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

Abstract

A spontaneous, autoreactive autoantibody called SN5–18 (IgG2b, κ) binds to a complex of H2A/H2B/dsDNA in chromatin, but erroneously appears to bind dsDNA when the Ab is used in a form that is not highly purified. Because of this finding, we evaluated the antigenic specificity of a prototypic anti-dsDNA Ab, 3H9/Vκ4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vκ4 binds chromatin and specifically a complex of H2A/H2B/dsDNA, but not dsDNA in solid phase or in solution. When used in the form of culture supernatant or as a standard protein G preparation, mAb 3H9/Vκ4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of VHCDR3 Arg residues in dsDNA binding and the near identity of the SN5–18 sequence to other dsDNA-specific Ab, we tested the contributions of two VHCDR3 Arg residues in SN5–18 to chromatin specificity. We found that both these Arg residues at positions 104 and 106 were required for detectable chromatin binding. These results indicate a role for VHCDR3 Arg residues in chromatin specificity of lupus-derived autoantibodies. Further, they provide an explanation for a possible discrepancy in the form of tolerance observed in different anti-DNA Ig transgene models.

Intricacies of Anti-DNA Autoantibodies

“…Guth et al (5) confirm this finding and identify the H2A/H2B/DNA complex as the target of 3H9 binding. However, Guth et al further report that a "twice-purified" preparation of 3H9, also treated with DNaseI, failed to bind free DNA.…”

“…Multireactivity of anti-DNAs has been the focus of several studies including a recent study in this journal by Guth et al (5). Multireactivity of 3H9 derives from its asymmetric combining site: the DNA-binding surface is located in the H chain and can be enlarged by adding arginine residues to certain sites in V H complementarity-determining regions and FR3 (1).…”

γδ T cells affect IL-4 production and B-cell tolerance

“…All platebound HRP-conjugated antibodies were detected by tetramethylbenzidine substrate solution (Life Technologies), read using a VERSAmax tunable microplate reader (Molecular Devices, Sunnyvale, CA), and processed using SoftMax Pro-4.7.1 software (Molecular Devices, Sunnyvale, CA). Serum levels of anti-dsDNA/histone and anti-ssDNA antibodies were measured as previously described (47). Measurement of antibodies in the presence of histone alone (Fig.…”

“…Serum IgG2c was detected using cross-reactive HRP-conjugated goat anti-mouse IgG2a (Southern Biotechnology) (46). An antichromatin antibody ELISA was established using purified chromatin (47) to capture chromatin-specific antibodies in the serially diluted serum. Captured antibodies were then detected with polyclonal HRP-conjugated goat anti-mouse Ig(H+L).…”

Lonely killers

“…Although these are predominantly unmutated IgMs, the strongest binders display basic residuesenriched H-CDR3, with a predominance of arginines. 45 The importance of these latter residues for the binding of antibodies to several self-antigens, such as chromatin, 85 cardiolipin 86 and other phospholipids, 87 and gangliosides has been demonstrated. 88,89 Unlike the parental antibody, the arginine-enriched mutated P3 variant displayed cytotoxic activity over cells with high expression of the ganglioside.…”