Abstract
Objectives
Antinuclear antibodies (ANAs) are associated with several autoimmune diseases. Indirect immunofluorescence (IIF) on human epithelial type 2 (HEp-2) cells is the golden standard for ANA detection in the clinic. In case of a positive HEp-2 IIF test result, follow-up tests are done to determine autoantibody specificity. For a fraction of the HEp-2 IIF-positive samples, the nature of the autoantigens remains uncharacterized. Our objective was to characterize autoantigens in such samples.
Methods
To characterize autoantigens in an unbiased way, we combined protein immunoprecipitation with liquid chromatography (LC) tandem mass spectrometry (MS/MS) sequencing.
Results
Using such approach we detected the Ki antigen, also referred to as PA28γ, in the immunoprecipitate of serum samples of three individuals with an autoimmune disease. The HEp-2 nuclear speckled IIF fluorescent signal of all three serum samples was abolished after pre-absorption of the serum with recombinant Ki antigen, confirming that autoantibodies against Ki underlie the HEp-2 IIF signal.
Conclusions
Our data suggest that anti-Ki autoantibodies can underlie a nuclear speckled HEp-2 IIF pattern.
Funding source: KU Leuven
Award Identifier / Grant number: C3 (C3/20/042)
Acknowledgments
We thank SyBioMa, the Proteomics Core of the Biomedical Science Group, KU Leuven. We thank Kusay Arat for his technical assistance and Professor Sebastien Carpentier for the constructive discussions.
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Research funding: This study was funded by the Research Fund KU Leuven C3 (C3/20/042).
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Author contributions: All authors have accepted responsibility for the entire content of this manuscript and approved its submission.
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Competing interests: Authors declare no competing interests.
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Informed consent: This was a retrospective study using left-over material of samples submitted to the clinical laboratory (secondary use) for which an informed consent waiver was authorized by the ethics committee University Hospital Leuven. Controls gave informed consent (study S63708 Ethics committee UZ Leuven).
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Ethical approval: The study was approved by the Ethical Committees of the University Hospitals Leuven (S60347/B32220071204) (Leuven). An MTA/DTA between University Hospitals Leuven and Hôpital Henri Mondor was in place.
References
1. Mahler, M, Meroni, PL, Bossuyt, X, Fritzler, MJ. Current concepts and future directions for the assessment of autoantibodies to cellular antigens referred to as anti-nuclear antibodies. J Immunol Res 2014;2014:315179. https://doi.org/10.1155/2014/315179.Search in Google Scholar PubMed PubMed Central
2. Aringer, M, Costenbader, K, Daikh, D, Brinks, R, Mosca, M, Ramsey-Goldman, R, et al.. Ann Rheum Dis 2019;78:1151–9.10.1136/annrheumdis-2018-214819Search in Google Scholar PubMed
3. Meroni, PL, Schur, PH. ANA screening: an old test with new recommendations. Ann Rheum Dis 2010;69:1420–2. https://doi.org/10.1136/ard.2009.127100.Search in Google Scholar PubMed
4. Bossuyt, X, De Langhe, E, Borghi, MO, Meroni, PL. Understanding and interpreting antinuclear antibody tests in systemic rheumatic diseases. Nat Rev Rheumatol 2020;16:715–26. https://doi.org/10.1038/s41584-020-00522-w.Search in Google Scholar PubMed
