Autoantibodies to double-stranded (ds)DNA immunoprecipitate 18S ribosomal RNA by virtue of their interaction with ribosomal protein S1 and suppress in vitro protein synthesis

Abstract

SUMMARYWe report that four systemic lupus erythematosus (SLE) patient sera containing anti-dsDNA antibodies, three affinity-purified anti-dsDNA IgG, and a human anti-dsDNA MoAb (33.H11) immunoprecipitate 18S ribosomal RNA from DNase-treated 32 P-labelled MOLT4 cell extract. This 18S RNA precipitation was inhibited completely by preincubating 33.H11 with calf thymus dsDNA or the recombinant human ribosomal protein S1, which was reported to cross-react with anti-dsDNA antibodies (J Immunol 1996; 156:1668-75). Whole IgG from three SLE sera with anti-dsDNA antibodies, 33.H11, and three affinitypurified anti-dsDNA IgG inhibited in vitro translation of globin mRNA (percent inhibition was 36-50%). This translation inhibition by anti-dsDNA antibodies was enhanced (67-79%) when the reticulocyte lysate was treated with DNase. Suppression of protein synthesis could be a pathogenic mechanism of anti-dsDNA antibodies, since it has also been shown that anti-dsDNA penetrates living cells (J Immunol 1995; 154:4857-64) in culture.

Cross-Reaction of Lupus Anti-dsDNA Antibodies with Protein Translation Factor EF-2

“…To our surprise, it has been shown that the cDNA library purchased commercially, which was used to select a cDNA encoding the ribosomal S1 protein, contained a cDNA encoding a ribosomal S1 protein from the bacterial species L. lactis (26). Nonetheless, a human analogue of these proteins appears to exist on the small ribosomal subunit, since anti-dsDNA antibodies immunoprecipitate 18S ribosomal RNA and suppress in vitro translation (19). In light of our previous observations demonstrating the reactivity of anti-dsDNA antibodies to ribosomal S1 protein (25) we utilized rabbit polyclonal antibodies toward the ribosomal S1 protein as a probe to identify analogous antigenically and functionally related proteins from eukaryotic cell lines.…”

“…Samples were then loaded onto an anion-exchange DE-52 column equilibrated with 0.01 M phosphate buffer, pH 7.2 (Whatman Biosystems Ltd., Maidstone, Kent, UK), and IgG was eluted with the same phosphate buffer. Purified anti-dsDNA IgG was eluted from DNA cellulose columns (Sigma, St. Louis, MO) as described previously (19). Nine affinity-purified anti-dsDNA Abs, three human monoclonal anti-dsDNA Abs, two affinity-purified anti-ribosomal P Abs, and Cohn fraction II IgG (Sigma) were used in translation assays (19).…”

“…Purified anti-dsDNA IgG was eluted from DNA cellulose columns (Sigma, St. Louis, MO) as described previously (19). Nine affinity-purified anti-dsDNA Abs, three human monoclonal anti-dsDNA Abs, two affinity-purified anti-ribosomal P Abs, and Cohn fraction II IgG (Sigma) were used in translation assays (19). Anti-dsDNA Abs were detected by ELISA (20).…”

“…What has been little investigated are the metabolic consequences of this binding and penetration except that with certain subsets of anti-dsDNA antibodies which are restricted to membrane binding, complement-dependent cytolysis can be shown to ensue (8). In this paper we pursue the observation that antibodies to dsDNA inhibit in vitro translation (19). An intracellular cross-reactive target has been identified that mediates the inhibition of in vitro translation.…”

Etiopathogenesis of Systemic Lupus Erythematosus

“…In addition, 33.H11 inhibited in vitro translation of globulin mRNA, which was enhanced when the reticulocyte lysate was treated with DNAse. From these data it might be speculated that suppression of protein synthesis could be regarded as a pathogenic mechanism of anti-dsDNA antibodies, since it had previously been shown that some anti-dsDNA antibodies were able to penetrate living cells in culture [67,68]. The different biological activities of monoclonal anti-dsDNA antibodies obtained from one patient give rise to the hypothesis that at least one possible mechanism leading to a specific organ targeting by anti-dsDNA antibodies in SLE could be due to a cross-reactivity of anti-dsDNA antibody clones with organ-specific antigens.…”