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.
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 histone-Trisacryl 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 signal 94-kDa protein from membrane fractions of CVI, 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.
Jacob
L
L
Institut National de la Santé et de la Recherche Médicale Unité 25, Paris, France.
Viard
J P
JP
Allenet
B
B
Anin
M F
MF
Slama
F B
FB
Vandekerckhove
J
J
Primo
J
J
Markovits
J
J
Jacob
F
F
Bach
J F
JF
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000936:Antigen-Antibody Complex / Q000032:analysis; D001323:Autoantibodies / Q000276:immunology*; D002460:Cell Line; D004247:DNA / Q000276:immunology* / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006657:Histones / Q000276:immunology / Q000378:metabolism*; D008565:Membrane Proteins / Q000276:immunology*; D008970:Molecular Weight; D009707:Nucleosomes / Q000276:immunology* / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D001323:Autoantibodies; D006657:Histones; D008565:Membrane Proteins; D009707:Nucleosomes; D004247:DNA