5. De Beeck, KO, Vermeersch, P, Verschueren, P, Westhovens, R, Mariën, G, Blockmans, D, et al.. Detection of antinuclear antibodies by indirect immunofluorescence and by solid phase assay. Autoimmun Rev 2011;10:801–8. https://doi.org/10.1016/j.autrev.2011.06.005.Search in Google Scholar PubMed
6. Bossuyt, X, Claessens, J, De Langhe, E, Belmondo, T, Westhovens, R, Hue, S, et al.. Antinuclear antibodies by indirect immunofluorescence and solid phase assays. Ann Rheum Dis 2020;79:e65. https://doi.org/10.1136/annrheumdis-2019-215443.Search in Google Scholar PubMed
7. Kurata, N, Tan, EM. Identification of antibodies to nuclear acidic antigens by counterimmunoelectrophoresis. Arthritis Rheum 1976;19:574–80. https://doi.org/10.1002/art.1780190309.Search in Google Scholar PubMed
8. Tan, EM, Kunkel, HG. Characteristics of a soluble nuclear antigen precipitating with sera of patients with systemic lupus erythematosus. J Immunol 1966;96:464–71.10.4049/jimmunol.96.3.464Search in Google Scholar
9. Tojo, T, Kaburaki, J, Hayakawa, M, Okamoto, T, Tomii, M, Homma, M. Precipitating antibody to a soluble nuclear antigen “Ki” with specificity for systemic lupus erythematosus. Ryumachi 1981;21(1 Suppl):129–40.Search in Google Scholar
10. Harmon, C, Peebles, C, Tan, EM. SL a new precipitation system. Arthritis Rheumatol 1981;24:S122.Search in Google Scholar
11. Ahn, JY, Tanahashi, N, Akiyama, K, Hisamatsu, H, Noda, C, Tanaka, K, et al.. Primary structures of two homologous subunits of PA28, a gamma-interferon-inducible protein activator of the 20S proteasome. FEBS Lett 1995;366:37–42. https://doi.org/10.1016/0014-5793(95)00492-r.Search in Google Scholar PubMed
12. Wilk, S, Chen, WE, Magnusson, RP. Properties of the nuclear proteasome activator PA28gamma (REGgamma). Arch Biochem Biophys 2000;383:265–71. https://doi.org/10.1006/abbi.2000.2086.Search in Google Scholar PubMed
13. Whitby, FG, Masters, EI, Kramer, L, Knowlton, JR, Yao, Y, Wang, CC, et al.. Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 2000;408:115–20. https://doi.org/10.1038/35040607.Search in Google Scholar PubMed
14. Nikaido, T, Shimada, K, Shibata, M, Hata, M, Sakamoto, M, Takasaki, Y, et al.. Cloning and nucleotide sequence of cDNA for Ki antigen, a highly conserved nuclear protein detected with sera from patients with systemic lupus erythematosus. Clin Exp Immunol 1990;79:209–14. https://doi.org/10.1111/j.1365-2249.1990.tb05180.x.Search in Google Scholar PubMed PubMed Central
15. Jiang, H, Monaco, JJ. Sequence and expression of mouse proteasome activator PA28 and the related autoantigen Ki. Immunogenetics 1997;46:93–8. https://doi.org/10.1007/s002510050246.Search in Google Scholar PubMed
16. Zannini, L, Lecis, D, Buscemi, G, Carlessi, L, Gasparini, P, Fontanella, E, et al.. REGgamma proteasome activator is involved in the maintenance of chromosomal stability. Cell Cycle 2008;7:504–12. https://doi.org/10.4161/cc.7.4.5355.Search in Google Scholar PubMed
17. Murata, S, Kawahara, H, Tohma, S, Yamamoto, K, Kasahara, M, Nabeshima, Y, et al.. Growth retardation in mice lacking the proteasome activator PA28gamma. J Biol Chem 1999;274:38211–5. https://doi.org/10.1074/jbc.274.53.38211.Search in Google Scholar PubMed
18. Levy-Barda, A, Lerenthal, Y, Davis, AJ, Chung, YM, Essers, J, Shao, Z, et al.. Involvement of the nuclear proteasome activator PA28γ in the cellular response to DNA double-strand breaks. Cell Cycle 2011;10:4300–10. https://doi.org/10.4161/cc.10.24.18642.Search in Google Scholar PubMed PubMed Central
