Structure–function analysis and molecular modeling of DNase catalytic antibodies

Abstract

There is great interest in the antibodies-to-DNA transformation, since this change is characteristic of autoimmune diseases and contributes to its pathology. After immunization and fusions, 14 hybridomas bearing DNA-hydrolysis activity against pUC19 plasmid DNA were obtained. Genes coding for V H and V L regions of the 14 monoclonal antibodies (mAbs) were cloned and sequenced. The sequences were compared with sequences of the Ig-Blast database to determine their germline and to identify potential mutations responsible for DNA binding and DNase activity. V genes of the H chains’ genes expressed four genes of the V H1/J558 family, three of V H5/V H7183, and three of V H8/V H3609. The genetic repertoire of these mAbs was examined by determining the nucleotide sequences of their H chain V regions. This V H and V L domain was most similar to an anti-ssDNA (DNA-1) antibody as well as to catalytic autoimmune mAb (m3D8). Computer-generated models of the three-dimensional structures of V H and V L (VHL4) of the IgG4 combinations were used to define the positions occupied by the important sequence motifs at the binding sites. The modeling structure showed that VHL4 binds to oligo (dT3) primarily by sandwiching thymine bases between Tyr L32, Tyr L49 and Tyr H97 side-chains. Superposing VHL4 with anti-nucleic acid m3D8 catAbs revealed a common ssDNA recognition module consisting of His L93, His H35 residues which are critical for DNA-hydrolyzing antibodies. This study demonstrates the potential usefulness of the protein DNA surrogate in the investigation of the origin of anti-DNA antibodies’ hydrolyzing activities.