Human Proteins with Affinity for Dermatan Sulfate Have the Propensity to Become Autoantigens
2011; Elsevier BV; Volume: 178; Issue: 5 Linguagem: Inglês
10.1016/j.ajpath.2011.01.031
ISSN1525-2191
AutoresJung-hyun Rho, Wei Zhang, Mandakolathur R. Murali, Michael H. A. Roehrl, Julia Y. Wang,
Tópico(s)Immunotherapy and Immune Responses
ResumoThe mystery of why and how a small, seemingly disparate subset of all self molecules become functional autoantigens holds a key to understanding autoimmune diseases. Here and in a companion article in this issue, we show that affinity of self molecules to the glycosaminoglycan dermatan sulfate (DS) is a common property of autoantigens and leads to a specific autoreactive B-1a cell response. Autoimmune ANA/ENA reference sera react preferentially with DS affinity-fractionated cellular proteins. Studying patients with autoimmune diseases, we discovered patient-specific complex autoantigen patterns that are far richer and more diverse than previously thought, indicating significant pathological heterogeneity even within traditionally defined clinical entities, such as systemic lupus erythematosus. By shotgun sequencing of DS affinity-enriched proteomes extracted from cell lines, we identified approximately 200 autoantigens, both novel and previously linked to autoimmunity, including several well-known families of autoantigens related to the nucleosome, ribonucleoproteins, the cytoskeleton, and heat shock proteins. Using electron microscopy, we recognized direct interaction with dead cells as an origin of autoantigenic association of DS with self molecules. DS affinity may be a unifying property of the human autoantigen-ome (ie, totality of self molecules that can serve as functional autoantingens) and thus provides a promising tool for discovery of autoantigens, molecular diagnosis of autoimmune diseases, and development of cause-specific therapies. The mystery of why and how a small, seemingly disparate subset of all self molecules become functional autoantigens holds a key to understanding autoimmune diseases. Here and in a companion article in this issue, we show that affinity of self molecules to the glycosaminoglycan dermatan sulfate (DS) is a common property of autoantigens and leads to a specific autoreactive B-1a cell response. Autoimmune ANA/ENA reference sera react preferentially with DS affinity-fractionated cellular proteins. Studying patients with autoimmune diseases, we discovered patient-specific complex autoantigen patterns that are far richer and more diverse than previously thought, indicating significant pathological heterogeneity even within traditionally defined clinical entities, such as systemic lupus erythematosus. By shotgun sequencing of DS affinity-enriched proteomes extracted from cell lines, we identified approximately 200 autoantigens, both novel and previously linked to autoimmunity, including several well-known families of autoantigens related to the nucleosome, ribonucleoproteins, the cytoskeleton, and heat shock proteins. Using electron microscopy, we recognized direct interaction with dead cells as an origin of autoantigenic association of DS with self molecules. DS affinity may be a unifying property of the human autoantigen-ome (ie, totality of self molecules that can serve as functional autoantingens) and thus provides a promising tool for discovery of autoantigens, molecular diagnosis of autoimmune diseases, and development of cause-specific therapies. Autoimmune diseases are among the most poorly understood medical conditions, although it is well accepted that they are caused by aberrant immune responses directed at endogenous molecules and tissues of the body. Autoimmune diseases encompass a wide spectrum of clinical presentations, and more than 80 types have been classified, based primarily on systemic or organ-specific involvement.1Plotz P.H. The autoantibody repertoire: searching for order.Nat Rev Immunol. 