DNA Hydrolysis by Monoclonal Autoantibody BV 04-01

Abstract

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+. Catalysis was associated with BV 04-01 IgG, Fab, and single-chain-antibody (SCA) proteins. Cleavage of both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of A7C7ATATAGCGCGT2, as well as a preference for cleaving within CG-rich regions of dsDNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA, and two SCA mutants were used to model the catalytically active antibody site using the previously resolved X-ray structure of BV 04-01. The resulting model suggested that the target phosphodiester bond is activated by induction of conformational strain. In addition, the antibody-DNA complex contained a Mg2+ coordination site composed of the L32Tyr and L27dHis side chains and a DNA 3'-phosphodiester group. Induction of strain along with the metal coordination could be part of the mechanism by which this antibody catalyzes DNA hydrolysis. Sequence data for BV 04-01 V(H) and V(L) genes suggested that the proposed catalytic-antibody active site was germline-encoded. This observation suggests that catalytic activity might represent an important-rarely examined-function for some antibody molecules.

Comparison and Analysis of Different Methods for Purification of Autoimmune Antibody Reactive with Single Stranded DNA: A Pilot Study

“…They stressed the possibility that the Fc fragment, eliminated in papain hydrolysis, could have inhibitory effects upon this reaction. 7,21,34 Our results, to our knowledge for the first time, confirm the hydrolytic activity of pure, polyclonal, human anti-ssDNA lupus antibody. Future work is necessary to increase the sensitivity levels of the hydrolytic assays, locate the specific position of the hydrolytic component of the antibody and determine its reaction kinetics in comparison to DNAse1 in order to prove the antibody's intrinsic hydrolytic capacity.…”

“…substrate binding, hydrolytic, and cytotoxic activity). 1,7,16,17,21,33 Our two-step affinity purification method was designed to target anti-ssDNA IgG antibodies. 19,20 However, our attempts to purify anti-ssDNA antibodies using other oligomers (dC, dG, and dA) bound to streptavidin (SA) coated Dynabeads® were unsuccessful (data not shown).…”

“…Other hypotheses implicate viral DNA, 7,18,47,50 microbial DNA, and/or unmethylated CpG motifs within the DNA of any species 56 all of which may be interesting to the innate immune system. Could a new mechanism of antigen recognition be taking place, omitting the classical antigen-presentation loop to the T-cells and their helper response?…”

“…7,18,47,50 Furthermore, all of the same questions that we have about anti-ssDNA antibodies can also be applied to the dsDNA category.…”

“…However, since antissDNA antibody has been shown in a limited number of experimental and clinical studies to be both hydrolytic and nephritogenic, it is suggested that it may serve as a strong flare predictor. [2][3][4] The important role of anti-ssDNA antibody is supported by studies in mouse models of nephritogenic lupus in which only anti-ssDNA antibodies were found 6,7 as well as by the findings of Swanson et al, 1 & Spatz et al, 8 that some anti-dsDNA human antibodies are not pathogenic at all. The ultimate goal of this study was to determine the optimal conditions for analysis of the types and activity of highly purified human antissDNA (IgG antibody) in order to gain deeper insight into functional characteristics such as binding, DNA hydrolysis, cytotoxicity and the role of these antibodies in SLE pathogenesis.…”