Synovial Fluid Cell Proteomic Analysis Identifies Upregulation of Alpha-Taxilin Proteins in Rheumatoid Arthritis: a Potential Prognostic Marker
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Hindawi Journal of Immunology Research Volume 2020, Article ID 4897983, 10 pages https://doi.org/10.1155/2020/4897983 Research Article Synovial Fluid Cell Proteomic Analysis Identifies Upregulation of Alpha-Taxilin Proteins in Rheumatoid Arthritis: A Potential Prognostic Marker Ashish Sarkar,1 Shivani Sharma,1 Prachi Agnihotri,1 Tanmoy Sarkar,1 Pooja Kumari,1 Rajesh Malhotra,2 Barun Datta,3 Vijay Kumar,2 and Sagarika Biswas 1 1Council of Industrial Research (CSIR)-Institute of Genomics and Integrative Biology, Mall Road, Delhi University Campus, 110007, Delhi, India 2All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110029, India 3Army Hospital Research And Referral (RR Hospital), Dhaula Kuan, New Delhi, India Correspondence should be addressed to Sagarika Biswas; [email protected] Received 7 November 2019; Accepted 13 March 2020; Published 23 April 2020 Academic Editor: Eirini Rigopoulou Copyright © 2020 Ashish Sarkar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Rheumatoid arthritis (RA) is a chronic autoimmune inflammatory disease affecting the joints and surrounding tissue. Identification of novel proteins associated with the progression of a disease is a prerequisite for understanding the pathogenesis of RA. The present study was undertaken to identify the potential biomarkers from a less explored biological sample such as synovial fluid (SF) cells which is specific for RA and to analyze their functional aspects using proteomic approach. Two-dimensional gel electrophoresis (2-DE) was performed using synovial fluid cells of RA and osteoarthritis (OA) patients, and 7 differentially expressed proteins were identified using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF MS/MS). Αlpha-Taxilin (α-Taxilin) has been found as one of the novel, significantly up regulated protein in RA. It has been validated in the synovium, synovial fluid (SF), SF cells, and plasma samples by Western blot, enzyme-linked immunosorbent assay (ELISA), fluorescence-activated cell sorting (FACS), immunohistochemistry (IHC), and real-time PCR. The identification of autoantibody against α-Taxilin and in silico studies has further helped us to understand its involvement in disease mechanism. The present study will therefore provide knowledge towards the etiology of RA that pave the way for suitable prognostic marker identification along with other clinical parameters. 1. Introduction citrullinated peptide (anti-CCP), rheumatoid factor (RF), and antimannose binding lectin [9] (anti-MBL) are helpful Rheumatoid arthritis (RA) is the most common chronic, sys- in diagnosing the disease to a certain extent. However, these temic inflammatory autoimmune disease affecting around antigens are not specific whether they initiate autoimmune 1% of the population worldwide [1, 2]. It is often accompa- reactions, thus making the diagnosis critical [8, 10]. As the nied by systemic manifestation such as anemia, fatigue, and early stage of RA is characterized by nonspecific clinical osteoporosis [3]. In spite of many efforts, the etiology of RA symptoms, a delayed diagnosis leads to irreversible joint is still not clear [4, 5]. As the understanding about the disease damage [11, 12]. Thus, there is an urgent and strong need is very limited, symptomatic treatment is mainly by the use of for novel and more precise biomarkers with higher sensitivity disease-modifying antirheumatic drugs (DMARDs), bio- and specificity for early diagnosis of disease. The study can logics, steroids, or combinations thereof [6, 7]. therefore complement the conventional measures which RA is believed to be mediated by an immune complex may provide a more efficient marker to diagnose the disease that does not clarify the acute inflammatory features of the for timely initiation of available therapies. Since the specific disease [8]. Tumor Necrosis Factor (TNF), IL-6, anticyclic site of inflammation plays a vital role in the disease 2 Journal of Immunology Research ° progression, synovial fluid cell (SF cell) has been used as pri- 5 min in a swinging bucket rotor at 4 C. Cells were collected mary source to study the differentially expressed proteome and washed 3 times with phosphate-buffered saline (PBS) at ° profile. Additionally, synovium and plasma samples were 300 × g for 5 min and incubated for 30 min at 4 C in SF cell also used to strengthen our findings. lysis buffer (25 mM Tris, 1% Nonidet P-40, 150 mM Sodium We used two-dimensional gel electrophoresis (2-DE) Chloride (NaCl), 1.5 mM Ethylenediaminetetraacetic acid followed by matrix-assisted laser desorption/ionization (EDTA), 0.5% Sodium dodecyl sulfate (SDS), 1 mM phenyl- time-of-flight mass spectrometry (MALDI-TOF MS/MS) methane sulfonyl fluoride (PMSF), and 1% v/v