Anti-A2/RA33 autoantibodies are directed to the RNA binding region of the A2 protein of the heterogeneous nuclear ribonucleoprotein complex. Differential epitope recognition in rheumatoid arthritis, systemic lupus erythematosus, and mixed connective tissue disease.

The recently described anti-A2/RA33 autoantibodies occur in 20-40% of patients with RA, SLE, and mixed connective tissue disease (MCTD). They are directed to the A2 protein of the heterogeneous nuclear ribonucleoprotein complex (hnRNP-A2), an abundant nuclear protein associated with the spliceosome. The NH2-terminal half of the antigen contains two conserved RNA binding domains whereas its COOH-terminal part is extremely glycine-rich. The aim of this study was to characterize the autoepitopes of hnRNP-A2 and to investigate the effects of anti-A2/RA33 autoantibodies on possible functions of the antigen. Using bacterially expressed fragments, two major discontinuous epitopes were identified. One containing the complete second RNA binding domain was recognized by the majority of patients with RA and SLE but not by patients with MCTD. The second epitope contained sequences of both RNA binding domains and was preferentially targeted by patients with MCTD. When the RNA binding properties of the antigen were investigated, oligoribonucleotides containing the sequence motif r(UUAG) were found to bind to a site closely adjacent or overlapping with the epitope targeted by autoantibodies from patients with RA and SLE. Moreover, anti-A2/RA33 autoantibodies from patients with RA or SLE, but not from patients with MCTD, inhibited binding of RNA. Thus, anti-A2/RA33 autoantibodies recognize conformation-dependent epitopes located in a functionally important region of the antigen. Furthermore, the specific recognition of an epitope by MCTD patients may be used as another argument in favor of considering MCTD a distinct connective tissue disease.

Anti-RA33 autoantibodies may recognize epitopes in the N-terminal region of hnRNP-A2 (RA33).

The nuclear autoantigen RA33, which is identical to the A2 protein of the heterogeneous nuclear ribonucleoprotein (hnRNP-A2), is a nucleic acid binding protein of 341 amino acids. The N-terminal part contains two RNA binding domains whereas the C-terminal part consists of a long glycine-rich region starting around amino acid 192. Autoantibodies to hnRNP-A2/RA33 can be detected in 20-40% of sera from RA, SLE and MCTD patients. So far, it has not been known which regions of A2/RA33 are recognized by these autoantibodies. To address this issue, tryptic fragments of natural A2/RA33 were investigated by immunoblotting using 14 sera from anti-RA33 positive patients with RA (n = 5), SLE (n = 5) and MCTD (n = 4). Most sera reacted with a 22 kD fragment comprising the N-terminal part of the protein. However, a smaller 18 kD fragment was recognized only by 3 RA and 3 MCTD sera whereas two of five SLE sera were reactive with two larger fragments of 26 and 29 kD. In order to further characterize the epitope(s) C-terminal deletion mutants of recombinant A2/RA33 were investigated by immunoblotting employing 27 sera from anti-RA33 positive patients with RA (n = 10), SLE (n = 8), and MCTD (n = 9). All sera recognized a fragment terminating at amino acid 212 which contained the complete N-terminal region as well as 20 amino acids of the glycine-rich section. Thus, these data indicate that the N-terminal part of A2/RA33 contains epitopes for antiA2/RA33 autoantibodies.(ABSTRACT TRUNCATED AT 250 WORDS)