19. Baldin, V, Militello, M, Thomas, Y, Doucet, C, Fic, W, Boireau, S, et al.. A novel role for PA28gamma-proteasome in nuclear speckle organization and SR protein trafficking. Mol Biol Cell 2008;19:1706–16. https://doi.org/10.1091/mbc.e07-07-0637.Search in Google Scholar PubMed PubMed Central
20. Huang, L, Haratake, K, Miyahara, H, Chiba, T. Proteasome activators, PA28γ and PA200, play indispensable roles in male fertility. Sci Rep 2016;6:23171. https://doi.org/10.1038/srep23171.Search in Google Scholar PubMed PubMed Central
21. Shen, M, Wang, Q, Xu, S, Chen, G, Xu, H, Li, X, et al.. Role of oncogenic REGγ in cancer. Biomed Pharmacother 2020;130:110614. https://doi.org/10.1016/j.biopha.2020.110614.Search in Google Scholar PubMed
22. Cascio, P. PA28γ: new insights on an ancient proteasome activator. Biomolecules 2021;11:228. https://doi.org/10.3390/biom11020228.Search in Google Scholar PubMed PubMed Central
23. Bernstein, RM, Morgan, SH, Bunn, CC, Gainey, RC, Hughes, GR, Mathews, MB. The SL autoantibody-antigen system: clinical and biochemical studies. Ann Rheum Dis 1986;45:353–8. https://doi.org/10.1136/ard.45.5.353.Search in Google Scholar PubMed PubMed Central
24. Sakamoto, M, Takasaki, Y, Yamanaka, K, Kodama, A, Hashimoto, H, Hirose, S. Purification and characterization of Ki antigen and detection of anti-Ki antibody by enzyme-linked immunosorbent assay in patients with systemic lupus erythematosus. Arthritis Rheum 1989;32:1554–62. https://doi.org/10.1002/anr.1780321209.Search in Google Scholar PubMed
25. Yamanaka, K, Takasaki, Y, Nishida, Y, Shimada, K, Shibata, M, Hashimoto, H. Detection and quantification of anti-Ki antibodies by enzyme-linked immunosorbent assay using recombinant Ki antigen. Arthritis Rheum 1992;35:667–71. https://doi.org/10.1002/art.1780350610.Search in Google Scholar PubMed
26. Cavazzana, I, Franceschini, F, Vassalini, C, Danieli, E, Quinzanini, M, Airò, P, et al.. Clinical and serological features of 35 patients with anti-Ki autoantibodies. Lupus 2005;14:837–41. https://doi.org/10.1191/0961203305lu2226oa.Search in Google Scholar PubMed
27. Fredi, M, Cavazzana, I, Quinzanini, M, Taraborelli, M, Cartella, S, Tincani, A, et al.. Rare autoantibodies to cellular antigens in systemic lupus erythematosus. Lupus 2014;23:672–7. https://doi.org/10.1177/0961203314524850.Search in Google Scholar PubMed
28. Matsushita, M, Takasaki, Y, Takeuchi, K, Yamada, H, Matsudaira, R, Hashimoto, H. Autoimmune response to proteasome activator 28alpha in patients with connective tissue diseases. J Rheumatol 2004;31:252–9.Search in Google Scholar
29. Arribas, J, Rodríguez, ML, Forno, RAD, Castaño, JG. Autoantibodies against the multicatalytic proteinase in patients with systemic lupus erythematosus. J Exp Med 1991;173:423–7. https://doi.org/10.1084/jem.173.2.423.Search in Google Scholar PubMed PubMed Central
30. Feist, E, Dörner, T, Kuckelkorn, U, Schmidtke, G, Micheel, B, Hiepe, F, et al.. Proteasome alpha-type subunit C9 is a primary target of autoantibodies in sera of patients with myositis and systemic lupus erythematosus. J Exp Med 1996;184:1313–8. https://doi.org/10.1084/jem.184.4.1313.Search in Google Scholar PubMed PubMed Central
31. Feist, E, Brychcy, M, Hausdorf, G, Hoyer, B, Egerer, K, Dörner, T, et al.. Anti-proteasome autoantibodies contribute to anti-nuclear antibody patterns on human larynx carcinoma cells. Ann Rheum Dis 2007;66:5–11. https://doi.org/10.1136/ard.2006.055152.Search in Google Scholar PubMed PubMed Central
Supplementary Material
The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2022-0858).
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