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 October 2, 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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2017 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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

tion of IFN-a (6–10), and accumulated apoptotic bodies (11–13), responses from phagocytes and other immune cells (16). However, by guest on October 2, 2021 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 because serum C1q, C1s, C4, and C2 levels appeared to be reduced in these patients (34–36). In the Japanese population, Human cervical adenocarcinoma HeLa cells were cultured in DMEM containing 10% (v/v) HyClone FBS, 100 U/ml penicillin, 100 mg/ml C3-deficient patients were reported with clinical SLE-like pre- streptomycin, and 2 mM L-glutamine at 37˚C and 5% CO2. Mouse anti- sentations, although immunological presentations were either NPM1 (B0556) and rabbit anti-DDX5 (HPA020043) Abs were purchased absent or mild (37, 38). Genetic deficiencies in most other com- from Sigma-Aldrich (St. Louis, MO). Rabbit Abs for NCL (ab22758), plement proteins are not significant risk factors for SLE (39). A fibrillarin (ab5821), hnRNPC (ab97541), DDX21 (ab126968), BOP1 Downloaded from (ab86982), EIF2S1 (ab26197), RRP9 (ab168845), RPL30 (ab170930), possible context to understand the strong causal association be- SFPQ (ab38148), and hnRNPA2B1 (ab6102) were obtained from Abcam tween C1/C4 deficiency and SLE pathogenesis revolves around (Cambridge, U.K.). Goat anti-C1q (A301) and anti-C1s (A302) Abs were the ability of C1q to recognize dead cells (30, 31, 40) and the purchased from Quidel (San Diego, CA). A rabbit anti-human histone H3 ability of both C1q and activated C4 to opsonize dead cells for Ab (9715), the C1 complex (204873), and the C1-inh (204883) were purchased immune clearance (13, 41). Because apoptotic bodies are known from Merck Millipore (Billerica, MA). For most Ags, gene symbols are used

with full names being detailed in Tables I and II. http://www.jimmunol.org/ to accumulate in SLE patients and, in mice, injection of dead cells causes antinuclear autoimmunity (11, 13, 41, 42), the clearance Isolation of nucleoli functions of C1q and C4 are clearly relevant to SLE pathogenesis. Nucleoli were isolated from HeLa cells (48). In brief, cells in a 0.25-M However, other mechanisms may also be important by which these sucrose buffer (0.25 M sucrose, 5.0 mM MgCl2, and 10 mM Tris [pH 7.4]) complement proteins increase host tolerance to autologous nuclear were homogenized and, after centrifugation for 10 min at 600 3 g, crude Ags, for example, C1q regulation of dendritic cells (DCs) and C4- nuclei were further centrifuged in a 2.2-M sucrose buffer (2.2 M sucrose, 5.0 mM MgCl2, and 10 mM Tris [pH 7.4]) for 30 min at 50,000 3 g (20,000 rpm). mediated B cell inhibition or tolerance (13, 43–46). How C1r/C1s Nuclei were sonicated and centrifuged at 2500 3 g for 2 min, to collect the deficiency causes SLE has been mainly intuited based on studies supernatant as nuclear ghost 1 (NG1) and, after resuspension in 0.88 M of C1q and C4 (28, 47). sucrose containing 5.0 mM MgCl2 and 10 mM Tris (pH 7.4), centrifuged for by guest on October 2, 2021

FIGURE 1. Anti-nucleolus autoantibodies are broadly detected in SLE patients. (A) Schematic illustration of nucleoli and nuclear ghost fractions isolated from HeLa cells. (B) Two nucleoplasmic fractions, derived during nucleolus isolation (NG1 and NG2), were compared with isolated nucleoli (nucleolus) in protein profiles (total protein). Samples were normalized to OD280 reading of 2.0; then each sample was loaded at 12 ml. Autoantigens in these three fractions were detected by Western blotting using 14 SLE patient IgG autoantibodies (SLE37-50). IgG was also isolated from a normal human serum and used as a control. Representative data are shown for at least two independent experiments. The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/

FIGURE 2. Fluorescent and scanning electron microscopy images of isolated nucleoli. (A–C) Nucleoli were fixed in 1% (w/v) paraformaldehyde and stained for 30 min consecutively with a mouse anti-NPM1 Ab followed by goat anti-mouse IgG (Cy3, red). (A) Reconstructed three-dimensional light images; (B) 0.38-mm section images of NPM1 signals. (C) Section images of merged NPM1 and chromatin signals. Scale bar, 3 mm. (D and E) Nucleoli on by guest on October 2, 2021 coverslips were fixed for 2 h in 2.5% (w/v) glutaraldehyde, oxidized, gold-sputtered, and analyzed by scanning electron microscopy. (D) The image of a representative singular nucleolus (original magnification 327,000). (E) The images of three interconnected nucleoli (original magnification 320,000). Representative fluorescent and scanning electron microscopy images are shown for three and two independent experiments, respectively.

