title: The constant region affects antigen binding of antibodies to DNA by altering secondary structure.
abstract: We previously demonstrated an important role of the constant region in the pathogenicity of anti-DNA antibodies. To determine the mechanisms by which the constant region affects autoantibody binding, a panel of isotype-switch variants (IgG1, IgG2a, IgG2b) was generated from the murine PL9-11 IgG3 autoantibody. The affinity of the PL9-11 antibody panel for histone was measured by surface plasmon resonance (SPR). Tryptophan fluorescence was used to determine wavelength shifts of the antibody panel upon binding to DNA and histone. Finally, circular dichroism spectroscopy was used to measure changes in secondary structure. SPR analysis revealed significant differences in histone binding affinity between members of the PL9-11 panel. The wavelength shifts of tryptophan fluorescence emission were found to be dependent on the antibody isotype, while circular dichroism analysis determined that changes in antibody secondary structure content differed between isotypes upon antigen binding. Thus, the antigen binding affinity is dependent on the particular constant region expressed. Moreover, the effects of antibody binding to antigen were also constant region dependent. Alteration of secondary structures influenced by constant regions may explain differences in fine specificity of anti-DNA antibodies between antibodies with similar variable regions, as well as cross-reactivity of anti-DNA antibodies with non-DNA antigens.
authors.affiliation: Department of Microbiology & Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
authors.forename: Yumin
authors.initials: Y
authors.lastname: Xia
authors.forename: Alena
authors.initials: A
authors.lastname: Janda
authors.forename: Ertan
authors.initials: E
authors.lastname: Eryilmaz
authors.forename: Arturo
authors.initials: A
authors.lastname: Casadevall
authors.forename: Chaim
authors.initials: C
authors.lastname: Putterman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000276:immunology* / Q000378:metabolism; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D000941:Antigens / Q000276:immunology* / Q000378:metabolism; D001666:Binding Sites, Antibody / Q000235:genetics / Q000276:immunology*; D001667:Binding, Competitive / Q000276:immunology; D015153:Blotting, Western; D002942:Circular Dichroism; D004247:DNA / Q000276:immunology / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000276:immunology / Q000378:metabolism; D006657:Histones / Q000276:immunology / Q000378:metabolism; D007127:Immunoglobulin Constant Regions / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D011485:Protein Binding / Q000276:immunology; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence; D020349:Surface Plasmon Resonance
chemical_list: D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D000941:Antigens; D004277:DNA, Single-Stranded; D006657:Histones; D007127:Immunoglobulin Constant Regions; D007074:Immunoglobulin G; D004247:DNA