Groove-type recognition of Chlamydiaceae-specific lipopolysaccharide antigen by a family of antibodies possessing an unusual variable heavy chain N-linked glycan [Immunology]
March 28th, 2014 by Haȷi–Ghassemi, O., Muller–Loennies, S., Saldova, R., Muniyappa, M., Brade, L., Rudd, P. M., Harvey, D. J., Kosma, P., Brade, H., Evans, S. V.
The structure of the antigen-binding fragment of mAb S25-26 determined to 1.95 Å resolution in complex with the Chlamydiaceae family-specific trisaccharide antigen Kdo(2→8)-Kdo(2→4)Kdo (Kdo=3-deoxy-α-D-manno-oct-2-ulopyranosonic acid) displays a germline-coded paratope that differs significantly from previously characterized Chlamydiaceae-specific mAbs, despite being raised against the identical immunogen. Unlike the terminal Kdo recognition pocket that promotes cross-reactivity in S25-2-type antibodies, S25-26 and the closely related S25-23 utilize a groove composed of germline residues to recognize the entire trisaccharide antigen and so confer strict specificity. Interest in S25-23 was sparked by its rare high μM affinity and strict specificity for the family-specific trisaccharide antigen; however, only the related antibody S25-26 proved amenable to crystallization. The structures of three unliganded forms of S25-26 have a labile complementary determining region H3 adjacent to significant glycosylation of the variable heavy chain on asparagine 85 in Framework Region 3. Analysis of the glycan reveals a heterogeneous mixture with a common root structure that contains an unusually high number of terminal αGal-Gal moieties. One of the few reported structures of glycosylated mAbs containing these epitopes is the therapeutic antibody Cetuximab; however, unlike Cetuximab, one of the unliganded structures in S25-26 shows significant order in the glycan with appropriate electron density for nine residues. The elucidation of the three dimensional structure of an αGal containing N-linked glycan on a mAb variable heavy chain has potential clinical interest, as they have been implicated in allergic response in patients receiving therapeutic antibodies.