Prohibitin Is Involved in Patients with IgG4 Related Disease

Abstract

Objective: IgG4-related disease (IgG4-RD) is a chronic systemic disease involved in many organs and tissues. As only limited autoantigens have been found since the beginning of this century, the aim of this study was to reveal new candidate autoantigens of IgG4-RD.

Methods: Multiple cell lines including HT-29, EA.hy926, HEK 293 and HepG2 were used to test the binding ability of circulating autoantibodies from IgG4-RD sera. The amino-acid sequence was then analyzed by matrix-assisted laser desorption/ionization time-of-flight tandem (MALDI-TOF/TOF) mass spectrometry. After the cloning and expression of recombinant putative autoantigen in a bacterial expression system, the corresponding immuno assay was set up and utilized to observe the prevalence of serum autoantibodies in a large set of confirmed clinical samples.

Results: One positive autoantigen was identified as prohibitin. ELISA analysis showed that a majority of patients with IgG4-RD have antibodies against prohibitin. Anti-prohibitin antibodies were present in the sera of patients with definite autoimmune pancreatitis (25/34; 73.5%), Mikulicz's disease (8/15; 53.3%), retroperitoneal fibrosis (6/11; 54.5%), other probable IgG4-RD (26/29; 89.7%) and Sjögren's syndrome (4/30; 13.3%) but not in apparently healthy donors (1/70; 1.4%).

Conclusions: An association between prohibitin and patients with some IgG4-RD was observed, although the results were quite heterogeneous among different individuals within autoimmune pancreatitis, Mikulicz's disease and retroperitoneal fibrosis.

Fig 1. Immunofluorescence analysis.

(A-D) Immunofluorescence was performed on HT-29 by confocal laser microscopy, then compared with other cells including EA.hy926, HEK 293 and HepG2. Total cell fluorescence was analyzed by Image J software and significant differences were found between HT-29 and three other cell lines (p<0.0001), indicating positive reactions in HT-29 cells with IgG4-RD sera. (E-F) Control samples including SjS and HC were then tested on HT-29 cells, and the sera of patients with SjS were found to have a weaker specific reaction and no positive signal on HC.

Fig 2. Identification of target antigen.

(A) The value of cell fluorescence was analyzed by Image J software. *** indicates P<0.0001. (B) Five of 20 patients with IgG4-RD presenting relatively higher optical density values on ELISA for HT-29 cell were selected for Western blotting. (C) Western blotting of HT-29 cell extracts with the sera from 5 IgG4-RD patients showed a positive band with a molecular weight of approximately 30 kDa in 3 patients but not in healthy controls. (D) Immunoprecipitation was performed by incubating the extracts of HT-29 with IgG4-RD patient sera and an approximately 30 kDa protein band reacted with antibodies from IgG4-RD patients.

Fig 3. Verification of prohibitin.

(A) The cloning, expression and purification of recombinant PHB protein. M, protein markers; lane 1, cell extracts of pET-28a (+)-PHB/BL21 after IPTG induction for 6 hour at 37°C; lane 2, cell extracts of pET-28a (+)-PHB/BL21 before IPTG induction; lane 3, cell extracts of pET-28a (+)-BL21 after IPTG induction. lane 4, cell extracts of BL21 after IPTG induction. (B) Western blot using purified PHB protein showed that only the sera from patients with IgG4-RD (lane 1) rather than HC (lane 2) contain antibodies against a 30 kDa cellular protein. (C) The expressed protein was purified and further identified by MS, which revealed its identity as PHB. (D) PHB protein was also identified in immunoprecipitates; lane 1, supernatant of immunoprecipitation; lane 2, immunoprecipitates; lane 3, control sample (the purified rhPHB). (E) The protein band on lane 2 was excised and identified by MALDI-TOF/TOF MS, which again revealed its identity as PHB.

Fig 4. Anti-PHB autoantibodies induced in IgG4-RD patients.

(A) The prevalence of autoantibodies against human PHB in sera from patients was observed. ELISA was used to detect the reactivity of serum IgG4 against recombinant human PHB protein. The anti-PHB antibodies were detected in 65 of 89 RA patients (73%), 4 of 30 SjS patients (13.3%) and 1 of 70 healthy donors (1.4%). The reactivity of anti-PHB antibodies was significantly higher than HC (***P<0.0001). (B) The patients with IgG4-RD were then divided into the following confirmed subtypes: AIP, definite autoimmune pancreatitis (25/34, 73.5%); MD, Mikulicz’s disease (8/15, 53.3%); RPF, retroperitoneal fibrosis (6/11, 54.5%); MIX, affect multiply organs (26/29, 89.7%).