Label-free proteomics reveals decreased expression of CD18 and AKNA in peripheral CD4+ T cells from patients with Vogt-Koyanagi-Harada syndrome

Abstract

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.

Figure 1. Representative flow cytometric map of CD4⁺ T cells isolated from peripheral blood of active VKH patients.

The PBMC and freshly isolated CD4⁺ T cells were stained with the indicated markers using fluorescence-labeled mAb and analyzed by flow cytometry. Before sorting, the ratio of CD4⁺ T cells in PBMC was 38.11% (A). After microbeads based sorting, the purity of CD4⁺ T cells was as high as 98.06% (B).

Figure 2. Decyder MS intensity graphs of CD18 derived from LC-MS/MS analysis of a total membrane protein extracted from both normal group (A) and VKH group (B).

The location of peptide ALNEITESGR in both samples was labeled with square frames in the total graphs and the magnified graphs (C and D). Statistical analysis showed that the different expression of peptide ALNEITESGR was significant between VKH group and normal group (E).

Figure 3. Validation of CD18 and AKNA by the Western blot technique.

Antibodies were used at a dilution of 1∶1000 for the anti-human CD18 monoclonal antibody and anti-human AKNA monoclonal antibody. Proteins were detected using the Phototope-HRP Western blot detection system(A). The immunoreactive band intensities were quantitated and were presented as intensity volumes (vol%). The results showed that both CD18 and AKNA were significantly down-regulated in VKH patients as compared to normal controls (B). VKH: VKH patients, NC: normal controls.