Label-Free Proteomics Reveals Decreased Expression of
CD18 and AKNA in Peripheral CD4+ +T Cells from Patients
with Vogt-Koyanagi-Harada Syndrome
Liming Mao1, Peizeng Yang1*, Shengping Hou1, Fuzhen Li1, Aize Kijlstra2
1Laboratory of Ophthalmology, Chongqing Eye Institute, The First Affiliated Hospital of Chongqing Medical University, Chongqing, People’s Republic of China, 2The
Department of Ophthalmology, University of Maastricht, Maastricht, The Netherlands
Vogt-Koyanagi-Harada (VKH) syndrome is a systemic autoimmune disease. CD4+T cells have been shown to be involved in
autoimmune diseases including VKH syndrome. To screen aberrantly expressed membrane proteins in CD4+T cell from
patients with active VKH syndrome, blood samples were taken from five patients with active VKH syndrome and five healthy
individuals. A label-free quantitative proteomic strategy was used to identify the differently expressed proteins between the
two groups. The results revealed that the expression of 102 peptides was significantly altered (p,0.05) between two groups
and matched amino acid sequences of proteins deposited in the international protein index (ipi.HUMAN.v3.36.fasta). The
identified peptides corresponded to 64 proteins, in which 30 showed more than a 1.5-fold difference between the two
groups. The decreased expression of CD18 and AKNA transcription factor (AKNA), both being three-fold lower than controls
in expression identified by the label-free method, was further confirmed in an additional group of five active VKH patients
and six normal individuals using the Western blot technique. A significantly decreased expression of CD18 and AKNA
suggests a role for both proteins in the pathogenesis of this syndrome.
Citation: Mao L, Yang P, Hou S, Li F, Kijlstra A (2011) Label-Free Proteomics Reveals Decreased Expression of CD18 and AKNA in Peripheral CD4+T Cells from
Patients with Vogt-Koyanagi-Harada Syndrome. PLoS ONE 6(1): e14616. doi:10.1371/journal.pone.0014616
Editor: Wasif N. Khan, University of Miami, United States of America
Received July 23, 2010; Accepted January 3, 2011; Published January 28, 2011
Copyright: ? 2011 Mao et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by National Natural Science Foundation Project (30973242), Program for the Training of a Hundred Outstanding S&T Leaders
of Chongqing Municipality, Key Project of Health Bureau of Chongqing (2008115), Project of Medical Science and Technology of Chongqing (2008-1-15), Key
Project of Natural Science Foundation of Chongqing (CSTC, 2009BA5037), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), and PAR-EU
SCHOLARS PROGRAM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune
disorder mainly affecting systemic melanocytes including those in
the eyes, meninges, ears, skin, and hair . It is one of the most
common uveitis entities in China as well as in the Far East of Asia.
The uveitis seen in this syndrome is mostly characterized by a
chronic granulomatous inflammation with recurrent episodes .
Although the mechanisms involved in VKH syndrome are not
fully elucidated, previous reports have showed that CD4+T cells
sensitive to melanocytes are responsible for the development of
VKH syndrome [3,4]. Recent studies have shown that molecules
related to CD4+T cell function, including Fas/FasL [5,6], T-bet
, IFN-c, RORct and IL-17 [7,8], are involved in the
pathogenesis of this syndrome. As CD4+ T cells exert their role
principally through a variety of receptors, adhesion molecules,-
transport proteins and costimulatory molecules,which have been
found mainly in the plasma membrane or endomembrane systems,
our study focused on the differentially expressed proteins in the
CD4+ T cell membrane of VKH patients.
Proteomics provides important tools for identifying molecules
involved in both normal and pathological processes [9,10,
11,12,13,14,15]. A recently established label-free strategy [16,17]
has been shown to offer a more robust protein identification and
quantitation, easy automation and large-scale analysis with greater
efficiency as compared to conventional two dimensional electro-
phoresis (2-DE) approaches [18,19,20,21]. In the present study, we
investigated the differentially expressed membrane proteins in
active VKH patients using this label-free proteomic method.
Western blot technique was used to validate the proteomic results.
Our results showed a significantly decreased expression of CD18
and AKNA in CD4+T cells from patients with active VKH
Materials and Methods
Ten active VKH patients and eleven healthy individuals were
included in the present study. CD4+T cells from five active VKH
patients and five healthy individuals were used for label-free
proteomics analysis. CD4+T cells from another group of five
active VKH patients and six controls were used for a validation
study.The diagnosis of VKH syndrome was made according to the
criteria revised for VKH syndrome by an international nomen-
clature committee . The patients included in our study had not
been systemically treated with any immunosuppressive agent for at
least one week prior to blood sampling. The mainly clinical
features of VKH patients were shown in Table 1. The healthy
individuals are sex and age matched with VKH patients showing
not any past history of infectious or chronic diseases. An informed
PLoS ONE | www.plosone.org1 January 2011 | Volume 6 | Issue 1 | e14616
consent (written) was obtained from all patients and the
procedures had been approved by the Ethics Committee of the
First Affiliated Hospital of Chongqing Medical University.
