Purification and Partial Sequencing of the Nuclear Autoantigen RA33 Shows That It Is Indistinguishable from the A2 Protein of the Heterogeneous Nuclear Ribonucleoprotein Complex Gunter Steiner, * Klaus Hartmuth, * Karl Skrmner, * Ingrid Maurer-Fogy, Alexandra Sinski, * Eva Thalmann,t Wolfgang Hassfeld,11 Andrea Barta,t and Josef S. Smolen*II *Ludwig Boltzmann Institute for Rheumatology and Balneology, A-1130 Vienna; Institute of Biochemistry, University of Vienna, A- 1090 Vienna; 1Ernst Boehringer Institute, A-1120 Vienna; and 112nd Department ofMedicine, Lainz Hospital, A-1130 Vienna, Austria Abstract erative rheumatic disorders. Therefore, the antigen was termed RA33; this was the first description of a nuclear antigen appar- RA33 is a nuclear autoantigen with an apparent molecular ently specific for RA. mass of 33 kD. Autoantibodies against RA33 are found in about Disease specific immune responses to nuclear antigens 30% of sera from RA patients, but only occasionally in sera have been found for various autoimmune diseases: anti-Sm from patients with other connective tissue diseases. To charac- and anti-dsDNA in systemic lupus erythematosus (SLE), anti- terize RA33, the antigen was purified from HeLa cell nuclear topoisomerase (ScI70) in scleroderma, or anti-tRNA synthe- extracts to more than 90% homogeneity by affinity chromatog- tase in dermato/polymyositis are just a few examples. Other raphy on heparin-Sepharose and by chromatofocusing. Se- autoantibodies, such as anti-U 1 small nuclear ribonucleopro- quence analysis of five tryptic peptides revealed that their se- tein (snRNP),' anti-Ro/SS-A, anti-La/SS-B, or antihistone, quences matched corresponding sequences of the A2 protein of are less specific but nevertheless, they do form valuable tools the heterogeneous nuclear ribonucleoprotein (hnRNP) com- for the diagnosis of connective tissue diseases (for a review, see plex. Furthermore, RA33 was shown to be present in the 40S 7). In general, the target antigens are molecules conserved in hnRNP complex and to behave indistinguishably from A2 in evolution, many of which have the capacity to bind DNA or binding to single stranded DNA. In summary, these data RNA, such as histones, topoisomerase, or snRNP particles in- strongly indicate that RA33 and A2 are the same protein, and volved in posttranscriptional processing of pre-mRNA (8). thus identify on a molecular level a new autoantigen. (J. Clin. In contrast to snRNPs (9, 10), components ofthe heteroge- Invest. 1992. 90:1061-1066.) Key words: rheumatoid arthritis X neous nuclear ribonucleoprotein (hnRNP) complex have only autoimmune diagnostics * antinuclear antibodies * RNA binding rarely been described as target structures for human autoanti- protein* mRNA processing bodies ( 1 1-13). So far, only the Al protein has been identified as autoantigen in a variety of autoimmune diseases (14, 15). Introduction HnRNPs are large nuclear structures composed of heteroge- neous nuclear RNA and 30 different proteins with molecu- RA is the most common autoimmune disease and affects 1-2% lar masses between 34 and 120 kD ( 16). Six of these proteins of the population. Despite many years of intensive research, (Al, A2, B 1, B2, C 1, C2) have been referred to as the hnRNP the etiology of the disease is still poorly understood, and no "core" proteins ( 17), and the sequences of their cDNAs have definite cure is known ( 1). Sera from patients with RA are been recently elucidated ( 18-22). They belong to a group of primarily characterized by the presence of rheumatoid factor, nucleic acid binding proteins that contain an evolutionarily an autoantibody directed against the Fc portion of IgG (2). In highly conserved RNA binding domain of - 90 amino acids, addition, autoantibodies against components of the cell nu- the so-called consensus sequence-type RNA binding domain cleus (antinuclear antibodies) and against EBV-related struc- (CS-RBD [23]). Little is known about the detailed structure tures are frequently observed (3-5). However, neither rheu- and function of the hnRNP complex, but there is some evi- matoid factor nor any of the other autoantibodies have been dence for a functional relationship to the snRNPs (24). found to be specific for RA. Recently, autoantibodies against a In this report, we describe the purification and partial se- nuclear protein with an apparent molecular mass of 33 kD quencing of RA33. Furthermore, it is demonstrated that RA33 have been described (6). These autoantibodies were detected is present in the 40S hnRNP complex, and that it is indistin- in 35% of RA patients but apart from a few exceptions not in guishable from the A2 protein in affinity chromatography on sera from patients suffering from other autoimmune or degen- single stranded DNA (ssDNA). Taken together, the data strongly suggest that RA33 and A2 are identical. Portions of this paper have appeared in abstract form in FASEB (Fed. Am. Soc. Exp. Biol.) J. 1990. 