Structure-function analysis of a lupus anti-DNA autoantibody: central roleof 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 th e complementarity-determining region (CDR)3 Arg to DNA binding, we engineer ed the germ line V-H and V-kappa gene revertant and site-mutagenized the CD R3 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 tw o H-CDR3 Arg, Arg105 and Arg107, encoded by N segment additions, and one ka ppa-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: 4 10-423). The germ-line revertant bound double-stranded (ds) DNA and single- stranded (ss) DNA as effectively as its wild-type counterpart (relative avi dity: 6.4x10(-7) and 9.9x10(-9) vs. 6.7x10(-7) and 9.1x10(-9) g/mu l), rais ing 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 Arg10 7 was sufficient for ssDNA binding, The central role of Arg105 and Arg107 i n DNA binding reflected their solvent-exposed orientation at the apex of th e H-CDR3 main loop. Consistent with its inward orientation afar from the an tigen-binding surface, the kappa-CDR3 Arg97 played no role in either dsDNA or ssDNA binding.