Structure-function analysis of a human anti-DNA autoantibody: Role of H chain CDR3 Arg residues in DNA binding

Somatic hypermutation is thought to be a crucial process in the maturation of the anti-DNA autoantibody response. To analyze the role of somatic point-mutations in DNA binding, we have constructed, by in vitro gene recombination and site-directed mutagenesis, a V_H and V_κ germline revertant of the anti-DNA IgG1 κ mAb412.67.F1.3 generated frpm B-1 cells of a SLE patient. mAb412.67.F1.3 is encoded by a V_H and V_κ gene segments that bear a number of R mutations with significantly high R:S mutation ratio in the CDRs. The ÒgermlineÓ revertant of mAb412.67.F1.3 bound specifically to ssDNA and ds-DNA with an efficiency similar to that of the wildtype IgG1 progenitor (Kd ssDNA 9.05 x 10^-9 g/ul dsDNA 6.66 x 10^-7 g/ul Vs Kd ssDNA 9.95 x 10^-9 g/ul dsDNA 6.35 x 10^-7 g/ul). Stepwise site-directed mutagenesis analysis showed that both H chain CDR3 Arg 105 and 107 (both possibly encoded by N nucleotide additions), but not the κ chain CDR3 Arg 97 (a somatic point mutation) were necessary for the high affinity binding of dsDNA and ssDNA. Either Arg 105 or Arg 107 of H chain was sufficient for binding ssDNA (Kd 5.2 x 10^-9 g/ul). These findings suggest that DNA selection of R mutations in the CDR may not be a major mechanism in the generation of human high affinity autoantibodies to DNA.