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D V Schourov

Publications and source records attributed to D V Schourov.

5 recordsLinked to original sources
PubMed1997

DNA hydrolysis by monoclonal anti-ssDNA autoantibody BV 04-01: origins of catalytic activity.

Monoclonal anti-DNA autoantibody BV 04-01 catalyzed hydrolysis of DNA in the presence of Mg2+ ions. DNA hydrolyzing activity was associated with BV 04-01 IgG, Fab, and SCA 04-01 proteins. Pronounced cleavage specificity for both ss and dsDNA was observed with efficient hydrolysis of the C-rich region of the oligonucleotide A7C7ATATAGCGCGT7 as well as preference for cleavage within CG-rich regions of double-stranded DNA. Data on specificity of ssDNA hydrolysis and kinetic data obtained from wild-type SCA 04-01 and two SCA 04-01 mutants (L32Phe and L27dHis) were used to model the catalytically active antibody site utilizing the previously resolved X-ray structure of (dT)3 liganded Fab 04-01. The resulting model suggested that BV 04-01 activates the target phosphodiester bond by induction of conformational strain. In addition, the antibody-DNA complex contained a potential Mg2+ ion coordination site composed of the L32Tyr and L27dHis amino acid side chains and a DNA 3'-phosphodiester group. Induction of strain and metal coordination could be constituents of a mechanism by which this antibody catalyzed DNA hydrolysis. Sequence data for BV 04-01 VH and VL genes suggested that the proposed catalytic antibody active site was germ-line encoded. This observation suggests the hypothesis that catalytic activity might represent an important but unspecified function of some antibody molecules.

Animals↗
PubMed1995

Cleavage of supercoiled plasmid DNA by autoantibody Fab fragment: application of the flow linear dichroism technique.

A highly effective method consisting of two affinity chromatography steps and ion-exchange and gel-filtration chromatography steps was developed for purification of autoantibodies from human sera with DNA-hydrolyzing activity. Antibody Fab fragment, which had been purified 130-fold, was shown to catalyze plasmid DNA cleavage. The flow linear dichroism technique was used for quantitative and qualitative studying of supercoiled plasmid DNA cleavage by these autoantibodies in comparison with DNase I and EcoRI restriction endonuclease. The DNA autoantibody Fab fragment was shown to hydrolyze plasmid DNA by Mg(2+)-dependent single-strand multiple nicking of the substrate. Kinetic properties of the DNA autoantibody Fab fragment were evaluated from the flow linear dichroism and agarose gel electrophoresis data and revealed a high affinity (Kobsm = 43 nM) and considerable catalytic efficiency (kappcat/Kobsm = 0.32 min-1.nM-1) of the reaction.

Autoantibodies↗
PubMedDate not supplied

DNA-hydrolyzing autoantibodies.

Catalysis by antibodies could be a frequent phenomenon if the immune system generates a sufficiently diverse number of antibody-active sites, some of which may possess catalytic activity. A catalytic antibody can be expected to do more damage than one that simply binds antigen. The best biochemical marker of systemic lupus erythematosus (SLE) is presence of autoantibodies to DNA. In the present article, we describe the DNA-hydrolyzing activity of DNA-binding autoantibodies purified from SLE patients. The substrates employed were supercoiled plasmid, radiolabeled plasmid fragments, and oligonucleotides. Hydrolysis of DNA by the antibodies was indicated by the appearance of fragments visualized by ethidium bromide staining of agarose gels or autoradiography of polyacrylamide gels. Changes in linear dichroism values were also indicative of DNA hydrolysis. The antibody activity was purified by protein A-sepharose chromatography, high-performance liquid chromatography gel filtration, and DNA-affinity chromatography. Scrupulous control studies were done to demonstrate that DNA-hydrolyzing activity really belongs to the antibodies. Purified Fab fragments showed hydrolyzing activity, whereas the Fc fragment was inactive. The specificity of DNA cleavage was investigated, and the rate parameters of hydrolysis by antibodies and conventional nucleases were compared.

Antibodies↗
PubMedDate not supplied

DNA hydrolysis by monoclonal autoantibody BV 04-01.

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.

Animals↗