RESIDUES THAT MEDIATE DNA-BINDING OF AUTOIMMUNE ANTIBODIES

Citation
Mz. Radic et al., RESIDUES THAT MEDIATE DNA-BINDING OF AUTOIMMUNE ANTIBODIES, The Journal of immunology, 150(11), 1993, pp. 4966-4977
Citations number
39
Categorie Soggetti
Immunology
Journal title
The Journal of immunology
ISSN journal
00221767 → ACNP
Volume
150
Issue
11
Year of publication
1993
Pages
4966 - 4977
Database
ISI
SICI code
0022-1767(1993)150:11<4966:RTMDOA>2.0.ZU;2-5
Abstract
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-d sDNA antibodies in autoimmune MRL/lpr mice. To examine the consequence s of such amino acid substitutions on DNA binding, we reverted three s omatic mutations of a prototypic anti-dsDNA H chain gene, V(H)3H9, 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 comple te germ-line revertant of V(H)3H9 retained a low level of DNA binding, the substitution of R96, a product of N base addition in the third co mplementarity determining region (CDR3), with glycine (G) was sufficie nt to abolish measureable DNA specificity. Antibodies with higher affi nity for DNA were generated by introducing arginines into V(H)3H9 at a ny 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 dir ect involvement in DNA binding, although one such mutant, K64R, requir ed the simultaneous reversion of an adjacent aspartic acid (D) to the germ-line glycine. Two variants with three nongerm-line arginines show ed further improvements in DNA affinity suggesting that their contribu tions to DNA binding may be additive. Molecular modeling of antibody a nd mutant F(ab) structures and calculations of their electrostatic pot entials were used as an aid in interpreting the results and in predict ing the location and size of possible combining sites.