Protease [13] to study the differential proteome profile of RASF com- Inhibitor cocktail (PI cocktail) followed by sonication at pared to OASF cells. OA SF cells were used as control SF cells 20% amplitude for 5 min. The cell lysate was then centrifuged since OA is an old age inflammatory joint disease but lacks at 15000 × g for 30 min, and the supernatant was collected for autoimmune response [4]. OA is characterised by accumula- further experiments. tion of synovial fluid and inflammation; thus, synovium and synovial fluid samples from OA patients have been widely 2.3. Two-Dimensional Gel Electrophoresis (2-DE). Blood RA = 12 OA = 12 50 y ± 5 used as control for RA study [14, 15]. Following the study, plasma samples ( , , , male : female, fi a total of 17 differentially expressed protein spots were 1 : 1) were pooled and quanti ed by the Bradford assay observed. Among them, 7 spots have been identified success- [22]. Three sets of 2-DE gels were run by using a pooled fi fully by mass spectrometry analysis. “TXLNA” or “Alpha- plasma sample from RA and OA after small modi cation fl μ Taxilin/α-Taxilin” also known as interlukin-14 (IL-14) [16] [23]. Brie y, 150 g protein was added to the rehydration ff or “High molecular growth factor” was found to be the most bu er, loaded on immobilized pH gradient (IPG) strips – significantly upregulated protein in RA. Alpha-Taxilin (17 cm, pH 4 7 (Bio-Rad Laboratories), and was separated fi belongs to the Taxilin family, has three members which in the rst dimension by isoelectric focusing (IEF) followed include Alpha- (α-), Beta- (β-), and Gamma- (γ-) Taxilin. by the second dimension using sodium dodecyl sulfate- While α-Taxilin and γ-Taxilin are globally expressed, β- polyacrylamide gel electrophoresis [23] (SDS-PAGE). Taxilin (MDP77) is only expressed in the heart and skeletal 2.4. Silver Staining. After 2-DE, gels were kept in fixative muscles [16, 17]. α-Taxilin binds to proteins of the syntaxin (50% methanol and 12% acetic acid) for an hour [24], family that are localized on the plasma membrane [18, 19]. followed by washing with 50% ethanol and 30% ethanol for The role of α-Taxilin has been reported earlier in exocytosis 30 min each. Gels were then sensitized with 0.2% Na S O and cytokine activity which leads to inflammation via B-cell 2 2 3 for 1 min, washed with Milli-Q thrice, and stained with 1 M activation [20]; interestingly, literature support that an AgNO and 700 μl/lit formaldehyde (HCHO) solution for increased level of α-Taxilin has been found responsible for 3 20 min followed by the development of gels using a developer development of autoimmunity in transgenic mice [21], thus consisting Na CO (60 gm/lit), HCHO (500 μl/lit), and making our finding more relevant and important for under- 2 3 Na S O (25 mm) [23]. The reactions were then stopped by standing the etiology of RA. 2 2 3 adding 6% acetic acid. 2. Material and Methods 2.5. In-Gel Digestion of Proteins and Preparation for MALDI- TOF MS/MS Analysis. For identification, differentially RA = 100 2.1. Sample Collection. Whole blood samples ( (Age expressed protein spots were digested with trypsin gold (Pro- 50 y ± 5 28 = 6 , male+female, Disease activity score (DAS) mega, USA) as earlier [4]. Briefly, protein spots were excised ±0:5 OA = 100 50 y ± 5 ), (Age , male+female) were collected into 1 mm cubes, washed with Milli Q, destained with 30 mM hygienically in the EDTA-coated vacutainer (Ptech) from All potassium ferricyanide and 100 mM sodium thiosulfate India Institute of Medical Sciences (AIIMS, Ansari Nagar, (1 : 1) at RT, and dehydrated by incubation with 1 : 1 acetoni- New Delhi, India) and Army Hospital Research and Referral trile (ACN)/water. Followed by washing with ACN, gel Hospital (Dhaula Kuan, New Delhi, India). All enrolled par- pieces were rehydrated (10 mM NH HCO equal volume of fi 4 3° ticipant patients had ful lled the 2010 American College of ACN), vacuum dried, and digested at 37 C for 18 h with Rheumatology criteria for RA and OA diagnosis, having 20 μl of trypsin (20 ng/μl, Trypsin Promega). Peptides were radiological evidence and clinical history (detail clinical extracted by varying concentrations of ACN and trifluoroa- “ ” parameter is given in Supplementary table 1 ). Healthy cetic acid (TFA, Sigma-Aldrich, St. Louis, MO, USA). Peptide HC = 64 volunteers ( ) also requited in this study having no concentrate (1 μl) and sinapinic acid (1 μl) were mixed and radiographic evidence of joint degradation and other air dried and run using MALDI-TOF MS/MS (Applied Bio- fl related clinical history. Synovial uid (6 ml to 8 ml) were systems, Life Technologies, USA) as earlier [4]. MS/MS spec- n =16 n =16 collected from RA ( ) and OA ( ) patients, and tra were procured in reflector positive mode over the mass n =6 the synovium was collected after biopsy from RA ( ) scope of 10,000-20,000 Da, and proteins having more than n =6 and OA ( ), respectively.