2003; 3: 73-78Crossref PubMed Scopus (166) Google Scholar Common systemic autoimmune diseases include systemic lupus erythematosus (SLE), rheumatoid arthritis, and Sjögren syndrome; localized diseases include type 1 diabetes, multiple sclerosis, and Graves disease. Both precise diagnosis and development of cause-directed therapies remain challenging. A major hurdle has been the lack of understanding of etiologically inciting molecular and cellular events and key pathophysiologic mechanisms that lead to autoimmunity. A central component of autoimmunity consists of autoantigens, against which the immune system raises autoantibodies and destructive autoreactive cells. Rules that determine the repertoire of possible autoantigens and that differentiate autoantigens from the majority of nonautoantigenic self molecules are at present unknown. Among the tens of thousands of human molecules, only a small fraction (<1%) have been observed to serve as autoantigens.1Plotz P.H. The autoantibody repertoire: searching for order.Nat Rev Immunol. 2003; 3: 73-78Crossref PubMed Scopus (166) Google Scholar, 2Amital H. Shoenfeld Y. Natural autoantibodies, heralding, protecting and inducing autoimmunity.in: Shoenfeld Y. Gershwin M.E. Meroni P.L. Autoantibodies. ed 2. Elsevier, Amsterdam2007: 7-12Crossref Scopus (6) Google Scholar At first glance, these autoantigens appear to be a Wunderkammer—a collection of curiosities without apparent unifying properties, such as physicochemical properties or functional attributes.1Plotz P.H. The autoantibody repertoire: searching for order.Nat Rev Immunol. 2003; 3: 73-78Crossref PubMed Scopus (166) Google Scholar On the other hand, many autoimmune diseases share autoantigen markers and display overlapping clinical symptoms. Certain molecules, such as nuclear proteins and proteins involved in the synthesis and processing of DNA and RNA, are more prone to becoming autoantigens.3Gilbert D. Brard F. Jovelin F. Tron F. Do naturally occurring autoantibodies participate in the constitution of the pathological B-cell repertoire in systemic lupus erythematosus?.J Autoimmun. 1996; 9: 247-257Crossref PubMed Scopus (13) Google Scholar Autoimmune diseases also occur more frequently in females.4Fairweather D. Frisancho-Kiss S. Rose N.R. Sex differences in autoimmune disease from a pathological perspective.Am J Pathol. 2008; 173: 600-609Abstract Full Text Full Text PDF PubMed Scopus (395) Google Scholar Notably, autoantibody repertoires appear similar in both human patients and mouse models.5Cohen P.L. Maldonado M.A. Animal models for SLE.Curr Protoc Immunol. 2003; PubMed These facts and other evidence suggest that autoantigenic properties of self molecules are governed by rules yet to be uncovered. Several theories attempt to explain the origin of autoantigens. One popular theory is that autoantigens are derived from apoptotic cells, and that, during cell apoptosis, certain self molecules undergo programmed or spontaneous modifications that render them different from the native self and thus autoantigenic.6Casciola-Rosen L. Andrade F. Ulanet D. Wong W.B. Rosen A. Cleavage by granzyme B is strongly predictive of autoantigen status: implications for initiation of autoimmunity.J Exp Med. 1999; 190: 815-826Crossref PubMed Scopus (428) Google Scholar Another theory is that of molecular mimicry,7Fourneau J.M. Bach J.M. van Endert P.M. Bach J.F. The elusive case for a role of mimicry in autoimmune diseases.Mol Immunol. 