Identification of prohibitin as an antigen in Behcet’s disease

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Highlights

  • HUVEC was firstly tried as a new target to identify antigen in Behcet’s disease (BD).

  • Prohibitin was successfully screen out as a new autoantigen of BD.

  • It’s about 28% patients have circulating antibodies to prohibitin in Han Chinese BD.

Abstract

Objective

This study is intended to screen potential antigen for Behcet’s disease (BD) by using human microvascular endothelial cells (HUVEC).

Methods

Following cell-based indirect immunofluorescence assay with sera from BD patients, proteins extracted from HUVEC were separated and detected by Western blotting. Then the target protein was identified by LC-MALDI-TOF/TOF, the recombinant target protein was expressed, purified and then used as coating antigen to test the prevalence of autoantibodies in patient’s sera.

Results

The Western blotting result showed that some patients’ sera could react with a protein band with about 30 kDa of molecular weight, which was further identified as prohibitin by mass spectrometry. The prevalence of serum antibodies against recombinant human prohibitin was detected in 16 of 58 BD patients (28%) but none in healthy controls.

Introduction

Behcet’s disease (BD), or Silk Route disease [1], is a recurrent and multi-systemic inflammatory disorder with variable clinical manifestations including mucous membrane ulceration and ocular problems [2], [3], [4]. It involves in almost every organs in human body such as blood vessels, gastrointestinal tract, skin and neurological systems, thus several conditions including nervous system problems, vasculitis, fibromyalgia, eyesight problems, joint pain and swelling are also considered to have relationship with BD [5], [6]. Typically BD arises in both genders young adults and has a worldwide distribution, which is more prevalent in countries of the ancient Silk Route including Japan, Korea, China and so on [7], [8]. The etiology of BD is still unknown, while both genetic background and environmental factors are thought to have contributed to the pathogenesis of BD, besides the autoimmune reaction triggered by an infectious agent has been suggested.

Recently, endothelial cells and anti-endothelial cell antibodies (AECA) are emphasized involved in the pathogenesis of BD, and moreover, some new autoantigens were discovered from various primary vascular endothelial cells [9], [10]. For example, α-enolase and hnRNP-A2/B1 were successively identified as autoantigens of BD in human dermal microvascular endothelial cells (HDMEC) [11], [12]. It was also found new autoantigens in human microvascular endothelial cells (HMVEC) by using the method of cDNA expression library [13]. In this study, we speculate human umbilical vein endothelial cells (HUVEC) might be a substituted target to screen novel autoantigens in BD patients, which had been successfully used to screen the target antigens in patients with anti-neutrophil cytoplasmic antibody-associated vasculitides [14].

Section snippets

Subjects

164 serum samples from patients with Behcet’s disease (BD, 58 cases), systemic lupus erythematosus (SLE, 50 cases) or healthy controls (HC, 56 cases) were enrolled and tested in this study. The institutional review board of the Chinese PLA General Hospital approved the study, and written informed consent was obtained from all the subjects. In addition, diagnosis was based on the criteria proposed by the International Study Group for BD (Criteria for diagnosis of Behcet’s disease, 1990) [15].

Indirect immunofluorescence

By using indirect immunofluorescence on HUVEC, it was displayed a stronger reactivity in cells incubated with sera from BD patients than from healthy volunteers. Despite there has some individual variation, the specific binding immunofluorescence signal was a good indicator to show the autoantibodies in blood circulation from BD patients can recognize the antigen in HUVEC (Fig. 1A and B).

Detection of target autoantigen in HUVEC

The result of ELISA to screen autoantigens in HUVEC with serum samples from 58 BD patients was performed in

Discussion

Prohibitin, which is ubiquitously located in mitochondria, nucleus, and other cellular compartments, is highly conserved and one of the most important members in membrane protein families [18], [19]. Several evidences suggest that PHB participated in some important biological functions, including suppressed tumor, regulated cell proliferation [20], and prevented infection [21]. Furthermore, it is also referred to participate in the development process of many diseases, while its overexpression

Acknowledgments

The authors wish to express their great gratitude to the help of Prof. Dr. Yaping Tian and Dr. Chunyan Zhang from Chinese PLA General Hospital, and Dr. Shutao Sun from Institute of Microbiology Chinese Academy of Sciences for MS analysis.

This work was supported by the Fundamental Research Funds for the Central Universities, Program for New Century Excellent Talents in University and the National Natural Science Foundation of China (No. 31371203).

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