A Common NH53K Mutation in the Combining Site of Antibodies Raised against Chlamydial LPS Glycoconjugates Significantly Increases Avidity

Abstract

The crystal structures of the antigen-binding fragment of the murine monoclonal antibody (mAb) S25-39 in the presence of several antigens representing chlamydial lipopolysaccharide (LPS) epitopes based on the bacterial sugar 3-deoxy-α-D-manno-oct-2-ulosonic acid (Kdo) have been determined at resolutions from 2.4 to 1.8 Å. The antigen-binding site of this antibody differs from the well-characterized antibody S25-2 by a single mutation away from the germline of asparagine H53 to lysine, yet this one mutation results in a significant increase in avidity across a range of antigens. A comparison of the two antibody structures reveals that the mutated Lys H53 forms additional hydrogen bonds and/or charged-residue interactions with the second Kdo residue of every antigen having two or more carbohydrate residues. Significantly, the NH53K mutation results from a single nucleotide substitution in the germline sequence common among a panel of antibodies raised against glycoconjugates containing carbohydrate epitopes of chlamydial LPS. Like S25-2, S25-39 displays significant induced fit of complementarity determining region (CDR) H3 upon antigen binding, with the unliganded structure possessing a conformation distinct from those reported earlier for S25-2. The four different observed conformations for CDR H3 suggest that this CDR has evolved to exploit the recognition potential of a flexible loop while minimizing the associated entropic penalties of binding by adopting a limited number of ordered conformations in the unliganded state. These observations reveal strategies evolved to balance adaptability and specificity in the germline antibody response to carbohydrate antigens.

Groove-type Recognition of Chlamydiaceae-specific Lipopolysaccharide Antigen by a Family of Antibodies Possessing an Unusual Variable Heavy Chain N-Linked Glycan

“…The differences in the sequences corresponding to the combining sites were so striking that it was clear that the two groups must use markedly different strategies (Table 1). Interestingly, even the weakest binder among the latter group (S25-26) shows higher avidity than all S25-2-type antibodies except S25-39 (26), and that difference is marginal; see Table 1.…”

“…These ligands contained the lipid A backbone in addition to Kdo. To determine the cross-reactive potential, these binding assays were performed at low (2 pmol/well) and high (20 pmol/well) coated antigen concentrations (Table 1), and the results were compared with previously published binding data using oligosaccharides of synthetic origin without the lipid A backbone (18,25,26,31). ITC experiments carried out for S25-23 and S25-26 mAbs (Fig.…”

“…Antibodies raised against chlamydial LPS showed strong V region restriction (11,25,26), with two heavy and two light chain V genes accounting for almost all clones isolated. These groups were named after their prototypic clones, the S25-2-type and the S25-23-type.…”

“…S25-2-type antibodies (i.e. antibodies that shared V gene usage with S25-2 but had different D and J genes) displayed various avidities to a panel of chlamydial LPS antigens and included antibodies that ranged from cross-reactive to specific (11,14,18,20,(25)(26)(27)(28)(29)(30)(31). The structures of their Fabs (fragment antigen binding), determined unliganded and in complex with a number of antigens, provided molecular insight into the effects of V-gene restriction, DJ gene influence, and affinity maturation (11,14,18,20,(25)(26)(27)(28)(29)(30)(31).…”

“…We now report binding data and crystal structures of liganded and unliganded S25-26 Fab as a step toward elucidating the recognition mechanism of S25-23-type antibodies. avidities of mAbs S25-23, S25-26, S25-2, and S25-39 were determined by ELISA as described (25,26). BSA-neoglycoconjugates of oligosaccharides isolated from recombinant bacteria (33,34) and from synthetic origin (18,31,(35)(36)(37)(38)(39) were prepared as published (28,40).…”