Structure-function analysis of a lupus anti-DNA autoantibody: central role of the heavy chain complementarity-determining region 3 Arg in binding of double- and single-stranded DNA.
To determine the contribution of the somatic point mutations and that of the complementarity-determining region (CDR)3 Arg to DNA binding, we engineered the germline V(H) and V(kappa) gene revertant and site-mutagenized the CDR3 Arg residues of the mutated and "antigen-selected" mAb 412.67. This anti-DNA autoantibody was derived from B-1 cells of a lupus patient and bore two H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one kappa-CDR3 Arg, Arg97, resulting from a point mutation (Kasaian et al. 1994. J. Immunol. 152: 3137-3151; Kasaian et al. 1995. Ann. N.Y Acad. Sci. 764: 410-423). The germ-line revertant bound double-stranded (ds) DNA and single-stranded (ss) DNA as effectively as its wild-type counterpart (relative avidity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1 x10(-9) g/microl), raising the possibility that an antigen other than DNA was responsible for the selection of the mAb 412.67 V(H) and V(kappa) point mutations. H-CDR3 Arg105 and Arg107 were both required for dsDNA binding, but either Arg105 or Arg107 was sufficient for ssDNA binding. The central role of Arg105 and Arg107 in DNA binding reflected their solvent-exposed orientation at the apex of the H-CDR3 main loop. Consistent with its inward orientation afar from the antigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding.
Li
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Department of Pathology, Weill Medical College of Cornell University, New York, NY 10021, USA.
Schettino
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Padlan
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Ikematsu
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Casali
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D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D001120:Arginine / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D001483:Base Sequence; D002460:Cell Line; D022801:Complementarity Determining Regions; D004277:DNA, Single-Stranded / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry / Q000276:immunology; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007133:Immunoglobulin Joining Region / Q000235:genetics / Q000276:immunology; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D008177:Lupus Vulgaris / Q000276:immunology*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017354:Point Mutation; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D000974:Antibodies, Antinuclear; D000911:Antibodies, Monoclonal; D022801:Complementarity Determining Regions; D004277:DNA, Single-Stranded; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007133:Immunoglobulin Joining Region; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; C055966:immunoglobulin Fv; D001120:Arginine