5 min at 1000 3 g to collect the supernatant as NG2. The crude nucleoli Tris, 150 mM NaCl, and 3.3 mM CaCl2 (pH 7.4). C1 was stored at 270˚C were centrifuged through a 0.85- to 2.5-M sucrose gradient for 2 h at in an acidic buffer (300 mM NaCl, 50 mM sodium acetate, 50 mM ε amino 100,000 3 g (Fig. 1A). The pelleted nucleoli are resuspended in the 0.25-M caproic acid, 25 mM p-nitrophenyl-p9-guanido benzoate, 10 mM benzamidine, sucrose buffer, giving rise to A280 readings from 2.0 to 4.0. 10 mM EDTA, 40% [v/v] glycerol [pH 5.5]) and diluted immediately before use. After incubation at 37˚C for up to 2 h, reactions were stopped using Confocal microscopy SDS-PAGE sample buffer and analyzed by SDS-PAGE and Western blotting. m m Isolated nucleoli (5 ml) were incubated for 5 min on ice with 12-mm glass Where C1-inh was used, 12.5 lofC1(38.4 g/ml) was first premixed with 12.5 ml of C1-inh (320 mg/ml) on ice. This was then incubated with 25 ml coverslips and fixed for 30 min in 1% (w/v) paraformaldehyde. The cov- of nucleoli. erslips were briefly washed and immunostained for 30 min with anti-NPM1 and then for 30 min with goat anti-mouse IgG (Cy3). Coverslips were SLE patient autoantibodies mounted with DAPI-containing VECTASHIELD medium and viewed using the FluoView FV1000 confocal microscope equipped with a 1003 oil ob- Plasma samples were obtained from SLE patients in the Tan Tock Seng jective (aperture 1.45) and Cool/SNAP HQ2 image acquisition camera Hospital, Singapore, with Institutional Ethics approval. All patients fulfilled (Olympus). Images were captured with the FV-ASW 1.6b software and the diagnosis according to the 1982 American College of Rheumatology analyzed using the Imaris software (Bitplane AG). criteria for SLE (53). IgG was isolated from these plasma samples using the HiTrap Protein G columns (GE Healthcare) and used in Western blotting. Scanning electron microscopy Western blotting Coverslip-bound nucleoli were also fixed for 2 h in 2.5% (v/v) glutaraldehyde and, after washing in PBS, oxidized for 30 min in 1% (w/v) OsO4 (pH 7.4). Samples were separated on 12.5% (w/v) SDS-PAGE gels, and the blots Dehydration was performed at rising ethanol concentrations (i.e., 50, 75, 95, were, after blocking for 1 h in TBS-T (50 mM Tris, 150 mM NaCl [pH 7.4], and 100%). The samples were equilibrated with liquid CO2 using an EM and 0.1% [v/v] Tween 20) containing 5% [w/v] nonfat milk, incubated CPD 030 Critical Point Dryer (Leica Camera AG, Wetzlar, Germany). After overnight at 4˚C with specific Abs (10 mg/ml). After washing, the blots gold-sputtering with a Leica BAL-TEC SCD 005 Sputter Coater (100 s at 30 mA), were incubated for 2 h with HRP-conjugated secondary Abs. Signals were images were acquired at 10 KV and 20 mA emission current, using a JSM-6701F visualized using the West Dura Extended Duration substrate (Thermo Field Emission Scanning Microscope (JOEL, Tokyo, Japan). Scientific). C1 treatment of nucleoli Two-dimensional fluorescence difference gel electrophoresis Treatments were performed in 50-ml reactions, that is, 25 ml of purified These experiments were performed with assistance from Applied Biomics nucleoli (OD280 = 2.0) and 25 ml of C1 dilution (19.2 mg/ml) in 10 mM (Hayward, CA). Nucleoli were incubated with C1 for 2 h at 37˚C or 4˚C. 4 COMPLEMENT DEGRADATION OF NUCLEOLAR Ags

