ISOLATED V(H)4 HEAVY-CHAIN VARIABLE REGIONS BIND DNA CHARACTERIZATIONOF A RECOMBINANT ANTIBODY HEAVY-CHAIN LIBRARY DERIVED FROM PATIENT(S)WITH ACTIVE SLE

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibo dies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. Whil e some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V reg ions to bind DNA in isolation has not been investigated. We have utili zed a bacterial vector for cloning and expressing isolated antibody he avy chain variable regions. RNA was extracted from peripheral blood mo nonuclear cells of patients with active SLE, cDNA synthesized and heav y chain V regions amplified with V-H specific oligonucleotide primers. The V-H fragments were cloned into a bacterial expression plasmid inc luding the pelB leader peptide to direct appropriate expression. Recom binant antibodies were screened for binding to P-32-labeled double-str anded plasmid DNA and later also characterized for binding to single-s tranded DNA. Binding was confirmed by standard ELISA methodology. Sequ ence analysis of seven DNA binding V-H fragments revealed that they ut ilized the V-H gene family previously described to be associated with autoimmune responses, with a J(H)6 segment. On VH sequence analysis on ly one residue substitution in the consensus sequence is needed to for m a V(H)4 germline gene. Potential contact residues with DNA were deli neated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of V-H regions can be examined in the abse nce of light chain. DNA binding specificity appears to be a property o f the germline V(H)4 gene. Analysis of such V regions can aid in the i dentification of hypervariable region contact residues important for D NA binding.