Identification of the immunogenically active components of the Sm and RNP antigens.

Abstract

The spectrum of cellular targets in the autoimmune diseases is both large and varied and includes among the nuclear components the so-called Sm and RNP antigens associated with systemic lupus erythematosus. The use of immunoaffinity chromatography with dual specificity for the Sm and RNP antigens has allowed for their substantial purification from rabbit thymus in parallel and in quantity. In lieu of a functional assay, the use of a counterimmunoelectrophoresis assay provided a sensitive and rapid means ofmonitoring the distribution ofthe two antigens during purification and ensured the isolation of complexes containing the components required for antigenicity. The resulting purified complex consisted of nine polypeptides, having molecular weights of approximately 9000 to 44,000 and two small RNAs of similar size. However, .limited proteolysis of the isolated complex suggested that most of these polypeptides were not actually required forantigenic activity.,Unlike Sm. in crude thymus extracts, purified Sm was RNase sensitive. Thus, one ofthe major diagnostic criteria used to distinguish Sm and RNP antigens in crude extracts was shown to be invalid for purified material, suggesting that both antigens from rabbit thymus are actually ribonucleoprotein complexes.Autoimmune diseases are characterized by the presence of circulating antibodies that react against components ofthe affected individual's own tissues (1-3). Isolation of the antigens associated with specific autoimmune diseases would facilitate the diagnosis of these diseases and, perhaps, would help in understanding the etiology of these diseases. Among the highly conserved cell components that can be targets of the autoimmune reaction are the so-called Sm and RNP (ribonucleoprotein) antigens. The presence ofantibodies to Sm is diagnostic of the disease systemic lupus erythematosus; anti-RNP antibodies are found in patients suffering from lupus and, more specifically, mixed connective-tissue disease (4, 5). Sm and RNP are both located in the nucleus, giving similar speckled immunofluorescent staining patterns (5). They have been reported to have similar molecular weights (6, 7), to copurify during ion-exchange chromatography (7,8), and to show partial antigenic identity (6,9). In practice, they have been distinguished by differential sensitivity to RNase (4, 10).There have been several reports in recent years on attempts to purify Sm and RNP (8,(11)(12)(13)(14). Most recently Lerner and associates (9, 15), using the technique of immunoprecipitation, identified both Sm and RNP as ribonucleoprotein complexes. We report here a large-scale purification ofthe Sm and RNP antigens in quantities sufficient for physical and chemical characterization. Moreover, the isolation protocol allowed for continuous monitoring of antigenicity, thus ensuring that we were working with antigenically active material. The isolated complexes contained both protein and RNA moieties; and each moiety was composed of a subset of polypeptides and oligoribonucleotides. By using t...

Rapid inhibition of processing and assembly of small nuclear ribonucleoproteins after infection with vesicular stomatitis virus.

“…Thus, the Sm core proteins were synthesized and appeared to be available for association with the RNAs in VSV-infected cells; however, their assembly with the RNAs was inhibited. Because only a subset of the proteins precipitated by anti-Sm serum are antigenic (1,13,27,37,39), the data presented in Fig. 7 and 8 show that in VSV-infected cells, the anti-Sm-reactive core proteins appeared to form a complex in the absence of U RNAs, as shown previously with HeLa cells (6,7).…”

“…The small nuclear ribonucleoproteins (snRNPs) that are immunoprecipitable by the lupus Sm autoantibody specificity have been implicated in the processing of mRNA (14, 26). The snRNPs contain the snRNAs (Ul, U2, U4, U5, and U6) which are also thought to be products of RNA pol II and require trimethyl 5' capping, methylation, and excision of extraneous sequences (1,5,18,27,28,35,37). Previous work has suggested that the snRNAs are synthesized in the nucleus as precursors that are transported to the cytoplasm where they are processed to their mature sizes, assembled into RNPs, and then transported back to the nucleus (5, 6, 34, 42).…”

Autoimmune sera reactive with Sm antigen contain high levels of RNP-like antibodies.

“…In general, this has been accomplished by comparing the titers of antibody reactive with untreated (Sm plus RNP) and RNase-treated (Sm) antigens, as in the hemagglutination assay (2). However, the existence of other RNasesensitive or resistant antigens in nuclear extracts (18), as well as the observation that Sm may be partially sensitive (19,20) and RNP partly resistant (21) to RNase, make a more direct approach desirable. For this reason, we have attempted to distinguish between anti-Sm and anti-RNP antibodies using methods that are independent of the RNase sensitivity or resistance of the antigens to estimate more accurately the relative amounts of these antibodies in SLE and MCTD sera.…”

The U2 small nuclear ribonucleoprotein particle as an autoantigen. Analysis with sera from patients with overlap syndromes.

“…Structurally, the Ul snRNP consists of the U I small RNA and at least nine associated polypeptides (3,(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18). Three of these proteins, known as 68K (Mr = 68,000 D),2 A (Mr = 33,000 D), and C (Mr = 22,000 D) are unique to the U1 snRNP.…”