Cross-reactivity of anti-nDNA antibodies with nuclear envelope proteins. Isolation of a cDNA encoding the 70 kDa annular protein recognized by autoantibodies from patients with systemic lupus erythematosus.
OBJECTIVE
To determine whether annular rDNA is complexed with the nuclear envelope proteins.


METHODS
From a batch of lupus sera with anti-nDNA, we selected a lupus serum containing annular anti-nDNA autoantibodies resistant to DNase digestion and used it to isolate several cDNA clones from a lambda gt11 HeLa cell library.


RESULTS
The cloned fusion protein immunoadsorbed the annular anti-nDNA autoantibodies, and the immunoaffinity autoantibodies eluted from the recombinant filters produced an annular pattern around the nucleus in fluorescent assays on HEp-2 cells; by Western blot, they also recognized a 70 kDa protein from HEp-2 cell extracts. Annular-lambda gt11 lysogens generated in E. coli Y1089 produced a fusion protein that recognized annular anti-nDNA autoantibody-containing lupus sera by Western blot. The recombinant filters and annular fusion protein were also recognized by a prototype anti-lamin serum. To determine whether the annular recombinant protein bound DNA, an interaction assay was performed in vitro using DNA minicircles and DNA from HEp-2 cells; this assay resulted in a slowing of the electrophoretic mobility of the DNA.


CONCLUSIONS
1) The annular DNA in eukaryotic cells is complexed with nuclear envelope proteins. 2) Annular anti-nDNA autoantibodies from lupus patients cross-react with perinuclear proteins. 3) Perinuclear proteins recognized by anti-nDNA are lamins. 4) An interaction between DNA and the 70 kDa protein is inducible in vitro. Whether this interaction affects cell function is still unknown.
Herrera-Diosdado
R
R
Department of Immunology, Universidad Autónoma de Zacatecas, Mexico. herrerar@cantera.reduaz.mx
Avalos-Diaz
E
E
Herrera-Esparza
R
R
D000974:Antibodies, Antinuclear / Q000276:immunology*; D001323:Autoantibodies / Q000276:immunology; D003429:Cross Reactions; D004247:DNA / Q000276:immunology*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D008180:Lupus Erythematosus, Systemic / Q000276:immunology; D008970:Molecular Weight; D009685:Nuclear Envelope / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology*; D011994:Recombinant Proteins
D000974:Antibodies, Antinuclear; D001323:Autoantibodies; D018076:DNA, Complementary; D009687:Nuclear Proteins; D011994:Recombinant Proteins; D004247:DNA