2004; 40: 1095-1102Crossref PubMed Scopus (43) Google Scholar which postulates that an immune response initially aimed at a foreign antigen from a microbial infection also targets a self antigen with shared or very similar epitopes. A similar theory is that of epitope spreading.8Powell A.M. Black M.M. Epitope spreading: protection from pathogens, but propagation of autoimmunity?.Clin Exp Dermatol. 2001; 26: 427-433Crossref PubMed Scopus (73) Google Scholar A primary immune response triggered by a dominant epitope of a foreign antigen may prompt subsequent responses to other epitopes of the same molecule, and through molecular mimicry of these latter epitopes with self antigens, the immune response would spread from the exogenous antigen to self antigens. A less commonly broached theory is that autoantigens may share certain physicochemical features (not necessarily the conventional epitope per se). For example, long charge-rich coiled-coil segments have been found in various autoantigens.9Dohlman J.G. Lupas A. Carson M. Long charge-rich alpha-helices in systemic autoantigens.Biochem Biophys Res Commun. 1993; 195: 686-696Crossref PubMed Scopus (56) Google Scholar, 10Nozawa K. Fritzler M.J. Chan E.K. Unique and shared features of Golgi complex autoantigens.Autoimmun Rev. 2005; 4: 35-41Crossref PubMed Scopus (35) Google Scholar Here and in a companion article in this issue, we propose that affinity to dermatan sulfate (DS) is a unifying property of autoantigens. In the companion article, we demonstrate that DS physically interacts with dead cells and that the resulting DS∙autoantigen complexes drive autoreactive B-1a cell responses and autoantibody production in mouse models.11Wang J.Y. Lee J. Yan M. Rho J. Roehrl M.H.A. Dermatan sulfate interacts with dead cells and regulates CD5+ B-cell fate: implication for a key role in autoimmunity.Am J Pathol. 2011; 178: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Here, we provide further support for our hypothesis with data from human patients and report the identification of approximately 200 human autoantigens with DS affinity. QUANTA Chek ANA (antinuclear antigen) and ENA (extractable nuclear antigen) reference panels (INOVA Diagnostics, San Diego, CA) comprise well-characterized sera from autoimmune disease patients that are known to react with a variety of specific autoantigens. Additional sera from 36 patients with known autoimmune diseases (such as SLE and Sjögren syndrome) were obtained from the Department of Pathology at Massachusetts General Hospital (Boston, MA) after Institutional Review Board approval (see Supplemental Table S1 at http://ajp.amjpathol.org). The presence of autoantibodies in patient sera was initially surveyed with an INNO-LIA ANA Update test kit (Innogenetics, Gent, Belgium) that detects antibodies against the following autoantigens: Sm (SmB and SmD), RNP (RNP-70k, RNP-A, RNP-C), SS-A (Ro52 and Ro60), SS-B/La, centromere (Cenp-B), Scl-70 (DNA topoisomerase I), Jo-1, ribosomal P, and histones. A549 and HFL-1 cells were cultured in F-12K medium (Invitrogen, Carlsbad, CA). HEp-2 cells were cultured in Eagle's minimum essential medium. HS-Sultan and WIL2-NS cells were cultured in RPMI-1640 medium. All media were supplemented with 10% fetal bovine serum and a penicillin-streptomycin-glutamine mixture (Invitrogen). Harvested cells were resuspended in 50 mmol/L phosphate buffer (pH 7.4) containing Roche cOmplete Mini protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Cells were homogenized on ice and then sonicated on ice for 5 minutes. The homogenate was centrifuged, and the supernatant was used as total protein extract. Protein concentrations were measured with the RC DC Protein Assay (Bio-Rad Laboratories, Hercules, CA). All samples were stored at −80°C. DS-Sepharose resin was prepared by coupling DS (Sigma-Aldrich, St. Louis, MO) to