Chromatin specificity of anti-double-stranded DNA antibodies and a role for Arg residues in the third complementarity-determining region of the heavy chain.
A spontaneous, autoreactive autoantibody called SN5-18 (IgG2b, kappa) 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/Vkappa4, now used widely in transgenic studies of tolerance and autoimmunity. We found that the purified mAb 3H9/Vkappa4 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/Vkappa4 appears to bind dsDNA, apparently due to nuclear proteins in the preparation that assemble on target DNA. Because of the reported role of V(H)CDR3 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 V(H)CDR3 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 V(H)CDR3 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.
Guth
Amanda M
AM
Integrated Department of Immunology, National Jewish Medical and Research Center and University of Colorado School of Medicine, Denver, CO 80206, USA.
Zhang
Xianghua
X
Smith
Diana
D
Detanico
Thiago
T
Wysocki
Lawrence J
LJ
D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000502:physiology; D000918:Antibody Specificity / Q000235:genetics; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D001324:Autoantigens / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics; D001667:Binding, Competitive / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D002474:Cell-Free System; D002843:Chromatin / Q000276:immunology* / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000378:metabolism; D022801:Complementarity Determining Regions / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D000939:Epitopes / Q000276:immunology*; D005260:Female; D006657:Histones / Q000737:chemistry; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000378:metabolism; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000378:metabolism; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D051379:Mice; D008814:Mice, Inbred NZB; D016297:Mutagenesis, Site-Directed
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D001324:Autoantigens; D002843:Chromatin; D022801:Complementarity Determining Regions; D000939:Epitopes; D006657:Histones; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D004247:DNA; D001120:Arginine