25,000 3 g. Thirty micrograms of each protein lysate was labeled in the dark for 30 min on ice with 1.0 ml of 0.2 mM Cy2 (4˚C sample) and Cy5 (37˚C sample). After adding 1.0 ml of 10 mM lysine and 15-min incubation on ice, the samples were combined and then mixed with 23 2D sample buffer (8 M urea, 4% CHAPS, 20 mg/ml DTT, 2% pharmalytes, and trace amount of bromophenol blue). One hundred microliters of DeStreak solution and rehy- dration buffer (7 M urea, 2 M thiourea, 4% CHAPS, 20 mg/ml DTT, 1% pharmalytes, and trace amount of bromophenol blue) was added for the 18-cm immobilized pH gradient strip. After mixing and centrifugation, these samples were loaded into the strip holder for isoelectric focusing and SDS-PAGE separation. Images were scanned using Typhoon TRIO and analyzed using the Image QuantTL software (GE Healthcare). Gels were analyzed using the DeCyder software version 6.5 (GE Healthcare). MALDI-TOF and TOF/TOF tandem mass spectrometry Spots were picked using the Ettan Spot Picker facilitated by the DeCyder software and, after washing, in-gel digested using the protease Trypsin Gold (Promega). A spot was considered C1-cleaved if it disappeared or was markedly reduced in the 37˚C sample. After desalting on Zip-tip C18 (Millipore), the tryptic peptides were eluted with 0.5 ml of matrix solution (a-cyano-4-hydroxycinnamic acid, 5 mg/ml in 50% acetonitrile, 0.1% trifluoroacetic acid, 25 mM ammonium bicarbonate) and spotted on the FIGURE 3. C1 protease degradation of proteins in the nucleoli and

MALDI plate for MALDI-TOF mass spectrometry and TOF/TOF tandem Downloaded from A B NG2. Isolated nucleoli ( ) or the nuclear ghost fraction NG2 ( ) were mass spectrometry analyses using the 5800 Mass Spectrometer (AB Sciex). 2+ incubatedwithC1for0.5,1,and2hat37˚C.C1wasdilutedfromaCa -depleted The resultant peptide mass and fragmentation spectra were submitted to acidic buffer immediately before use. As controls, incubation was carried GPS Explorer (version 3.5) equipped with the MASCOT search engine out for 2 h at 4˚C. These samples were then analyzed by SDS-PAGE on (Matrix Science) to search the NCBInr database without constraining m.w. 12.5% (w/v) gels. As controls, C1, untreated nucleoli, or untreated NG2 or isoelectric point and with variable carbamidomethylation of cysteine and were also included in the gels. Protein bands that clearly diminished fol- oxidation of methionine residues. One missed cleavage was allowed in the lowing C1 incubation are indicated with arrowheads. Protein bands that search parameters. Candidates with either protein score confidence interval or ion confidence interval .95% were considered significant. http://www.jimmunol.org/ appeared after C1 incubation are marked by asterisks (*). Molecular mass standards are labeled on the left. Representative data are shown for at least three independent experiments. Ghost, NG2. Results Anti-nucleolus autoantibodies are common in SLE patients The two samples (∼200 mg each) were each dissolved in 0.2 ml of a cell lysis buffer (30 mM Tris-HCl [pH 8.8] containing 7 M urea, 2 M thiourea, In clinical diagnosis, a common method to detect total autoanti- and 4% CHAPS). The samples were sonicated on ice, mixed for 30 min at bodies is indirect immunofluorescence microscopy. In this study, room temperature, and cleared by centrifugation for 30 min at 4˚C and we use isolated nucleoli and isolated nucleoplasmic fractions, by guest on October 2, 2021

FIGURE 4. 2D-DIGE analysis of C1-digested nucleoli. (A) Schematic illustration of nucleolus digestion with C1 complex. One C1 complex consists of one C1q, two C1r proteases, and two C1s proteases. At 37˚C, C1q binding to nucleolus causes C1r activation, which then activates C1s to cause nucleolar protein degradation. C1 was diluted from a Ca2+-depleted acidic buffer immediately before use. Nucleoli were incubated with C1 at 37˚C or, as a control, 4˚C for 2 h and then precipitated with trichloroacetic acid. Samples were then labeled with Cy2 (green) and Cy5 (red), respectively, and analyzed by 2D electrophoresis. (B) Based on merged signals, protein spots that exhibited orange color are considered insignificantly cleaved by C1 proteases. Those spots that exhibited dominant green signals are considered cleaved at 37˚C, but not 4˚C. Those exhibited dominant red signals are considered cleavage products. Spots thatwere picked for mass spectrometry are numerically labeled. pH standards are indicated at the bottom (pH 4–9). Molecular mass standards are indicated on both sides. The 2D-DIGE experiment was performed twice, but spots were picked in only one experiment. The Journal of Immunology 5

