Antibodies against RNA hydrolyze RNA and DNA

Abstract

Immunization of animals with DNA leads to the production of anti-DNA antibodies (Abs) demonstrating both DNase and RNase activities. It is currently not known whether anti-RNA Abs can possess nuclease activities. In an attempt to address this question, we have shown that immunization of three rabbits with complex of RNA with methylated BSA (mBSA) stimulates production of IgGs with RNase and DNase activities belonging to IgGs, while polyclonal Abs from three non-immunized rabbits and three animals immunized with mBSA are catalytically inactive. Affinity chromatography of IgGs from the sera of autoimmune (AI) patients on DNA-cellulose usually demonstrates a number of fractions, all of which effectively hydrolyze both DNA and RNA, while rabbit catalytic IgGs were separated into Ab subfractions, some of which demonstrated only DNase activity, while others hydrolyzed RNA faster than DNA. The enzymic properties of the RNase and DNase IgGs from rabbits immunized with RNA distinguish them from all known canonical RNases and DNases and DNA- and RNA-hydrolyzing abzymes (Abzs) from patients with different AI diseases. In contrast to RNases and AI RNA-hydrolyzing Abs, rabbit RNase IgGs catalyze only the first step of the hydrolysis reaction but cannot hydrolyze the formed terminal 2',3'-cyclophosphate. The data indicate that Abzs of AI patients hydrolyzing nucleic acids in part may be Abs against RNA and its complexes with proteins.

Generation of humanized anti-DNA hydrolyzing catalytic antibodies by complementarity determining region grafting

“…These kinetic parameters documented that there were no significant differences in DNA hydrolyzing efficiency between m3D8 and h3D8 scFvs. Though lower than for canonical DNases [4], the DNA hydrolyzing kinetic parameters of m3D8 and h3D8 scFvs were similar to those of other DNA hydrolyzing Abs [5,7].…”

“…4 legend for details). For in situ assays of DNase activity [7], purified m3D8 and h3D8 Abs at 2 lg were applied to 15% SDS-PAGE gels containing 20 lg/ml of one of the following substrates, dsDNA pUC19 and ssDNA from calf thymus (Sigma). Fig.…”

“…After washing three times with A buffer (10 mM Tris, pH 7.4, containing 10 mM MgCl 2 and 50 mM NaCl) plus 0.1% (v/v) TritonX-100 and then three times with A buffer, the gels were incubated for 5 h at 37°C in the A buffer to allow renaturation of the protein [7]. The gels were stained with ethidium bromide to identify regions of substrate hydrolysis, then with Coomassie Blue, to determine the position of the Abs.…”

“…To exclude the possibility of false-positive nuclease activity due to any traces of contaminating enzymes from E. coli, in situ DNA hydrolysis assays of h3D8 Abs were performed on SDS-PAGE gels containing dsDNA or ssDNA as substrates [7]. DNA hydrolyzing activity was observed for the substrates solely at the gel positions corresponding to the m3D8 scFv, h3D8 scFv, VH, and VL Abs, but not to the irrelevant anti-DR5 HW20 scFv (Fig.…”

“…For anti-DNA Abs originated from human, only some polyclonal Abs, not mAbs, have been reported for the DNA hydrolyzing activity [6,7]. Mouse-derived anti-DNA mAbs in various formats, such as intact IgG and their fragments of antigen binding fragment (Fab), single chain variable fragment (scFv), and single domains of VH and VL, exhibited DNA hydrolyzing activity, suggesting the DNA binding and hydrolyzing active sites are located at least in the variable domain of VH and/or VL [4,5,8].…”