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 of diversity generating mechanisms in the etiology of autoantibodies to nuclear antigens, the heavy (H) chain of a murine autoantibody, 3H9, was used in 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 Escherichia coli and assayed for binding to DNA, nucleosomes, or cardiolipin by enzyme-linked immunosorbent assay. All recombinant scFv exhibited nearly identical binding to cardiolipin. In contrast, the binding to nuclear antigens was 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, double-stranded DNA and nucleosomes. The results illustrate diverse interactions between a single combining site and different autoantigens. The analysis of these interactions suggests that the 3H9 VH domain, as encoded by the germ line, directs binding to cardiolipin, whereas structural determinants of H3, in concert with the remainder of the combining site, guide the maturation of antibody binding toward nuclear autoantigens.
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Department of Microbiology and Immunology, MCP-Hahnemann University, Philadelphia, USA.
Monestier
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D000595:Amino Acid Sequence; D002308:Cardiolipins / Q000378:metabolism*; D003001:Cloning, Molecular; D022801:Complementarity Determining Regions / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000378:metabolism*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000378:metabolism*
D002308:Cardiolipins; D022801:Complementarity Determining Regions; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D009707:Nucleosomes; C055966:immunoglobulin Fv; D004247:DNA