Purification and partial sequencing of the nuclear autoantigen RA33 shows that it is indistinguishable from the A2 protein of the heterogeneous nuclear ribonucleoprotein complex.

Abstract

RA33 is a nuclear autoantigen with an apparent molecular mass of33 kD. Autoantibodies against RA33 are found in about 30% of sera from RA patients, but only occasionally in sera from patients with other connective tissue diseases. To characterize RA33, the antigen was purified from HeLa cell nuclear extracts to more than 90% homogeneity by affinity chromatography on heparin-Sepharose and by chromatofocusing. Sequence analysis of five tryptic peptides revealed that their sequences matched corresponding sequences of the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex. Furthermore, RA33 was shown to be present in the 40S hnRNP complex and to behave indistinguishably from A2 in binding to single stranded DNA. In summary, these data strongly indicate that RA33 and A2 are the same protein, and thus identify on a molecular level a new autoantigen. (J. Clin.

B and T Cell Responses to the Spliceosomal Heterogeneous Nuclear Ribonucleoproteins A2 and B1 in Normal and Lupus Mice

“…Thus, Abs to Sm and U1 snRNP Ags were described already many years ago to occur in up to 50% of the patients. More recently, Abs to the spliceosome-associated hnRNPs A1 and A2/B1 were found in SLE patients but also in other rheumatic autoimmune disorders, particularly in RA (11)(12)(13)(14)(15)(16). To study the pathological significance of the anti-hnRNP autoimmunity in SLE, we investigated several murine models for the presence of anti-A2/B1 Abs.…”

“…The natural hnRNPs A2/B1 were prepared from HeLa nuclear extracts (20) and partially purified by heparin-Sepharose (Pharmacia, Uppsala, Sweden) chromatography as previously described (14). Recombinant Ags (rA2, rB1) were overexpressed in Escherichia coli either as a GST fusion protein (rA2) or as His-tagged protein (rB1) and affinity-purified using glutathioneSepharose (Pharmacia) or nickel-chelate (Qiagen, Valencia, CA) chromatography, respectively.…”

“…Abs to hnRNPs A2/B1 were detected by Western blotting using HeLa nuclear extracts as well as a preparation of natural hnRNPs as described (14,15,20). Briefly, 200 l of purified hnRNPs (1 mg/ml) were separated on a large preparative 12% polyacrylamide SDS gel and transferred to nitrocellulose membranes.…”

“…Subsequent studies showed hnRNPs A/B to form the major targets of these Abs, particularly hnRNP A2/B1 (11)(12)(13)(14)(15). Although anti-A2/B1 Abs are also present in SLE and MCTD, they might represent an important marker Ab for RA because Abs associated with SLE and/or MCTD, such as Abs to dsDNA and U1-small nuclear ribonucleoproteins (snRNPs), are absent in RA.…”

Current methods for laboratory diagnosis of rheumatoid arthritis

“…Другие аутоантитела Антитела к RA33 (анти-RA33) направлены против ге-терогенного нуклеарного рибонуклеопротеина (hnRNP-A2), участвующего в сплайсинге и транспорте мРНК [101]. При РА происходит гиперэкспрессия RA33 в синовиаль-ной оболочке, вызывающая аутоиммунный ответ [102].…”