Figure 1.
Analysis of the recombinant proteins.
(A) Recombinant THUB and THUC were resolved by SDS-PAGE (15%) and stained with Coomassie Brilliant Blue. Lane 1: THUC (2 μg) ; Lane 2: THUB (2 μg). (B) Recombinant THU, HUC, and HUB were resolved by 15% SDS-PAGE and stained with Coomassie Brilliant Blue. Lane 1: THU (2 μg); Lane 2: HUC (2 μg); Lane 3: HUB (2 μg). (C) The cargo peptide is cleaved from THUC intracellularly. C1R-B2704 cells (2 × 106 cells) in 1 ml IMDM were treated with 20 μg THUC for 4 h. The cells were pelleted by centrifugation. Proteins were extracted with 100 μl of 1% NP40, resolved by SDS-PAGE, and immunoblotted using an anti-His6 antibody. Lane 1: THUC (100 ng); Lane 2: Total protein extracts (20 μg); Lane 3: THU (100 ng).
Figure 2.
The BH2 monoclonal antibody recognizes the misfolded HLA-B27 HC (A) Trx-truncated HLA-B27 HC and truncated HLA-B27 HC were resolved by SDS-PAGE (12%) and stained with Coomassie Brilliant Blue.
Lane 1: Molecular weight markers. Lane 2: 2 μg of Trx-truncated HLA-B27 HC. Lane 3: 2 μg of truncated HLA-B27 HC. (B) BH2 recognizes the monomeric and oligomeric forms of HLA-B27 HC. The membrane proteins from C1R-B2704 cells were extracted, resolved by non-reducing SDS-PAGE (10%), and analyzed by western blot using the BH2 monoclonal antibody (1:1000 dilution). Lane 1: 50 μg of membrane protein extracted from HMy2.C1R cells. Lane 2: 50 μg of membrane protein extracted from C1R-B2704 cells. (C) The BiP (Grp 78)/misfolded HLA-B27 HC complex can be immunoprecipitated by BH2. Cell lysate (50 μg) and the immunoprecipitated product from cell lysate by BH2 were analyzed by western blotting and probed for BiP/Grp 78 or HLA-B27 HC (recognized by BH2). (D) The HLA-B27 immunoprecipitated by W6/32 does not bind to BiP/Grp 78. Cell lysate (50 μg) and the immunoprecipitated products from cell lysate by W6/32 were analyzed by western blotting and probed for BiP/Grp 78 or HLA-B27 HC.
Figure 3.
Treatment with either THUC or THUB reduces the formation of (B27-HC)2.
After treatment with each protein for the indicated time point, membrane proteins were extracted. 50 μg of extract was resolved by non-reducing SDS-PAGE (10%) and immunoblotted with a BH2 monoclonal antibody and anti-transferrin (Tf) receptor antibody. Tf receptor serves as an internal control. (A) THUC treatment for 12 h, but not THU or HUC treatment, significantly decreased the levels of (B27-HC)2. (B) The level of (B27-HC)2 is significantly reduced in C1R-B2704 cells treated with THUB. (C) The production of (B27-HC)2 is reduced when PBMCs isolated from AS patients are treated with THUC. (D) The results obtained in Figure 3C are plotted. The amount of immunostaining observed at 0 h was set to 100%. The results shown are the mean levels of (B27-HC)2 immunostaining observed in membrane proteins independently extracted from the PBMCs of five AS patients (mean ± SD, n = 5).
Figure 4.
THUC-treatment reducing the level of (B27-HC)2 in C1R-B2704 cells is TAP1-dependent.
(A) Stably expressed TAP1 shRNA reduces TAP1 expression. (B) Knockdown of TAP1 does not affect the expression of HLA-B27 HC. (C) TAP1-knockdown suppresses the THUC-induced reduction of (B27-HC)2. (D)The ratio of (B27-HC)2/Tf receptor averaged from three independent experiments in Figure 4C are plotted against THUC treatment time. The ratio of (B27-HC)2/Tf receptor extracted from cells without treatment with THUC was set to 100%.
Figure 5.
Analysis of HLA-B27 HC by Endo H digestion.
(A) SDS-PAGE analysis of Endo H-resistant and Endo H-sensitive HLA-B27 HC in C1R-B2704 cells. C1R-B2704 cells were treated with the indicated reagents. Membrane proteins (2 μg) digested with Endo H (one unit) were analyzed by reducing SDS-PAGE and immunoblotted by BH2 monoclonal antibody. (B) The results obtained in Figure 5A were plotted. The ratio of Endo H-sensitive HLA-B27 HC/total HLA-B27 HC is averaged from three independent experiments (mean ± SD, n =3).
Figure 6.
Treatment with either THUC or THUB increases the antigenic peptide targeted to the cell surface of C1R-B2704.
(A) Treatment with THUC, but not THU or HUC, increases the HLA-B27 HC/β2m/RRFKEGGRGGKY complex presented at the cell surface of C1R-B2704, as detected by flow cytometry. (B) Mean-channel fluorescence measured in Figure 6A is increased when C1R-B2704 cells were treated with THUC. Values (mean ± SD, n=3) are averaged from three independent experiments. (C) Treatment with THUB, but not THU or HUB, increases the HLA-B27 HC/β2m/ RRYLENGKETL complex presented at cell surface, as detected by flow cytometry. (D) Mean-channel fluorescence measured in Figure 6C is increased when C1R-B2704 cells were treated with THUB. Values (mean ± SD, n=3) are averaged from three independent experiments. (E) Knockdown of TAP1 impairs the antigenic peptides presented on the cell surface. (F) Mean-channel fluorescence measured in Figure 6E is not affected when C1R-B2704 cells with TAP1 knockdown were treated with THUC or THUB. Values (mean ± SD, n=3) are averaged from three independent experiments.
Figure 7.
Treatment with THUC, but not THUB or THU, enhances apoptosis mediated by CD8+ T-cell cytotoxicity.
(A) Treatment with THUC, but not THU, increases apoptosis mediated by CD8+ T-cell cytotoxicity. TAP1-knockdown abolishes the THUC-induced increase in apoptosis. PBMCs isolated from AS patients (n = 9) were treated with THUC, and stimulated with IL-2. CD8+ T cells were isolated from THUC-stimulated PBMCs. C1R-B2704 cells were stained with phycoerythrin-conjugated anti-CD19 antibodies and the apoptotic cells were stained with FITC-conjugated anti-active caspase 3 antibody. (B) The results obtained in Figure 7A are plotted. (C) The THUC-induced cell apoptosis mediated by CD8+ T-cell cytotoxicity is CD8-dependent. (D) The results obtained in Figure 7C are plotted. (E) Treatment with THUB cannot enhance apoptosis mediated by CD8+ T-cell cytotoxicity. (F) The resulted obtained in Figure 7E are plotted.