A monoclonal anti-double-stranded DNA autoantibody binds to a 94-kDa cell-surface protein on various cell types via nucleosomes or a DNA-histone complex.

Abstract

A crude supernatant of hybridoma secreting a monoclonal anti-double-stranded (ds)DNA antibody (PME77 mAb), used to stain fibroblasts (CVI cells) in immunofluorescence, gives a punctuated staining of variable intensity. We had suggested that anti-DNA antibodies bind to cell-surface protein(s) of several cells. When the mAb of this crude supernatant was purified on a dsDNA-cellulose column and a histoneTrisacryl column, the mAb no longer bound to the cell surface. Only when dsDNA plus purified histones was added to the purified antibody did the immune complex strongly and uniformly stain again the cell surface of CVI cells. No significant staining was observed if either DNA or histones were omitted. A single 94-kDa protein from membrane fractions ofCVI, Raji, and RINm cell lines was visualized in immunoblots when mAb-DNA-histone complexes were applied to the nitrocellulose strips. No polypeptide was seen if one component was omitted. This 94-kDa protein behaved like a plasma membrane protein since it required the use of detergent to be solubilized and was quantitatively recovered in the Triton X-114 detergent-rich phase. Moreover, a brief treatment of living cells with trypsin cleared off this protein. Purified nucleosomes could be substituted to DNA-histone complexes, giving rise to identical results. Finally, purified polyclonal anti-DNA antibodies from sera of systemic lupus erythematosus patients labeled a 94-kDa protein provided that DNA-histone complexes were added. Anti-DNA autoantibodies could be pathogenic when they are bound to nucleosomes.

DNA topoisomerase I binding to fibroblasts induces monocyte adhesion and activation in the presence of anti–topoisomerase I autoantibodies from systemic sclerosis patients

“…Anti-topo I are not the only autoantibodies that bind to the cell surface via a nuclear autoantigen. For example, nucleosomes, or DNA-histone complexes, have been shown to recruit anti-double-stranded DNA autoantibodies to the surface of various cell types (45). In that particular case, an unidentified 94-kd protein has been pinpointed as the target of nucleosomes on cell surfaces.…”

Presence of nucleosome‐restricted antibodies in patients with systemic lupus erythematosus

“…These IgGs were further depleted of anti-dsDNA antibodies by means of adsorption on DNA-cellulose columns (10). Briefly, dsDNA-cellulose (Sigma, St. Louis, MO) was equilibrated overnight in 10 mM Tris, 1 mM EDTA (TE) and 150 mM NaC1, pH 7.4 (TE buffer) at 4"C, washed extensively with TE and 2M NaCl, pH 8.0, and further equilibrated in TE buffer.…”

“…Briefly, dsDNA-cellulose (Sigma, St. Louis, MO) was equilibrated overnight in 10 mM Tris, 1 mM EDTA (TE) and 150 mM NaC1, pH 7.4 (TE buffer) at 4"C, washed extensively with TE and 2M NaCl, pH 8.0, and further equilibrated in TE buffer. Purified IgGs from each SLE serum were passed through individual columns, and the effluent was further depleted of antihistone antibodies by adsorption on histone columns made of a mixture of total histones (Sigma) covalently linked to p-nitrophenyl chloroformate-activated trisacryl beads (IBF, Villeneuve la Garenne, France) (10). The effluent from each histone column was taken as the source of purified adsorbed IgGs.…”

Chromatin Specificity of Anti-Double-Stranded DNA Antibodies and a Role for Arg Residues in the Third Complementarity-Determining Region of the Heavy Chain

“…The importance of accurately defining antigenic specificity is illustrated by the paradox that lupus autoantibodies often appear to bind multiple and seemingly disparate antigenic structures (3,(21)(22)(23)(24)(25)(26)(27)(28)(29)(30). For example, Bloom et al (3) reported one such Ab with affinity for both dsDNA and La, a common nuclear protein autoantigen.…”