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

Abstract

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.

Chromatin Specificity of Anti-Double-Stranded DNA Antibodies and a Role for Arg Residues in the Third Complementarity-Determining Region of the Heavy Chain

“…Distinguishing conclusively between these alternatives may require an x-ray structure. We cannot say with certainty why purified 3H9 is highly specific for chromatin in our hands but cross-reactive with other Ags when tested as an engineered scFV (41). The cross-reactions observed are unlikely to be due to contaminating bacterial nucleoid material, because we found this to be nonantigenic in our assay.…”

“…In view of a report (41) that an engineered 3H9 scFv fragment bound to dsDNA, ssDNA, nucleosomes, and cardiolipin, we wondered whether this bacterially derived version of the Ab might contain contaminating chromosomal proteins that substitute for histones. For this reason, we tested 2ϫ purified mAb 3H9/V4 for binding to bacterial chromosome-coated plates.…”

Exogenous and Endogenous TLR Ligands Activate Anti-Chromatin and Polyreactive B Cells

“…Thus, the V H 3H9 H chain appears particularly well-suited for binding to Ags, which include DNA, chromatin, histones, and may also predispose toward polyreactivity (60). Additionally, recent data from Radic and colleagues (61) suggested that the germline form of the V H 3H9 biases the repertoire toward cardiolipin binding, and that nuclear reactivity is obtained by somatic mutation and combination with permissive L chains. Consistent with the model put forth by Marshak-Rothstein and colleagues (6) that nuclear-reactive B cells may be activated by binding to endogenous Ags that also trigger TLR9, one hybridoma, 5361-3, binds chromatin as well as mitotic figures in the ANA assay.…”

Editing and escape from editing in anti-DNA B cells

“…By reverting the somatically mutated VH3H9 to its germ-line sequence, it was discovered that the precursor of 3H9 bound to phosphatidylserine (PS) (17). Subsequent mutations gave rise to 3H9, an Ab with reactivity toward dsDNA, nucleosomes, and additional phospholipids (18)(19)(20)(21). Thus, an incompletely edited B cell may have been the precursor of this pathogenic autoantibody.…”