Isolated VH4 Heavy Chain Variable Regions Bind DNA Characterization of a Recombinant Antibody Heavy Chain Library Derived from Patient(s) with Active SLE

Abstract

In many autoimmune diseases autoantibodies are intimately involved in disease manifestations. Molecular characterization of these autoantibodies should provide insights into the pathogenesis of these diseases, as well as suggest novel avenues for development of therapeutics. While some prior studies suggest that DNA binding may be a characteristic of individual heavy chain variable regions, the ability of these V regions to bind DNA in isolation has not been investigated. We have utilized a bacterial vector for cloning and expressing isolated antibody heavy chain variable regions. RNA was extracted from peripheral blood mononuclear cells of patients with active SLE, cDNA synthesized and heavy chain V regions amplified with V_H specific oligonucleotide primers. The V _H fragments were cloned into a bacterial expression plasmid including the pelB leader peptide to direct appropriate expression. Recombinant antibodies were screened for binding to³²P-labeled double-stranded plasmid DNA and later also characterized for binding to single-stranded DNA. Binding was confirmed by standard ELISA methodology. Sequence analysis of seven DNA binding V_H fragments revealed that they utilized the V_H gene family previously described to be associated with autoimmune responses, with a J_H6 segment. On V_H sequence analysis only one residue substitution in the consensus sequence is needed to form a V _H4 germline gene. Potential contact residues with DNA were delineated by three-dimensional structure analysis. We concluded that the DNA binding characteristics of V_H regions can be examined in the absence of light chain. DNA binding specificity appears to be a property of the germline V_H4 gene. Analysis of such V regions can aid in the identification of hypervariable region contact residues important for DNA binding.