Table I. Proteins diminished following nucleoli treatment with C1 at 37˚C

Spot IDa Accession Number Protein Name (Gene Name) Predicted Sizes (kDa) 2 C1S_HUMAN Complement C1s subcomponent (C1S) 77 5 NUCL_HUMAN NCL (NCL) 77 6 BOP1_HUMAN Ribosome biogenesis protein BOP1 84 14 NUCL_HUMAN NCL (NCL) 77 20 NUCL_HUMAN NCL (NCL) 77 21 NUCL_HUMAN NCL (NCL) 77 26 ZNF326_HUMAN DBIRD complex subunit ZNF326 66 28 CWC27_HUMAN Peptidyl-prolyl cis-trans isomerase CWC27 54 29 RED_HUMAN Protein red (IK) 66 39 U3IP2_HUMAN U3 small nucleolar RNA-interacting protein 52 2 RRP9 41 DDX5_HUMAN Probable ATP-dependent RNA helicase 69 DDX5 42 DDX47_HUMAN Probable ATP-dependent RNA helicase 51 DDX47 56 EEF1A3_HUMAN Putative elongation factor 1-a–like 3 50 (EEF1A1P5) 57 HNRNPC_HUMAN Heterogeneous nuclear ribonucleoproteins 34 C1/C2 62 IF2A_HUMAN Eukaryotic translation initiation factor 2 36 Downloaded from subunit 1 (EIF2S1) 65 SRSF1_HUMAN Serine/arginine-rich splicing factor 1 28 70 PP1G_HUMAN Serine/threonine-protein phosphatase PP1g 37 catalytic subunit (PPP1CC) 72 HNRNPC_HUMAN Heterogeneous nuclear ribonucleoproteins 34 C1/C2 (HNRNPC)

73 ROA2_HUMAN Heterogeneous nuclear ribonucleoproteins 37 http://www.jimmunol.org/ A2/B1 (HNRNPA2B1) 81 SRSF1_HUMAN Serine/arginine-rich splicing factor 1 28 (SRSF1) 115 NPM_HUMAN NPM1 (NPM1) (may contain 33 RPL12_human) 116 RPA49_HUMAN DNA-directed RNA polymerase I subunit 54 RPA49 127 RS10_HUMAN 40S ribosomal protein S10 (RPS10) 19 146 SFPQ_HUMAN Splicing factor, proline- and glutamine-rich 76 (SFPQ) 147 DDX21_HUMAN Nucleolar RNA helicase 2 (DDX21) 87 by guest on October 2, 2021 151 DDX5_HUMAN Probable ATP-dependent RNA helicase 69 DDX5 (may contain UTP18_human) 153 RBMX_HUMAN RNA-binding motif protein, X chromosome 42 (RBMX) aSpot ID corresponds to the number assigned to a picked spot from the 2D-DIGE analysis (Fig. 4). which are represented by the nuclear ghosts (NG1 and NG2) (48), to Structural features of nucleoli and the nuclear ghosts detect these Abs. The homogeneity of isolated nucleoli was esti- Isolated nucleoli were particles of ∼1–3 mm in diameter (Fig. 1A, mated based on the enrichment of two nucleolar proteins, NPM1 Supplemental Fig. 1A). These particles stained strongly for NPM1 and NCL (Fig. 1B). This was further validated by immunofluores- (Supplemental Fig. 1B). Based on the 0.38-mm section images, cence staining of the nucleolus particles (Supplemental Fig. 1). NPM1 formed a compact and continuous layer surrounding each Western blotting was performed to compare nucleoli with NG1 nucleolus, and it overlapped with a surface layer of dense chro- and NG2 for the presence of autoantigens. Fourteen SLE patient matins (Supplemental Fig. 1C, 1D). These morphological features Abs were used (SLE37-50), which reacted heterogeneously with were more clearly observed at higher magnifications (Fig. 2B, 2C). these three Ag sources (Fig. 1B). All 14 patient Abs reacted with the nucleoli and also appeared to react with more Ag species in the The nucleoli were also analyzed by scanning electron microscopy, nucleoli as compared with NG1 and NG2, except for patients which revealed consistent surface textures on different nucleolus SLE43, SLE46, and SLE48 (Fig. 1B). These three patient Abs particles (Fig. 2D, 2E, data not shown). These nonlipid surfaces of reacted more strongly with NG1, in which two autoantigens of 40 the nucleoli appeared dense and “capsid-like,” and both singular and 50 kDa, respectively, were recognized. These two nucleo- (Fig. 2D) and interconnected (Fig. 2E) nucleoli could be isolated. plasmic autoantigens were either scarce or absent in the nucleoli. In contrast, NG2 exhibited an irregular and fragmental mor- Normal human IgG also weakly reacted with a 50-kDa protein in phology in which chromatins appeared to be the backbone and NG1, which is absent in the nucleoli. This is consistent with SLE NPM1 appeared as sporadic or discontinuous nodules affiliated to patients being known to develop heterogeneous anti-nuclear au- the chromatins (Supplemental Fig. 2A, 2B). NG1 has similar toantibodies and the nucleoli being major targets of these Abs in morphology (data not shown). Through scanning electron mi- some patients (49, 50). These Western blotting results show that croscopy, similar fragmental morphology was observed with NG2 the nucleoli are broadly targeted by autoantibodies in SLE pa- (Supplemental Fig. 2C). Therefore, the nuclear ghost fractions, tients, although only in a subgroup of these patients are they the which represent the nucleoplasm, are clearly distinct from the dominant Abs (Fig. 1; data not shown). nucleoli in both composition and structure. 6 COMPLEMENT DEGRADATION OF NUCLEOLAR Ags

