Diverse roles for the third complementarity determining region of the heavy chain (H3) in the binding of immunoglobulin Fv fragments to DNA, nucleosomes and cardiolipin

Autoantibodies to DNA and chromatin employ junctional diversity and somatic mutations to generate or enhance antigen recognition. To define the role o f diversity generating mechanisms in the etiology of autoantibodies to nucl ear antigens, the heavy (H! chain of a murine autoantibody, 3H9, was used i n its somatically mutated or germ-line form in conjunction with its own or with heterologous CDR3 (H3) domains. The resulting H chains were expressed together with the 3H9 light (L) chain as single-chain Fv (scFv) in Escheric hia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enz yme-linked immunosorbent assay. An recombinant scFv exhibited nearly identi cal binding to cardiolipin. in contrast, the binding to nuclear antigens wa s drastically reduced by the reversion of mutations in 3H9 or the exchange of H3, such that only 3H9 itself bound strongly to single-stranded DNA, dou ble-stranded DNA and nucleosomes. The results illustrate diverse interactio ns between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the g erm line, directs binding to cardiolipin, whereas structural determinants o f H3, in concert with the remainder of the combining site, guide the matura tion of antibody binding toward nuclear autoantigens.