Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation This information is current as Yitian Cai, Seng Yin Kelly Wee, Junjie Chen, Boon Heng of September 25, 2021. Dennis Teo, Yee Leng Carol Ng, Khai Pang Leong and Jinhua Lu J Immunol published online 25 October 2017 http://www.jimmunol.org/content/early/2017/10/25/jimmun ol.1700728 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2017/10/25/jimmunol.170072 Material 8.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published October 25, 2017, doi:10.4049/jimmunol.1700728 The Journal of Immunology Broad Susceptibility of Nucleolar Proteins and Autoantigens to Complement C1 Protease Degradation Yitian Cai,*,1 Seng Yin Kelly Wee,*,1 Junjie Chen,* Boon Heng Dennis Teo,* Yee Leng Carol Ng,† Khai Pang Leong,† and Jinhua Lu* Anti-nuclear autoantibodies, which frequently target the nucleoli, are pathogenic hallmarks of systemic lupus erythematosus (SLE). Although the causes of these Abs remain broad and ill-defined, a genetic deficiency in C1 complex (C1qC1r2C1s2) or C4 is able to induce these Abs. Considering a recent finding that, in dead cells, nucleoli were targeted by C1q and two nucleolar autoantigens were degraded by C1r/C1s proteases, we considered that C1 could help protect against antinuclear autoimmunity by broadly degrading nucleolar proteins or autoantigens. Nucleoli were isolated to homogeneity and structurally defined. After C1 treatment, cleaved nucleolar proteins were identified by proteomic two-dimensional fluorescence difference gel electrophoresis and mass spectrometry, and further verified by Western blotting using specific Abs. The extent of nucleolar autoantigen degradation upon Downloaded from C1 treatment was estimated using SLE patient autoantibodies. The isolated nucleoli were broadly reactive with SLE patient autoantibodies. These nucleoli lacked significant autoproteolysis, but many nucleolar proteins and autoantigens were degraded by C1 proteases; >20 nucleolar proteins were identified as C1 cleavable. These were further validated by Western blotting using specific Abs. The broad autoantigenicity of the nucleoli may attribute to their poor autoproteolysis, causing autologous immune stimulation upon necrotic exposure. However, C1q targets at these nucleoli to cause C1 protease activation and the cleavage of many nucleolar proteins or autoantigens. This may represent one important surveillance mechanism against antinuclear auto- http://www.jimmunol.org/ immunity because C1 genetic deficiency causes anti-nuclear autoantibodies and SLE disease. The Journal of Immunology, 2017, 199: 000–000. ystemic lupus erythematosus (SLE) is a complex auto- of anti-nuclear autoantibodies remain unclear, and understanding immune disease with limited treatment options (1, 2). their origins can greatly expand therapeutic options. S Although progress has been made in understanding the In live cells, nuclear Ags are segregated from autologous im- underlying pathogenesis of this disease, including the hallmark mune cell recognition and responses. Early apoptotic cells similarly contributions of anti-nuclear autoantibodies (3–5), chronic eleva- conceal these Ags, and also actively suppress proinflammatory by guest on September 25, 2021 tion of IFN-a (6–10), and accumulated apoptotic bodies (11–13), responses from phagocytes and other immune cells (16). However, how these modular mechanisms are activated and rally toward exposure of these intracellular materials can occur when cell death SLE disease remains poorly understood. Anti-nuclear autoanti- takes the necrotic pathway (17, 18). The naive B cell repertoire bodies are early pathogenic factors that can manifest long before contains significant autoreactive B cells (19). The autoimmunogenicity SLE disease onset, whereas IFN-a and apoptotic cellular Ags of nuclear materials has been demonstrated when anti-nuclear au- appear to surge at disease flare (1, 3, 14, 15). The primary causes toantibodies were found induced following the injection of dead cells into mice (11, 20). This was especially prominent when ad- juvant was also injected (20). This reaction inevitably involves in- *Department of Microbiology and Immunology, Yong Loo Lin School of Medicine tracellular autoantigens and potentially intracellular adjuvants such and Immunology Programme, National