Table II. Proteins appeared following nucleoli treatment with C1 at 37˚C

Spot IDa Accession Number Protein Name (Gene Name) Predicted Size (kDa) Observed Size (kDa) Structural Integrity 30 C1s_HUMAN Complement C1s subcomponent (C1S) 77 55 ND 34 NUCL_HUMAN NCL (NCL) 77 53 Missing NH2 40 NUCL_HUMAN NCL (NCL) 77 53 Missing NH2 52 NUCL_HUMAN NCL (NCL) 77 43 Missing NH2 85 ROA2_HUMAN Heterogeneous nuclear ribonucleoproteins 37 31 ND A2/B1 (HNRNPA2B1) 87 C1S_HUMAN Complement C1s subcomponent (C1S) 77 27 ND 109 HNRNPC_HUMAN Heterogeneous nuclear ribonucleoproteins 34 22 ND C1/C2 (HNRNPC) 113 PSIP1_HUMAN PC4 and SFRS1-interacting protein (PSIP1) 60 21 NH2-fragment 118 NPM_HUMAN NPM1 (NPM1) 33 17 ND 130 LSM8_HUMAN U6 snRNA-associated Sm-like protein LSm8 10 12 ND 134 RL30_HUMAN 60S ribosomal protein L30 (RPL30) 13 12 ND 136 NPM_HUMAN NPM1 (NPM1) 33 11 Fragment 139 HNRNPC_HUMAN Heterogeneous nuclear ribonucleoproteins 34 11 NH2-fragment C1/C2 (HNRNPC) (may contain NPM1 fragment) 142 NPM_HUMAN NPM1 (NPM1) (may contain fragment of 33 9 Fragment K2C1_HUMAN) Downloaded from aSpot ID corresponds to the number assigned to a picked spot from the 2D-DIGE analysis (Fig. 4).

C1 proteases broadly degrade nucleolar proteins A total of 42 protein spots gave rise to conclusive data: 28 Cy2- To gauge the extent of nucleolar protein degradation by C1 pro- dominant and 14 Cy5-dominant proteins. Seven protein spots were teases, we incubated nucleoli with C1 for 0.5, 1.0, and 2.0 h at 37˚C. identified as NCL or its proteolytic fragments (Tables I, II). This As controls, nucleoli were either untreated or incubated with C1 was consistent with our earlier observation that NCL was cleaved http://www.jimmunol.org/ for 2.0 h at 4˚C. Based on Coomassie blue staining, nucleoli that by C1 proteases (48). Four protein spots were identified as NPM1 were treated at 4˚C exhibited an overall similar protein profile to or its fragments, which was also anticipated (48, 54). One Cy2- ∼ untreated nucleoli, suggesting insignificant degradation (Fig. 3A). dominant protein of 80 kDa (spot 2) was identified as C1s and, However, those incubated with C1 at 37˚C showed time-dependent based on the size, it corresponded to the proenzyme form of C1s ∼ degradation of some otherwise abundant proteins in the 110-, 65-, (Table I). Two Cy5-dominant proteins of 55 kDa (spot 30) and 27 36-, and 28-kDa regions (Fig. 3A). Those in the 110- and 36-kDa kDa (spot 87), respectively, were also identified as C1s, which were regions were predicted to contain NCL and NPM1, respectively (48). most likely to be the H and L chains of activated C1s (Table II). The remaining 28 spots represented 22 distinct protein identities, which