CD4+T Cell isolation and flow cytometry
The PBMCs were prepared from heparinized blood by Ficoll-
Hypaque density-gradient centrifugation and were rinsed for three
times in phosphate-buffered saline (PBS). Peripheral CD4+T cells
were purified using human CD4 microbeads according to the
manufacturer’s instructions (Miltenyi Biotec, Palo Alto, Calif).
Briefly, PBMCs were suspended in 80 ml of PBS containing 0.5%
bovine serum albumin (BSA) and 2 mM ethylenediamine tetra-
acetic acid (EDTA) per 107total cells. A volume of 20 ml of CD4
microbeads was added to this suspension and incubation was
performed for 15 min at 4uC. The cells were then washed in 2 ml
of PBS containing 0.5% BSA and 2 mM EDTA and applied to a
magnetic column on a MiniMACS separation unit (Miltenyi
Biotec, Palo Alto, Calif). The CD4+T cell fraction was collected
and used in subsequent experiments. For flow cytometric analysis,
aliquots of 16106PBMCs and isolated CD4+T cells were stained
with PE-cy7-conjugated monoclonal antibody (mAb) against
human CD4 and appropriate isotype controls (eBioscience, San
Diego, CA, USA) for 30 min at 4uC in the dark. Flow cytometric
analysis was performed using FACS Calibur and CellQuest
software (BD Biosciences, SanJose, CA).
Membrane Protein Preparation
Membrane proteins were extracted using a membrane protein
extraction kit (Merck KGaA, Darmstadt, Germany) according to
the manufacturer’s instructions. Briefly, 56106CD4+T cells were
pelleted by centrifugation at 300 g for five min. The supernatant
was carefully removed and discarded. Reagent A (150 ml) was
added to the cell pellet and a homogeneous cell suspension was
prepared by pipetting up and down and was then incubated for
10 min at room temperature with occasional vortexing. The lysed
cells were placed on ice. One part of reagent B with two parts of
reagent C were mixed. A total amount of 450 ml of the mixed
reagent B and C was added to each tube of lysed cells and then
vortexed. The tubes were incubated on ice for 30 min with
occasional vortexing. After centrifuging at 10,000 g for 3 min at
4uC, the supernatant was transferred to a new tube and was
incubated for 10 min at 37uC to separate the membrane protein
fraction. The tubes were then centrifuged at room temperature for
2 min at 10,000 g to isolate the hydrophobic fraction from the
hydrophilic fraction. The hydrophilic phase (top layer) was
removed carefully from the membrane-enriched phase (bottom
layer). The protein concentration of the hydrophobic protein was
determined using the protein assay kit (Bio-Rad, Hercules, CA).
The protein was aliquoted and stored at 280uC until use.
Protein digestion was performed as follows. The protein
concentration was adjusted to 5 ug/ul with lysis buffer. The
aforementioned membrane protein was chemically reduced for
2.5 h at room temperature by adding DTT to 10 mM, and then
carboxyamidomethylated in 50 mM iodoacetamide for 40 min at
room temperature in the dark. Endoprotease LysC (Roche,
Indianapolis, IN) was added to a final substrate: enzyme ratio of
100:1 (w/w), and the reaction was incubated at 37u for 3 h. The
urea concentration in protein samples was adjusted to 1.5 M with
25 mM NH4HCO3, and then 2 mg of trypsin was added to a final
substrate: enzyme ratio of 50:1 (w/w). The trypsin digest was
incubated at 37u for 20 h. The peptide mixture was acidified by
formic acid to 0.1% for further MS analysis.
Analysis by HPLC and MS
Separation of the trypsin-digested peptides was performed with
the Ettan MDLC system (GE Healthcare, Piscataway, NJ). Peptide
samples were first desalted through a Zorbax 300SB-C18peptide
traps (Agilent Technologies, Wilmington, DE) and then separated
by a 0.15 mm*150 mm (RP-C18) column (Column Technology
Inc, Fremont, CA) with micro spray mode. An aqueous 0.1%
formic acid solution was used as phase A and a solution of 0.1%
formic acid and 84% acetonitrile was used as phase B. The
Table 1. The clinical features of the investigated patients with VKH disease.
number SexAge Ocular Manifestation
2 Male 34present present0.5 0.5
3 Male 39 presentpresent present0.080.06-
4Male24 present presentpresentpresent0.060.05
5 Female34 presentpresent present0.81.0-
7 Female27 present presentpresent0.60.01
8 Female50 presentpresentpresent0.3 0.05
9Male 46presentpresent 1.00.8-
10Male 41 present presentpresent present0.20.5
Proteomics in VKH Syndrome
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