EAH Sepharose 4B (GE Healthcare, Piscataway, NJ). The resin (20 mL) was washed with distilled water and 0.5 mol/L NaCl and then was mixed with 100 mg of DS dissolved in 10 mL of 0.1 mol/L MES buffer (pH 5.0). N-ethyl-N-(3-dimethylaminopropyl) carbodiimide hydrochloride (Sigma-Aldrich) was added to a final concentration of 0.1 mol/L. The mixture was incubated at 25°C for 24 hours with end-over-end rotation. After the first 60 minutes, the pH was readjusted to 5.0. After the coupling, the resin was washed three times each with a low-pH buffer (0.1 mol/L acetate, 0.5 mol/L NaCl, pH 5.0) and a high-pH buffer (0.1 mol/L Tris, 0.5 mol/L NaCl, pH 8.0). The DS-Sepharose resin in 10 mmol/L phosphate buffer (pH 7.4) was packed into a C 16/20 column (GE Healthcare). Fractionation of proteins by DS affinity was performed using a BioLogic Duo-Flow system (Bio-Rad). Typically, 20 mg to 50 mg of protein extract in 40 mL of 10 mmol/L phosphate buffer (pH 7.4; buffer A) was loaded onto the DS affinity column at a rate of 1 mL/minute. The column was washed with 60 mL of buffer A to elute nonbinding proteins. DS-binding proteins were eluted with sequential 40-mL step gradients containing 0.2, 0.4, 0.6, and 1.0 mol/L NaCl. Fractions were desalted and concentrated to 0.5 mL each in 5-kDa cutoff Vivaspin 20 centrifugal filters (Sartorius Stedim Biotech, Aubagne, France). The final protein concentration of each fraction was measured. In some experiments, only two step gradients (0.5 and 1.0 mol/L NaCl) were used. For 1-D gel electrophoresis, 8 μg to 20 μg of proteins were loaded per lane and separated by SDS-PAGE with 4% to 12% NuPAGE Novex Bis-Tris gels (Invitrogen) with MES or MOPS (morpholine propane sulfonic acid) running buffer. For 2-D gel electrophoresis, 50 μg to 100 μg of proteins were cleaned with the ReadyPrep 2-D cleanup kit (Bio-Rad), redissolved in 185 μL of ReadyPrep rehydration/sample buffer (Bio-Rad) and separated on 11-cm IPG strips (pH 3 to 10; Bio-Rad) using a Protean isoelectric focusing cell (Bio-Rad). In some experiments, 7-cm IPG strips (pH 3 to 10 or pH 3 to 6) were used with 120 μL of rehydrated sample. The focused strips were equilibrated with SDS-PAGE equilibration buffers I and II (Bio-Rad). Proteins were separated along the second dimension using 8% to 16% Criterion Tris-HCl gradient gels (Bio-Rad). Fixed gels were stained with fluorescent SYPRO Ruby (Bio-Rad) for 16 hours and imaged on a Typhoon 9410 scanner (GE Healthcare). Images were analyzed with PDQuest 7.4.0 software (Bio-Rad). Gels subjected to Western blot analysis were stained with Bio-Safe Coomassie Blue G250 (Bio-Rad). Proteins from 1-D or 2-D gels were transferred to polyvinylidene difluoride membranes and blocked with 2% bovine serum albumin, 1% casein, and 0.5% Tween 20 in Tris-buffered saline (pH 7.4) at 25°C for 1 hour. Membranes were incubated with test serum (diluted 1:1000 or 1:2000) in blocking buffer at 25°C for 1 hour. After three washes with Tris-buffered saline containing 0.5% Tween 20, membranes were incubated with 0.2 μg/mL goat anti-human IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) at 25°C for 1 hour. Membranes were developed using enhanced chemiluminescence Western blotting substrate (Pierce; Thermo Fisher Scientific, Rockford, IL). Mass spectrometric (MS) sequencing was performed at the Taplin Biological Mass Spectrometry Facility (Harvard Medical School). Selected protein spots from 1-D or 2-D PAGE gels were excised and cut into 1-mm3 pieces.12Shevchenko A. Wilm M. Vorm O. Mann M. Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels.Anal Chem. 1996; 68: 850-858Crossref PubMed Scopus (7807) Google Scholar Gel pieces were dehydrated with acetonitrile, dried in a speed-vac concentrator, and rehydrated at 4°C for 45 minutes in 50 mmol/L NH4HCO3 containing 12.5 ng/μL modified sequencing-grade trypsin (Promega). Tryptic peptides were separated on a nano-scale C18 HPLC capillary column and analyzed after electrospray ionization in an LTQ linear ion-trap mass spectrometer (Thermo Fisher Scientific, West Palm Beach, FL).13Peng J. Gygi S.P. Proteomics: the move to mixtures.J Mass Spectrom. 2001; 36: 1083-1091Crossref PubMed Scopus (510) Google Scholar Peptide sequences and protein identities were assigned by matching protein or translated nucleotide databases with the measured fragmentation pattern using SEQUEST software, version 27 (revision 12) (Therma Fisher Scientific). Peptides were required to be fully tryptic peptides with XCorr values of at least 1.5 (1+ ion), 1.5 (2+ ion), or 3.0 (3+ ion). All data were manually inspected. Only proteins with ≥2 independent peptide matches were considered positively identified. Patient serum samples demonstrating strong and well-separated bands on Western blots were used to determine the identities of the recognized autoantigens. Protein extracts from HEp-2 or WIL2-NS cells were fractionated by DS-affinity chromatography. Patient-specific autoantigens present in these fractions were enriched by immunoprecipitation with patient serum, separated by 1-D or 2-D electrophoresis in duplicate gels, and reactivity with patient serum was verified using Western blot analysis. Immunoprecipitation was performed with a ProFound coimmunoprecipitation kit (Pierce; Thermo Fisher Scientific), and antibodies in patient sera were covalently immobilized onto the resin. Before coupling, antibodies were typically purified from 100 μL of patient serum using a Melon gel IgG purification kit (Pierce; Thermo Fisher Scientific). In some experiments, protein A-Sepharose resin was used to noncovalently immobilize antibodies in patient sera. Protein extracts were absorbed with protein A-Sepharose to remove proteins that bind to protein A and then were incubated with autoantibody-bound protein A-Sepharose at 25°C for 1 hour. The mixture was centrifuged to precipitate autoantigen∙autoantibody∙protein A-Sepharose complexes. The complexes were dissociated by SDS-PAGE and examined using Western blot analysis. Reactive protein spots or bands were excised from duplicate gels and sequenced by tandem MS. DS-specific IgM and IgG in patient sera were measured by enzyme-linked immunosorbent assay.11Wang J.Y. Lee J. Yan M. Rho J. Roehrl M.H.A. Dermatan sulfate interacts with dead cells and regulates CD5+ B-cell fate: implication for a key role in autoimmunity.Am J Pathol. 2011; 178: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Nunc MaxiSorp 96-well plates were coated with 0.1 mg/mL DS or 2.5 μg/mL goat anti-human F(ab′)2 at 4°C overnight. Wells were blocked with 1% bovine serum albumin at 25°C for 1 hour. Patient sera were diluted 100-fold for measurement of DS-specific antibodies and 2000-fold for measurement of total Ig. IgG and IgM were detected by alkaline phosphatase-conjugated goat anti-human IgG and IgM, respectively. WIL2-NS cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (DMEM-10) with 40 μg/mL DS-biotin conjugate (in-house synthesized) for 3 days. Cells were fixed with 4% paraformaldehyde at 25°C for 30 minutes and processed as described.11Wang J.Y. Lee J. Yan M. Rho J. Roehrl M.H.A. Dermatan sulfate interacts with dead cells and regulates CD5+ B-cell fate: implication for a key role in autoimmunity.Am J Pathol. 