In contrast, a prominent 23-kDa protein species appeared after C1 by guest on October 2, 2021 incubation, which may correspond to a proteolytic fragment. It was were not previously known to be cleavablebyC1proteases.Thisdata also noted that many nucleolar proteins were not significantly de- set focused only on more abundant proteins, and the actual number graded, including core histones and other proteins that migrated in of C1-degradable nucleolar proteins is likely to be greater. Our re- the 10- to 15-kDa region. sults resonate with an earlier prediction that, besides its complement NG2 showed different protein profiles in which the 110-, 65-, and substrates, C1s potentially cleaves many intracellular proteins (55). 36-kDa regions were not particularly abundant with proteins In fact, C1s is also known to cleave cell surface receptors and the (Figs. 1B, 3B). However, the 28-kDa region in NG2 was similarly secreted insulin-like growth factor binding protein 5 (56). abundant with protein as in the nucleoli and was also cleaved by C1 degradation of nucleolar proteins attributes to its C1 proteases (Fig. 3B). This experiment revealed the broad but C1r/C1s proteases selective susceptibility of nucleolar proteins to C1 degradation. However, it lacked the resolution for protein identification and the To validate these newly identified nucleolar substrates for the C1 sensitivity to view less abundant proteins. proteases, C1-treated nucleoli were subjected to Western blotting using specific Abs. Initial experiments showed that different nucleolar Proteomic profiling of C1-cleavable nucleolar proteins proteins exhibited varying sensitivities to C1 cleavage. Therefore, To determine the identities of these C1-cleavable nucleolar pro- nucleoli were again treated with C1 for 0.5, 1.0, or 2.0 h at 37˚C as teins, we resolved the C1-treated nucleoli in two dimensions through shown in Fig. 3A. As controls, nucleoli were also incubated at 37˚C two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). without C1. Abs were obtained for BOP1, RRP9, DDX5, DDX21, As illustrated in Fig. 4A, nucleoli were treated for 2.0 h with C1 at hnRNPC, hnRNPA2B1, EIF2S1, SFPQ, and RPL30. In addition, Abs 4˚C, which were then labeled with Cy2 (green). Those treated at for C1q and C1s, the nucleolar proteins NPM1, NCL, and fibrillarin, 37˚C were subsequently labeled with Cy5 (red). The two samples histones H3, H1.2, and H1.x, and heterogeneous nuclear ribonu- were combined equivalently and resolved by 2D electrophoresis. cleoprotein U (hnRNPU) were also included in this experiment. Most proteins were found similarly represented by Cy2 and Cy5 as As previously observed (48), NPM1 and NCL were degraded by orange spots, suggesting insignificant degradation at 37˚C (Fig. 4B). C1 in a time-dependent manner (Fig. 5). In these reactions, the However, some protein spots were overrepresented by either Cy2 or inputs of C1 were identified based on the levels of C1q and C1s. Cy5. We proposed that those overrepresented by Cy2 (green) indi- The activation status of C1s was apparent because it was con- cated their degradation at 37˚C and therefore were underrepresentative verted from its larger proenzyme form to its activated H (55 kDa) of their Cy5-labeled forms in the expected spots. Those overrep- and L (27 kDa) chains (Fig. 5). Without C1, incubation of nu- resented by Cy5 (red) were likely to be new proteolytic fragments. cleoli alone caused undetectable NPM1 or NCL degradation Greater than 200 proteins were collected, and some were further (Fig. 5). When C1-inh was included in these reactions, NPM1 and analyzed by MALDI-TOF and TOF/TOF. NCL degradation was inhibited to varying extents, showing the The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/ by guest on October 2, 2021

FIGURE 5. Validation of nucleolar protein cleavage by C1 proteases. After 2D-DIGE analysis, selected protein spots were analyzed by MALDI-TOF and TOF/TOF. The identified proteins were validated by Western blotting. In brief, nucleoli were incubated with C1 for 0.5, 1, and 2 h at 37˚C. C1 was diluted from a Ca2+-depleted acidic buffer immediately before use. As controls, nucleoli were incubated alone. Nucleoli were also incubated with C1 in the presence of the C1-inh. The protein profiles of each sample were shown by Coomassie blue staining (Supplemental Fig. 4). Samples were then analyzed by Western blotting with Abs specific for BOP1, DDX5, DDX21, SFPQ, EIF2S1, HNRNPC, HNRNPA2B1, RRP9, RPL30, and, as controls, with Abs specific for C1q, C1s, NCL, and NPM1. Molecular mass standards are indicated on the left. Two representative H1 histones H1.2 and H1.x and hnRNPU, which were not among the picked spots, were also included. Representative data are shown for at least two independent experiments. involvement of C1 proteases (Fig. 5). We noticed that the anti-C1s Histones H1.2 and H1.x and the nucleoplasmic protein hnRNPU, Ab recognized, besides the three major bands corresponding to which were not identified in the 2D-DIGE experiment, were also proenzyme C1s and its H and L chains, numerous other minor degraded. Where a protein was found degraded by C1, this was bands. This cannot be simply explained because, with human always inhibited by C1-inh (Fig. 5), suggesting pivotal contributions serum and the isolated C1 complex, the Ab only recognized C1s of the C1r/C1s proteases in the observed proteolytic degradation. (Supplemental Fig. 3). The Ab also showed no recognition of The nucleoli are poorly autoproteolytic nucleolar proteins (Fig. 5). With C1 treatment, SFPQ, BOP1, EIF2S1, and hnRNPC1 di- Cells contain abundant intracellular proteases and are therefore minished, leaving no proteolytic fragments that remained detect- intrinsically autoproteolytic (57). However, a careful inspection of able by the Abs used (Fig. 5). DDX5, DDX21, hnRNPA2B1, the Western blotting data revealed that, unless C1 was added, the RRP9, and RPL30 were also degraded with C1 incubation and, for nucleoli exhibited little protein degradation after incubation for up these new C1 substrates, proteolytic fragments were detectable by to 2 h at 37˚C (Fig. 5). It suggests that the nucleoli contain little the Abs used (Fig. 5). For most of these new C1 substrates, active proteases and are therefore poorly autoproteolytic. This cleavage was apparent at 0.5 h, but BOP1, hnRNPA2B1, and may preserve their antigenicity in dead cells. DDX21 was an RRP9 appeared more resistant to C1 degradation. A significant exception because, without C1, it still underwent degradation, fraction of BOP1 remained intact after incubation with C1 for 2.0 which reduced its molecular mass from 90 to 70 kDa. C1 prote- h. In contrast, SFPQ, DDX5, and EIFS1 were completely cleaved ases increased DDX21 degradation, but neither C1-mediated nor within 1.0 h (Fig. 5). Fibrillarin and histone H3 appeared to resist self-proteolytic degradation reduced DDX21 below a 70-kDa frag- C1 degradation. ment (Fig. 5). It appears that the two types of cleavage sites are 8 COMPLEMENT DEGRADATION OF NUCLEOLAR Ags

