystallography of the antibody combining site (Michon et al, 1987;Yamasaki and Bacon, 1991;Brisson et al, 1992;Evans et al, 1995), the actual mechanisms of antibody interaction have remained unclear.…”

“…Due to their polyanionic nature, PSA epitopes are suggested to employ marked electrostatic components in their binding events (Häyrinen et al, 1989;Evans et al, 1995).…”

“…The PSA-Ab binding reaction has been suggested to involve electrostatic interaction (Häyrinen et al, 1989;Evans et al, 1995). According to previous studies (Aubin and Prestegard, 1993), the pK a -values of carboxyl groups in PSA are exceptionally high compared to Neu5Ac-monoand oligomers: the carboxyl groups are assumed to be 100% protonated at pH 2.5 and 30% at pH 7.0.…”