Residues that mediate DNA binding of autoimmune antibodies.
Somatic mutations to arginine (R) are a common feature of a subset of J558 H chain genes that code for the majority of high-affinity, anti-dsDNA antibodies in autoimmune MRL/lpr mice. To examine the consequences of such amino acid substitutions on DNA binding, we reverted three somatic mutations of a prototypic anti-dsDNA H chain gene, VH3H9, and assayed the effect of those reversions by expression in a V lambda 1 L chain-only plasmacytoma line. Reversion of R53 eliminated virtually all dsDNA binding and sharply reduced ssDNA affinity. While the complete germ-line revertant of VH3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third complementarity determining region (CDR3), with glycine (G) was sufficient to abolish measureable DNA specificity. Antibodies with higher affinity for DNA were generated by introducing arginines into VH3H9 at any one of four positions where somatic mutations to arginine had been identified by sequencing other anti-dsDNA J558 H chain genes. All four arginine mutants showed affinity increments consistent with their direct involvement in DNA binding, although one such mutant, K64R, required the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines showed further improvements in DNA affinity suggesting that their contributions to DNA binding may be additive. Molecular modeling of antibody and mutant F(ab) structures and calculations of their electrostatic potentials were used as an aid in interpreting the results and in predicting the location and size of possible combining sites.
Institute for Cancer Research, Fox Chase Cancer Center, Philadelphia, PA 19111.
M Z
MZ
Radic
J
J
Mackle
J
J
Erikson
C
C
Mol
W F
WF
Anderson
M
M
Weigert
D000595:Amino Acid Sequence; D000974:Antibodies, Antinuclear / Q000378:metabolism*; D001120:Arginine / Q000235:genetics; D001483:Base Sequence; D001666:Binding Sites, Antibody; D004247:DNA / Q000276:immunology / Q000378:metabolism*; D004563:Electrochemistry; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D013329:Structure-Activity Relationship
D000974:Antibodies, Antinuclear; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D004247:DNA; D001120:Arginine