2011; 178: 2168-2176Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar Sections of cells were stained with goat anti-biotin conjugated to 20-nm gold particles and examined with a JEOL 1200EX transmission electron microscope (JEOLO USA, Peabody, MA). To test the hypothesis that autoantigens are characterized by affinity to DS, we first examined a panel of well-known ANA (antinuclear antigen) and ENA (extractable nuclear antigen) autoantigens. Autoantibodies against ANAs and ENAs are hallmarks of systemic autoimmune diseases, and their detection is a major screening test in clinical practice.14Damoiseaux J.G. Tervaert J.W. From ANA to ENA: how to proceed?.Autoimmun Rev. 2006; 5: 10-17Crossref PubMed Scopus (98) Google Scholar We extracted the proteomes of various human cell lines, fractionated them on a DS-affinity column with increasing concentrations of salt, and interrogated for abundance of ANA or ENA autoantigens in these fractions by blotting with ANA or ENA reference sera. Proteins eluted from the DS-affinity column with step gradients of 0.2 mol/L, 0.4 to 0.6 mol/L, or 1 mol/L NaCl were considered not/weakly DS-binding, DS-binding, or tightly DS-binding, respectively (Figure 1; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Because a single cell line may not express a complete repertoire of autoantigens, we examined the proteomes from five human cell lines representing different tissue types: HEp-2 (epithelial cell line used diagnostically for ANA immunofluorescence assays), A549 (epithelial), HFL-1 (mesenchymal), HS-Sultan (lymphoid), and WIL2-NS (lymphoid). Protein extracts from the different cell lines typically revealed distinct autoreactive patterns, illustrating the differential presence of various autoantigen repertoires in these cells (Figure 1; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Of the five cell lines, WIL2-NS cells gave typically rise to the highest number of autoantigens and associated autoreactive bands (Figure 1A). Remarkably, all autoantigens recognized by ANA and ENA reference antisera were present in the DS-binding or tightly DS-binding fractions (Figure 1; see also Supplemental Figure S1 at http://ajp.amjpathol.org). Some autoantigens were detectable only after DS-affinity fractionation; for example, PCNA (proliferating cell nuclear antigen) was detectable only in DS-binding fractions, not in unfractionated or nonbinding proteins. Similarly, autoantigens reactive with anti-RNP or anti-centromere serum were detected only in DS-binding, not in total unfractionated or nonbinding fractions. Thus, DS-affinity fractionation significantly enriched these autoantigens to detectable levels. Total unfractionated protein extracts typically showed a single major autoantigen band corresponding to the expected specificity of each ANA or ENA reference serum (Figure 1). After DS-affinity fractionation, however, numerous additional autoantigen bands became detectable that had not previously been known to be recognized by these reference sera. For example, instead of one major band (in unfractionated extract), ANA reference serum (with speckled immunofluorescence reactivity pattern) recognized numerous bands in the 0.4 mol/L and 0.6 mol/L NaCl fractions (Figure 1). Similarly, additional autoantigens were revealed by reference antisera with nominal specificities for SS-B, SS-A/SS-B, Scl-70, and ribosomal P. In some cases, autoreactive bands were also detected in the 0.2 mol/L NaCl fraction (Figure 1), possibly because of weak affinity of these autoantigens to DS or, particularly when the same bands were also present in higher-salt fractions, because of capacity overloading of the DS-affinity column. The increased sensitivity of autoantigen detection by DS-affinity enrichment enabled further differentiation of traditional ANA/ENA immunofluorescence reactivity patterns. For example, two reference sera, each with homogeneous reactivity pattern in the HEp-2-based immunofluorescence assay and thus phenotypically indistinguishable, yielded very