Structure-function and gene analysis of anti-DNA autoantibodies from BW mice

Anti-DNA autoantibodies in both autoimmune murine strains and in humans have been implicated in the pathogenesis of the autoimmune disorder SLE (systemic lupus erythematosus). The primary goal was to examine the structural features of monoclonal anti-DNA autoantibodies derived from the BW mouse autoimmune strain in order to: (1) determine the IgV$\sb{\rm H}$ and V$\sb{\rm L}$ gene repertoire encoding these autoantibodies, and (2) define components of the antibody active-site involved in DNA binding.Initial studies to define the gene repertoire of five different monoclonal anti-DNA autoantibodies utilized a northern blot analysis and unique gene probes designed to maximize hybridization to CDR or active site residues. Results indicated that V$\sb{\rm H}$ active site residues of a single-strand specific DNA autoantibody (BV04-01) were unique, while V$\sb{\rm L}$ residues were shared among four of five autoantibodies. This suggested a restricted usage of genes of the V$\sb{\rm L}$ repertoire.The primary structures of the five anti-DNA autoantibodies was deduced from the complete V$\sb{\rm H}$ and V$\sb{\rm L}$ nucleotide sequences as determined from cloned cDNA. Results showed that two V$\sb{\rm H}$ genes shared complete identity, while homologies of the remaining three were 67% or less. Framework homologies allowed assignment of four of five V$\sb{\rm H}$ genes to existing murine heavy chain gene families, although CDR residues were relatively unique. The V$\sb{\rm L}$ genes showed restriction of four of five light chains to the V$\sb{\kappa}$1 subgroup confirming the northern blot analysis. Identical or nearly identical V$\sb{\kappa}$ subgroup light chains are utilized in a variety of responses to exogenous antigens.A system was devised to allow site-directed mutagenesis of V$\sb{\rm H}$ encoded DNA binding residues in the active-site of a ssDNA specific autoantibody and utilized an H chain loss variant that was characterized by northern blot analysis and PCR of genomic DNA. Transfection of this cell line with a mammalian expression vector carrying the genomic homolog of the V$\sb{\rm H}$ and C$\sb{\rm H}$ genes restored DNA binding activity. A PCR mutagenesis procedure allowed site-directed mutagenesis of two V$\sb{\rm H}$ encoded CDR2 residues thought to be important in DNA binding.U of I OnlyETDs are only available to UIUC Users without author permissio