DNA-binding property of Sm nuclear antigen.

Abstract

Antibodies to nuclear antigens are detected frequently in the sera of patients with systemic lupus erythematosus (SLE) and related autoimmune diseases. These antibodies have multiple immunochemical specificities and they react with different macromolecular components of cell nuclei (1), among which are nonhistone or nuclear acidic proteins. The first nuclear acidic protein characterized immunochemically with the aid of spontaneously occurring antibodies was called Sm nuclear antigen (2). Subsequently, other nuclear acidic protein antigens have been identified in a similar manner and include a nuclear ribonucleoprotein antigen reactive with sera of patients with SLE and mixed connective tissue disease (3) and other acidic nuclear proteins which are reactive with sera of patients with SjSgren's syndrome, scleroderma, and rheumatoid arthritis (4-6).Many nuclear acidic proteins have been shown to have the property of binding to DNA and to play a role in the control of transcription of DNA to RNA in prokaryotic and eukaryotic systems (7). In the studies reported here, we have shown that tissue extracts containing Sm nuclear antigen have the property of binding to single-strand DNA but not to double-strand DNA. Materials and MethodsSource of Sm Antigen The nuclear origin of the Sm antigen had been established m previous studies (2, 4). The antigen was present in rabbit thymus acetone powder (8) and because this was readily available commercially (Pel-Freez Bio-Animals, Inc., Rogers, Ark.) it was used as the source of Sm antigen. 60 mg of this powder was extracted in each 1 ml of phosphate-buffered saline (0.15 M NaC1, 0.01 M PO4, pH 7.3) by gently stirring the suspension at 4°C for 4 h After centmfugatlon at i0,000 rpm for 10 mm, the supernatant solution contained protein varying from 10 to 15 mg/ml Partml purification of Sm antigen was obtained by salting out between 35 and 60% saturation with ammonium sulfate. This fraction contained all Sm antigenic activity but only onehalf of the total protein and was used as the source material m further studies.Immunologwal Detectwn of Sin Antzgenic Actiwty Sera from patients with SLE containing high tlters of antibody to Sm antigen were used as reagents to detect presence of Sm antigen m isolated fractions of rabbit thymus extract. These sera had no detectable antibody activity to other known nuclear antigen-antibody systems, including DNA, deoxymbonucleoprotein, nuclear mbonucleoprotein, and nuclear antigens A and B reported in Sjogren's syndrome (4). In this sense, these SLE sera were "monospeclfiC" for antibodies to Sm. The immunological assays used were immunodiffusion and inhibition ofpasmve hemagglutmation In the latter test, tanned sheep erythrocytes were coated with rabbit thymus extract according to a procedure that resulted m complexing of Sm antigen to the erythrocytes (9) Presence of Sm antigen in isolated fractions was determined by inhibition of hemagglutmation.

Nonhistone nuclear antigens reactive with autoantibodies. Immunofluorescence studies on distribution in synchronized cells.

“…Some of the differences in the findings may be related to the fact that the nuclear antigens may be present in the native state as molecules associated with one another in larger-complexed particles . In a previous study, it was shown that the Sm protein had DNA-binding property and bound more tightly to single-strand DNA than to double-strand DNA (24). Although binding to RNA was not studied, it is possible that Sm protein might bind to RNA molecules in the cell nucleus, and at least some Sm molecules may be associated with RNA protein complexes.…”

The binding of lupus-derived autoantibodies to the C-terminal peptide (83-119) of the major SmD1 autoantigen can be mediated by double-stranded DNA and nucleosomes

“…It was shown previously that single-stranded or dsDNA can bind to Sm proteins. 10 Also, the reactivity of anti-dsDNA-positive sera from patients with SLE with the SmD1(83-119) peptide was much lower after DNaseI treatment of the blocking reagent, although in particular anti-dsDNA-negative anti-SmD-positive sera from patients with SLE retained their reactivity. These data may explain that the use of different ELISA methods may lead to great differences (36-70%) in the prevalence of anti-SmD1(83-119) reactivity in patients with SLE.…”

Identification and characterization of SmD183–119‐reactive T cells that provide T cell help for pathogenic anti–double‐stranded DNA antibodies

“…A precondition for these models is that the highly positively charged SmD1 83–119 or SmD1 molecules bind to DNA and do not critically impair the binding of DNA to anti‐dsDNA antibodies on the B cell surface. Several groups (26, 27) have hypothesized that such SmD1–dsDNA complexes are targets for both anti‐dsDNA and anti‐Sm responses. Our previous studies demonstrated that the addition of dsDNA to SmD1 increased the frequency of SmD1‐reactive T cells in human SLE (17).…”