different underlying autoantigen profiles (Figure 1). Similarly, two reference sera with nominal anti-Scl-70 specificity yielded complicated and distinct profiles (Figure 1B). These experiments also pointed to a potential mechanism for autoantigenicity of DNA. In addition to discrete protein bands, the two anti-Scl-70 sera reacted with smeared bands at high molecular weight in the DS-binding fractions from both HEp-2 and WIL2-NS proteome extracts (Figure 1B). Scl-70 (topoisomerase I) is an enzyme that unwinds supercoiled DNA by nicking and ligating DNA strands. Only DNase digestion, but not treatment with PNGase F or RNase A, caused the disappearance of the high molecular weight smears, identifying them as Scl-70∙DNA complexes (Figure 1B). Thus, DS interacts not only with Scl-70 alone, but also with Scl-70∙DNA complexes. In fact, it seems attractive to speculate that DS∙Scl-70∙DNA ternary complexes may be responsible for the production of autoantibodies to both Scl-70 and DNA. We next asked whether autoantigen affinity to DS could be used as a principle for comprehensive profiling of autoantigens in human autoimmune diseases. We collected sera from 36 patients with autoimmune diseases, including SLE and Sjögren syndrome, among others (see Supplemental Table S1 at http://ajp.amjpathol.org). Proteins extracted from HEp-2 or WIL2-NS cells were fractionated by DS affinity, separated by SDS-PAGE, and blotted with individual patient serum (Figure 2 and data not shown). The unexpectedly complex autoreactive patterns observed were largely unique for each patient (Figure 2). Compared with a limited spectrum of clinical diagnoses (see Supplemental Table S1 at http://ajp.amjpathol.org) and a commercial strip assay (see Supplemental Figure S2 at http://ajp.amjpathol.org) that demonstrated only a small set of autoantigens, our method uncovered a much larger repertoire of autoantigens. The patient-specific autoreactive profiles illustrate an autoantigenic diversity in autoimmune diseases that is much greater than previously thought or currently assayed for. Even within patient groups with similar clinical presentation (eg, patients diagnosed with SLE), autoantigen profiles were strikingly different (Figure 2; see also Supplemental Table S1 at http://ajp.amjpathol.org). When unfractionated protein extracts from either HEp-2 or WIL2-NS were used, typically only very few bands reacted with patient sera. After DS-affinity fractionation, however, numerous additional autoreactive bands emerged, particularly in DS-binding fractions. Many patients presented complex patterns with many distinct autoantigens present across the DS-binding fractions (eg, patients 1, 5, 10, 18, and 22) (Figure 2). Without DS-affinity enrichment, this large number of additional autoantigens would not have been detectable. WIL2-NS proteome extracts generally revealed more reactive bands, and so contained more autoantigens than HEp-2 extracts. For example, serum from patient 10 reacted with two bands using fractionated HEp-2 proteins, but many more bands when proteins from WIL2-NS cells were used (Figure 2). This observation is particularly noteworthy and relevant because virtually all current clinical patient testing, including immunofluorescence pattern description, is based on HEp-2 cells, and thus likely misses a significant number of autoantibodies. To verify that the newly detected autoreactive bands indeed resulted from specific recognition of autoantigens by patient sera, we characterized selected prominent bands in detail. Sera from patients 11, 12, and 14 (all with speckled HEp-2 ANA immunofluorescence pattern) reacted strongly with distinct bands in DS-binding fractions (Figure 2; see also Supplemental Figure S3, A–C at http://ajp.amjpathol.org). Proteins