University of Singapore, Singapore 117597, as IL-1a, IL-33, S100 proteins or high mobility group box 1 Singapore; and †Department of Rheumatology, Allergy and Immunology, Tan Tock Seng Hospital, Singapore 308433, Singapore (HMGB1), which are also known as danger-associated molecular 1Y.C. and S.Y.K.W. contributed equally to this work. patterns (DAMPs) or alarmins (18, 21, 22). Incidentally, HMGB1 ORCIDs: 0000-0002-5462-2118 (Y.C.); 0000-0001-9403-505X (S.Y.K.W.); 0000- is released in complex with chromatin fragments from necrotic 0003-4696-9813 (B.H.D.T.). cells, which induces lupus-like antinuclear responses in mice (21). Received for publication May 19, 2017. Accepted for publication October 5, 2017. Genetic studies have revealed .50 SLE risk genes, broadly This work was supported by a Singapore Ministry of Education Tier 2 grant related to inflammation, immune clearance, and IFN-a–producing (MOE2012-T2-2-122), a Singapore Ministry of Health National Medical Research or signaling pathways (23–25). These risk genes mostly exhibit Council grant (NMRC/OFIRG/0013/2016), and a National University Health System low-to-moderate impacts on SLE disease and are mostly not seed fund (T1-BSRG 2015-07). specific for SLE pathogenesis. More than 20 monogenic SLE risk Address correspondence and reprint requests to Dr. Jinhua Lu, Department of Mi- crobiology and Immunology, Yong Loo Lin School of Medicine, National University genes have also been reported, which also broadly impact on other of Singapore, Blk MD4, 5 Science Drive 2, Singapore 117597, Singapore. E-mail autoimmune diseases (26). However, genetic deficiencies in a address: [email protected] group of complement proteins (i.e., C1q, C1r/C1s, and C4) show The online version of this article contains supplemental material. strong and highly specific association with SLE pathogenesis (23, Abbreviations used in this article: C1-inh, C1 inhibitor; DAMP, danger-associated 27–29). These patients develop anti-nuclear autoantibodies and molecular pattern; DC, dendritic cell; 2D-DIGE, two-dimensional fluorescence dif- ference gel electrophoresis; HMGB1, high mobility group box 1; hnRNPU, hetero- manifest severe SLE disease characterized by early disease onset geneous nuclear ribonucleoprotein U; NCL, nucleolin; NG1, nuclear ghost 1; NPM1, and equal gender susceptibility. These complement proteins are nucleophosmin 1; SLE, systemic lupus erythematosus. intimately related; C1q, C1r, and C1s form the C1 (C1qC1r2C1s2) Copyright Ó 2017 by The American Association of Immunologists, Inc. 0022-1767/17/$35.00 complex in which C1q is a scaffold, and C1r and C1s are effector www.jimmunol.org/cgi/doi/10.4049/jimmunol.1700728 2 COMPLEMENT DEGRADATION OF NUCLEOLAR Ags serine proteases (30, 31). C1 complex defines the complement In determining C1q binding sites in dead cells, we recently classical pathway: binding of C1q to Ag-bound Abs causes acti- observed that C1q bound to the cell periphery in early apoptosis, vation of C1r followed by C1s, which subsequently cleaves C4 but it bound predominantly to the nucleoli during late apoptosis and C2 to trigger the complement cascade (32). (48). Isolated nucleoli were also recognized by C1q, causing ac- The strong causal association between C1 or C4 deficiency and tivation of the C1s protease and the cleavage of two major nu- SLE pathogenesis cannot be explained in the traditional paradigm cleolar proteins, nucleophosmin-1 (NPM1) and nucleolin (NCL) of the complement system, which consists of nearly 30 plasma (48). Because anti-nucleolus autoantibodies are prevalent in SLE proteins (23, 28). C2 deficiency is relatively common and also patients and are also significant autoantibody specificities in SLE contributes to SLE pathogenesis (27, 33). However, it lacks the mice and some cancer patients (49–52), in this study, we estimated type of strong and specific effect, early disease onset, and equal the extent of nucleolar protein and autoantigen degradation by C1 gender susceptibility found with C1 and C4 deficiencies. In some proteases through a proteomic approach. patients, C1 inhibitor (C1-inh) deficiency can manifest mild SLE- like conditions, and this could be explained by excessive C1r or Materials and Methods C1s activation and consumption of intimately related complement Cell culture and Abs elements