FIGURE 6. C1 degradation of nucleolar autoantigens. Nucleoli were incubated with C1 for 2.0 h at 4˚C (left lane) and 37˚C (right lane), respectively. C1 was diluted from a Ca2+-depleted acidic buffer immediately before use. Samples were then separated on 12.5% (w/v) SDS-PAGE gels and either stained with Coomassie blue (proteins) or analyzed by Western blotting using SLE patient IgG autoantibodies. A total of 14 patient Abs were used, and only those that clearly detected C1 cleavage of autoantigens are shown. Patient codes are labeled on the tops. Molecular mass standards are indicated on the left. Autoantigens that diminished after incubation with C1 at 37˚C are indicated by asterisks (*). Newly generated autoantigen fragments are indicated by arrowheads. Representative experiments are shown for at least two independent experiments. Downloaded from confined to a 20-kDa terminal region on DDX21. Nonetheless, from the recent finding that C1q specifically targeted nucleoli in these preserved nucleolar proteins may induce autoantibodies. The dead cells, which caused C1s activation and the degradation of two targeting of C1 proteases to these nucleolar Ags through C1q nucleolar autoantigens (48). Considering that the nucleoli are also could be one mechanism in the dismantling of nucleolar auto- major targets of autoantibodies (49, 50), we hypothesized that C1 antigenicity to reduce anti-nuclear autoantibodies induction. complexes broadly dismantle nucleolar proteins and autoantigens to diminish their autoantigenicity. Our data support this hypothesis.

Many nucleolar autoantigens are cleaved by C1 proteases http://www.jimmunol.org/ The context where C1q was first found to bind to the nucleoli was Because the isolated nucleoli were broadly recognized by SLE in late apoptotic cells (48). However, this cellular context was patient autoantibodies (Fig. 1B), we asked whether these auto- overly heterogeneous and complex for proteomic identification of antigens might be degraded by C1 proteases. After incubation C1-cleaved nucleolar proteins. Nucleoli were therefore isolated to with C1 for 2 h at 37˚C, nucleoli were then analyzed by Western incubate with C1. Using 2D-DIGE, we found many nucleolar blotting using SLE patient autoantibodies. As a control, nucleoli proteins to be degraded after C1 treatment, among which 22 nu- were incubated with C1 at 4˚C. With 10 SLE patient Abs, at least cleolar proteins represented novel substrates for the C1 proteases. 25 autoantigens were found degraded after C1 treatment at 37˚C, Abs were available to validate independently by Western blotting although the protein identities of these autoantigens were not

that some of these nucleolar proteins were indeed cleavable by C1 by guest on October 2, 2021 defined (Fig. 6). Besides, five autoantigen fragments were detected proteases. after C1 treatment at 37˚C. It was also noted that a significant In this study, it could not be concluded whether C1r or C1s number of autoantigens were not affected after C1 treatment. cleaved these nucleolar proteins. Within the C1 complex, C1r and Our results have revealed that nucleoli are intrinsically poor in self-proteolysis, and this can contribute to their broad auto- antigenicity in SLE patients. However, we also revealed that nu- cleolar proteins or autoantigens are broadly but selectively degraded by C1 proteases, which can be targeted to apoptotic nucleoli through C1q (48). Collectively, our data help explain the high autoantigenicity of the nucleoli and the strong association between C1 deficiency and the development of anti-nuclear autoantibodies and SLE disease (Fig. 7).

