Structural studies of polyspecific anti-carbohydrate antibodies

Abstract

The inheritance of germline immunoglobulin genes must ensure the recognition of prevalent pathogens and the ability to adapt to new pathogens. The current understanding of the generation of diversity of immunoglobulin binding sites involves the recombination of the B-lymphocyte germline genes during development. In general, the production of immunoglobulins proceeds with the help of T-lymphocytes through a process called "affinity maturation" involving mutations that increase antigen specificity. Carbohydrate epitopes, however, do not illicit such a response, and so are more dependent on the primary germline repertoire. Although anti-carbohydrate antibodies exhibit exquisite specificity, to date there have been few structural reports of complexes due to their low binding affinities. Here, high-resolution X-ray crystal structures of antigen-binding fragments from two highly homologous immunoglobulins S25-2 and S45-18 in their unliganded forms and bound to several carbohydrate epitopes of 3-deoxy- D-manno-oct-2-ulosonic acid (Kdo) found in Gram-negative bacteria including members of the family Chlamydiaceae. The combining sites of both immunoglobulins are similarly composed of a common germline-conserved "pocket" that binds a non-reducing terminal Kdo monosaccharide, and a "groove"-like remainder of the binding site that uses the same residues multiply in interactions with different epitopes in the case of the germline-conserved S25-2, or that has undergone mutations that enhance the specificity for a single epitope in the case of S45-18. The structural basis of this adaptability is accomplished firstly, by the combination of a specific set of germline VH and Vkappa genes that encodes a binding pocket that recognizes the non-reducing terminal Kdo; secondly, by a difference in the nature of the D and JH mini-genes between S25-2 and S45-18; and thirdly, by the contributions of conserved germline residues compared to regions of amino acid mutation. Furthermore, the strategy of coupling a strict binding pocket with a more adaptive region demonstrates how anti-carbohydrate immunoglobulins can bind a limited range of related epitopes without necessarily incurring the entropic penalty of immobilisation of flexible regions.