Isolation and characterization of nuclear ribonucleoprotein complexes using human anti-nuclear ribonucleoprotein antibodies
Douvas, A.S.; Stumph, W.E.; Reyes, P.; Tan, E.M.
Journal of Biological Chemistry 254(9): 3608-3616
ISSN/ISBN: 0021-9258 PMID: 429372 Accession: 005763108
The feasibility of using human autoimmune antibodies to isolate and characterize specific nuclear ribonucleoprotein (nRNP) complexes was investigated. High titers of anti-nRNP antibodies occur in a syndrome called mixed connective tissue disease. Ig[immunoglobulin]G antibodies from 2 mixed connective tissue disease patients were used to construct affinity columns to isolate the antigenic complexes from rat liver nuclei. A maximum of 2.9% of the nuclear RNA and 0.7% of the protein bound to anti-nRNP but not to control IgG columns. A fraction of the bound antigen, comprising less than 0.15% of the total nuclear protein, was isolated in antigenically active form. The protein moiety of this fraction consisted of 2 quantitatively major polypeptides of MW 30,000 (P30) and 13,000 (P13). Antigens isolated from both anti-nRNP columns possessed essentially the same 2 polypeptides. Immunological tests of crude and purified antigens against anti-nRNP sera from a total of 4 patients provided additional evidence that antibodies from different individuals are directed against the same nRNP antigen. There were no polypeptides in the isolated antigen which corresponded in MW to the core proteins of heterogeneous nuclear ribonucleoprotein (hnRNP) particles described by other investigators. The binding RNA and protein to anti-nRNP columns was greatly reduced by treating the crude antigen with pancreatic RNase A before chromatography. The binding of P13 was reduced to a fraction of the pre-RNase control. None of the 4 sera contained antibodies to DNA, histones, RNA, DNA histone complexes, or nonhistone chromosomal proteins.