Discussion Anti-nuclear autoantibodies contribute to diseases by forming injurious immune complexes (14, 15). Aberrant cell death and impaired immune clearance are believed to expose nuclear Ags to inflammatory or immunogenic contexts and induce these Abs (11, 13, 18, 20). How anti-nuclear autoantibodies are induced remains poorly understood, but cases of homozygous deficiencies of C1q, C1r/C1s, or C4 are informative because they mostly cause these FIGURE 7. Schematic illustration of C1 complex responses to nucleoli autoantibodies and the SLE disease (23, 28, 58). in live, apoptotic, and necrotic cells. In live cells, the surface membrane is In the quest to understand how these genetic deficiencies con- impermeable to extracellular C1q, which excludes C1 proteases (C1r and tribute to SLE, studies have mostly emphasized individual genes. C1s) from all intracellular protein substrates including nucleolar proteins. Early apoptotic cells remain impermeable to extracellular C1q and the C1r/ For C1q, previous articulations drew mainly from two lines of C1s proteases. These cells also conceal intracellular Ags from autologous investigation: C1q opsonizes apoptotic cells for effective clearance immune responses. In necrotic cells, nucleolar Ags are exposed to autol- (13, 40, 41), and C1q inhibits DC and macrophage responses (45, ogous immune responses, but they also become accessible to C1 and 46, 59–61). The fact that C1q complexes with C1r and C1s in vivo susceptible to C1q-mediated degradation by C1r/C1s proteases. C1s acti- was mostly not considered. How C1r/C1s deficiency also causes vation on nucleoli could also lead to C4b deposition and C4b-mediated these autoantibodies has not been investigated. This study stemmed clearance and tolerance induction. The Journal of Immunology 9

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2 3

4 5 6 Supplemental figure 1. Homogeneity of isolated nucleoli. Isolated nucleoli in the

7 0.25-M sucrose buffer were incubated with coverslips for 5 min. The nucleoli were

8 fixed for 30 min in 1% (w/v) PFA and stained for NPM1 (Cy3, red) and chromatins

9 (DAPI, blue). The top panels are reconstructed 3D light (A) and fluorescence (B)

10 images. The lower panels are 0.38-µm section images. (C) NPM1 signals. (D)

11 Merged NPM1 and chromatin signals. Scale bar, 10 µm. Representative data are

12 shown for at least three independent experiments.

13 14

1 15 16 17 18 19 20

21 22 23 Supplemental figure 2. Fluorescent and SEM images of a nucleoplasmic fraction. (A

24 and B) Reconstructed 3D images of the isolated NG2 fraction. NG2 was incubated

25 with coverslips for 5 min and, after fixation for 30 min in 1% (w/v) PFA, stained for

26 NPM1 (Cy3, red) and chromatin (DAPI, blue). (A) 0.38-µm section images of

27 chromatins. (B) 0.38-µm section images of merged NPM1 and chromatin signals.

28 Scale bar, 5 µm. (C) SEM image of a single NG2 fragment (magnifications, x

29 95,000). Representative data are shown for at least three independent experiments.

30

31

32

33

34

35

36

37

38

2 39 40

41 Supplemental figure 3. Examination of contaminating serum proteases in the

42 isolated C1 complex and C1-inh. Serum (1/100, 8 µl), C1 (9.6 µg/ml, 8 µl), and C1-

43 inh (160 µg/ml, 8 µl) were separated on 12.5% SDS-PAGE gels and were either

44 stained with Coomassie blue (left panel) or analyzed by Western blotting using the

45 following antibodies (right panels): goat anti-C1q (A301, Quidel), goat anti-C1s

46 (A302, Quidel), rabbit anti-thrombin (ab92621, Abcam), rabbit anti-plasmin

47 (ab48350, Abcam), mouse anti-factor XII (ab1007, Abcam), and mouse anti-

48 kallikrein 1B (Abcam). The amount of C1 was normalized so that equivalent amount

49 of C1q and C1s are detected. Data represent two independent experiments.

50

51

52

3 53

54 Supplemental figure 4. Loading controls for Figure 5. This is a representative SDS-

55 PAGE gel performed in parallel with the Western blotting experiments presented in

56 Figure 5 in the main manuscript. The same volumes of samples, as used for the

57 Western blots, were loaded on separate gels for Coomassie blue staining. C1,

58 equivalent C1 complex used in the reactions. C1-inh, equivalent C1 inhibitor used in

59 the reactions. Data represent at least three experiments.

60 61 62 63 64 65 66 67

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