Activation of DNA-hydrolyzing antibodies from the sera of autoimmune-prone MRL-lpr/lpr mice by different metal ions

摘要

We have shown previously that electrophoretically and immunologically homogeneous polyclonal IgGs from the sera of autoimmune-prone MRL mice possess DNase activity. Here we have analyzed for the first time activation of DNase antibodies (Abs) by different metal ions. Polyclonal DNase IgGs were not active in the presence of EDTA or after Abs dialysis against EDTA, but could be activated by several externally added metal (Me~(2+)) ions, with the level of activity decreasing in the order Mn~(2+) ≥ Mg~(2+) > Ca~(2+) ≥ Cu~(2+) > Co~(2+) ≥ Ni~(2+) ≥ Zn~(2+), whereas Fe~(2+) did not stimulate hydrolysis of supercoiled plasmid DNA (scDNA) by the Abs. The dependencies of the initial rate on the concentration of different Me~(2+) ions were generally bell-shaped, demonstrating one to four maxima at different concentrations of Me~(2+) ions in the 0.1–12 mM range, depending on the particular metal ion. In the presence of all Me~(2+) ions, IgGs pre-dialyzed against EDTA produced only the relaxed form of scDNA and then sequence-independent hydrolysis of relaxed DNA followed. Addition of Cu~(2+), Zn~(2+), or Ca~(2+) inhibited the Mg~(2+)-dependent hydrolysis of scDNA, while Ni~(2+), Co~(2+), and Mn~(2+) activated this reaction. The Mn~(2+)-dependent hydrolysis of scDNA was activated by Ca~(2+), Ni~(2+), Co~(2+), and Mg~(2+) ions but was inhibited by Cu~(2+) and Zn~(2+). After addition of the second metal ion, only in the case of Mg~(2+) and Ca~(2+) or Mn~(2+) ions an accumulation of linear DNA (single strand breaks closely spaced in the opposite strands of DNA) was observed. Affinity chromatography on DNA-cellulose separated DNase IgGs into many subfractions with various affinities to DNA and very different levels of the relative activity (0–100%) in the presence of Mn~(2+), Ca~(2+), and Mg~(2+) ions. In contrast to all human DNases having a single pH optimum, mouse DNase IgGs demonstrated several pronounced pH optima between 4.5 and 9.5 and these dependencies were different in the presence of Mn~(2+), Ca~(2+), and Mg~(2+) ions. These findings demonstrate a diversity of the ability of IgG to function at different pH and to be activated by different optimal metal cofactors. Possible reasons for the diversity of polyclonal mouse abzymes are discussed.