4:A3259; and Clin. Rheumatol. 1990. 9:576. Methods Address correspondence to Gunter Steiner, Ludwig Boltzmann In- stitut fur Rheumatologie und Balneologie, 2. Medizinische Abteilung, Protein purification. HeLa S3 cells or nuclei were obtained frozen from Krankenhaus Lainz, Wolkersbergenstrasse 1, A-I 130-Vienna, Austria. the Computer Cell Culture Center, (University of Mons, Belgium). Receivedfor publication 10 February 1992. J. Clin. Invest. 1. Abbreviations used in this paper: CS-RBD, consensus sequence-type © The American Society for Clinical Investigation, Inc. RNA binding domain; hnRNP, heterogeneous nuclear ribonucleopro- 0021-9738/92/09/1061/06 $2.00 tein; MCTD, mixed connective tissue disease; snRNP, small nuclear Volume 90, September 1992, 1061-1066 ribonucleoprotein; ssDNA, single-stranded DNA. Characterization ofthe Nuclear Autoantigen RA33 1061 Isolation of nuclei and preparation of nuclear extracts was performed rotor (SW40; Beckman Instruments, Fullerton, CA). For reference. as described (6). Briefly, 10 g of nuclei were incubated for 30 min in 4 dissociated 70S E. coli ribosomes were run in parallel. Gradients were ml hypotonic buffer (10 mM Hepes-KOH, 2.5 mM MgCl2, 25% glyc- fractionated from bottom to top into 0.7 ml fractions and their absor- erol, pH 7.9) and subsequently extracted for 40 min by addition of an bence at 260 nm was determined. Protein was recovered from gradient equal volume of the same buffer containing 0.88 M NH4C1. The slurry fractions by TCA precipitation and was analyzed by immunoblotting was centrifuged for 20 min at 20,000 g, and the NH4C1 concentration of with patient serum. the supernatant was adjusted to 0.3 M by diluting with 10 mM Hepes- Gel electrophoresis and immunoblotting. Samples were usually ana- KOH, pH 7.9. lyzed on 12% SDS-polyacrylamide minigels (8 X 5 cm x 0.75 mm); in 40 ml nuclear extract ( - 200 mg protein) was applied to a 2.5 x 20 some experiments electrophoresis was performed on 20 x 14 cm X 1.5 cm heparin-Sepharose CL-6B column (Pharmacia, Uppsala, Sweden) mm gels. Prestained protein markers from Bethesda Research Labora- equilibrated with buffer A (20 mM Hepes-NaOH, 0.3 M NaCl, pH tories were used as mol wt references. Electrophoretic transfer to nitro- 7.9). The column was washed with 3 vol of buffer A, and bound pro- cellulose (BAS 53; Schleicher und Schuell, Germany) was performed tein was eluted with 3 vol of buffer B (20 mM Hepes-NaOH, 1 M NaCl, for 40 min at 250 mA for minigels, and for 2 h at 400 mA for large gels. pH 7.9) followed by 3 vol of buffer C (20 mM Hepes-NaOH, 1 M For immunodetection, the nitrocellulose was blocked for 30 min with a NaCl, 6 M urea, pH 7.9). Protein concentration of each fraction (10 solution of 3% nonfat dried milk in PBS, pH 7.4. Subsequently, the ml) was measured in a microplate reader at 620 nm using the Coomas- blots were incubated for 40 min with serum diluted 1:25 in the same sie Protein Assay Reagent from Pierce Chemical Co. (Rockford, IL). buffer, washed 3 x 5 min with PBS containing 0.1% Triton X-100 Protein-rich fractions were analyzed for the presence ofRA33 by immu- (PBS-Triton) and incubated for 30 min with an alkaline phosphatase noblotting, as described below. RA33 containing fractions were pooled coupled anti-human IgG (Fc) 2nd antibody (Accurate Chemical & ( 10 mgtotal protein), precipitated with 0.4 g/ml ammonium sulphate, Scientific Corp., Westbury, NY) diluted 1:2,500 in PBS-Triton, and redissolved in chromatofocusing starting buffer (20 mM Tris- 3% nonfat dried milk. Finally, bound antibody was detected as de- CH3COOH, 3 M urea, pH 8.7). After dialyzing against the same buffer, scribed (6). proteins were further separated by chromatofocusing on a 15 X 0.6 cm PBE 94 ion exchanger column (Pharmacia). A 1:8 diluted Polybuffer Results 96-acetic acid, pH 6, containing 3 M urea, was used as eluent (flow rate 8 ml/h, fraction size 2 ml). Aliquots were precipitated with 10% TCA, Purification of RA33. A HeLa nuclear extract containing redissolved in Laemmli sample buffer and investigated by immunoblot- - 200 mg protein was chromatographed on heparin-Sepha- ting. Fractions containing RA33 were pooled, precipitated with ammo- rose as nium sulphate, and redissolved in either 20 mM Tris-HCl, pH 7.5, described in Methods. Immunoblot analysis showed containing 6 M urea, or trypsin sequencing buffer (see below). that RA33 was detected exclusively in three fractions of the 1 Protein sequencing. 20 ,ug of purified RA33 were mixed with 0. 1 ml M NaCl/6 M urea eluate (Fig. 1); as these fractions contained of 125 mM ammonium bicarbonate, pH 7.5, containing 2 ug trypsin about 10 mg protein, RA33 was - 20-fold enriched. In addi- sequencing grade (Boehringer Mannheim GmbH, Mannheim, Ger- tion to RA33, two proteins with estimated molecular masses many) and incubated for 6 h at 37°C; an extra 2 ,ug of trypsin was measuring 36 and 37 kD migrating slightly above RA33 were added and incubation was continued for another 16 h.