from HEp-2 cells were fractionated by DS affinity, and the specific reactive proteins were further enriched by serum immunoprecipitation and identified by MS sequencing (see Supplemental Figure S3 at http://ajp.amjpathol.org). Serum from patient 11 reacted with two bands in DS-binding fractions (0.6 mol/L NaCl), which corresponded to SPTA2 (spectrin α-II) and PRP8 (U5 snRNP) (upper band) and α-actinin-1 and α-actinin-4 (lower band) (see Supplemental Figure S3A at http://ajp.amjpathol.org). Serum from patient 12 reacted with LA (La/SS-B protein) (0.4 to 0.6 mol/L NaCl; see Supplemental Figure S3B at http://ajp.amjpathol.org. Serum from patient 14 reacted with BASP1 (brain acid soluble protein 1) and FETUA (fetuin A) (0.4 to 0.6 mol/L NaCl; see also Supplemental Figure S3C at http://ajp.amjpathol.org). Of note, all of these proteins serve as well-known autoantigens (Table 1; see also Supplemental Appendix and references therein at http://ajp.amjpathol.org), supporting the usefulness of DS affinity as a tool for enhanced profiling of autoantigens.Table 1Tandem MS Sequencing of Selected DS-binding Proteins from 1-D/2-D PAGE GelsUniProt IDProtein ID⁎Repeated listing of the same protein indicates that tandem MS sequencing of more than one gel band/spot yielded the same protein identity.Peptide matchesMS/MS coverage (aa)Cell linePAGENaCl⁎⁎NaCl concentrations used to wash and elute from DS-affinity resin (eg, 0.4-1.0 M indicates that proteins were eluted with 1.0 M NaCl after washing with 0.4 M NaCl).P05455LA (La autoantigen, SS-B)15148/408 (36.3%)HS-Sultan2-D0.4-1.0 MP12956KU70 (Ku autoantigen protein p70, XRCC6)345/608 (7.4%)HEp-21-D0.4-0.6 MKU7021257/608 (42.3%)HEp-21-D0.4-0.6 MP13010KU86 (Ku autoantigen protein p86, XRCC5)10109/731 (14.9%)HEp-21-D0.4-0.6 MP19338NUCL (nucleolin)12122/709 (17.2%)HEp-21-D0.4-0.6 MNUCL14161/709 (22.7%)A5492-D0.4-0.6 MP06748NPM (nucleophosmin)16172/294 (58.5%)A5492-D0.4-0.6 MP05388RLA0 (60S acidic ribosomal protein P0)10120/317 (37.9%)HS-Sultan2-D0.4-1.0 MQ9UNX3RL26L (60S ribosomal protein L26-like 1)433/145 (22.8%)A5492-D0.4-0.6 MP46777RL5 (60S ribosomal protein L5)16110/296 (37.2%)HEp-21-D0.5-1.0 MQ96A08H2B1A (histone H2B type 1-A)542/126 (33.3%)A5492-D0.4-0.6 MP33778H2B1B (histone H2B type 1-B)425/125 (20.0%)A5492-D0.4-0.6 MQ6FGB8Histone H4754/103 (52.4%)WIL2-NS2-D0.4-0.6 MQ01105SET (HLA-DR-associated protein II)9138/290 (47.6%)A5492-D0.4-0.6 MP39687AN32A (acidic leucine-rich nuclear phosphoprotein 32 family member A)16130/249 (52.2%)A5492-D0.4-0.6 MQ92688AN32B (acidic leucine-rich nuclear phosphoprotein 32 family member B)1157/251 (22.7%)A5492-D0.4-0.6 MP16989DBPA (DNA-binding protein A)440/372 (10.8%)HEp-22-D0.5-1.0 MO60812HNRCL (hnRNP core protein C-like 1)973/293 (24.9%)A5492-D0.4-0.6 MHNRCL541/293 (14.0%)WIL2-NS2-D0.4-0.6 MP07910HNRPC (heterogeneous nuclear ribonucleoproteins C1/C2)895/306 (31.0%)A5492-D0.4-0.6 MQ16629SFRS7 (splicing factor, arginine/serine-rich 7)330/238 (12.6%)WIL2-NS2-D0.4-0.6 MQ15393SF3B3 (splicing factor 3B subunit 3)11139/1217 (11.4%)HEp-21-D0.4-0.6 MP12814ACTN1 (alpha-actinin-1)28334/892 (37.4%)HEp-21-D0.4-0.6 MO43707ACTN4 (alpha-actinin-4)22270/911 (29.6%)HEp-21-D0.4-0.6 MQ13813SPTA2 (spectrin alpha chain, brain)17233/2472 (9.4%)HEp-21-D0.4-0.6 MP27797Calreticulin19209/417 (50.1%)HEp-22-D0.5-1.0 MCalreticulin15180/417 (43.2%)HEp-22-D0.2-0.4 MQ07021C1QBP (complement component 1 Q subcomponent-binding protein, mitochondrial)358/282 (20.6%)HEp-22-D0.5-1.0 MC1QBP12171/282 (60.6%)A5492-D0.4-0.6 MC1QBP8115/282 (40.8%)HS-Sultan2-D0.4-1.0 MQ13310PABP4 (poly(A)-binding protein 4)16136/644 (21.1%)HEp-21-D0.5-1.0 MQ9H361PABP3 (poly(A)-binding protein 3)977/631 (12.2%)HEp-21-D0.5-1.0 MP11021GRP78 (heat shock 70 kDa protein 5)51
Referência(s)