The structural basis for DNA binding by an anti-DNA autoantibody.
We have used single and multiple site-directed mutagenesis, and molecular modeling, to identify critical residues in the DNA binding site of MAb 2C10, an IgG anti-dsDNA autoantibody from an MRL/lpr lupus mouse. Simultaneous replacement of four Arg residues in the CDR3H abolished binding activity. With one exception, replacement of any one of these Arg residues reduced the activity to 20-50% of the unmutated scFv activity. Arg to Asp replacements had a slightly greater effect than Arg to Ala replacements. In the one exceptional case, replacement of Arg99 with Ala actually increased DNA binding five-fold and replacement by Asp had little effect. Mutation of Phe32 and Asn35 to A1a in CDRIH decreased DNA binding, whereas replacement of Arg31 with A1a had negligible effect. Ala substitution of any one of a cluster of Asp residues in CDR1L increased DNA binding three to six-fold, confirming previous findings that the L-chain of MAb 2C10 is not favorable for DNA binding. The L-chain does participate in shaping the selectivity of antigen binding, and mutation of CDR3L residue Asp92 or Asn93 caused a decrease in DNA binding activity. Directed mutagenesis, consistent with a molecular model, indicates that: several CDR amino acids contribute to DNA binding, without one residue dominating; both VH and VL CDR3 domains contribute to specificity of binding whereas the CDR1L hinders DNA binding. The results suggest a significant role for electrostatics in the interaction of DNA with MAb 2C10.
Jang
Y J
YJ
Laboratory of Immunology, Institute for Medical Sciences, Ajou University Schools of Medicine, Suwon, Korea. jangyj@madang.ajou.ac.kr
Sanford
D
D
Chung
H Y
HY
Baek
S Y
SY
Stollar
B D
BD
D000595:Amino Acid Sequence; D000818:Animals; D000974:Antibodies, Antinuclear / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000915:Antibody Affinity / Q000235:genetics; D001323:Autoantibodies / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001666:Binding Sites, Antibody / Q000235:genetics; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000378:metabolism; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism; D051379:Mice; D019463:Mice, Inbred MRL lpr; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed
D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D007141:Immunoglobulin Fc Fragments; D007135:Immunoglobulin Variable Region; D004247:DNA