At the beginning of the experiment, B*27:05-Y84C still showed significantly higher cell surface levels than the wild type in TAP2-deficient cells (Fig 3A, left) since B*27:05-Y84C levels also increased after the 25C incubation (data not shown). STF1-TAP2 cells. TAP function was confirmed by measuring the surface levels of endogenous HLA levels by using the monoclonal anti-HLA antibody, W6/32 and anti-mouse IgG conjugated to AlexaFluo-488 in flow cytometry. In peptide-deficient STF1 cells, only low cell surface expression of endogenous HLA molecules could be detected (dashed line), in peptide-proficient STF1-TAP2 cells, however, the surface expression was strongly enhanced (solid line) confirming functionality of the reconstituted TAP transporter. (C) Cell surface expression of B*27:05 and B*27:05-Y84C. Cells were stained with W6/32 and anti-mouse IgG conjugated with AlexaFluor-488 and subjected to flow cytometry. Surface signal intensities from B*27:05 (blue) and B*27:05-Y84C (orange) are displayed as histograms. Grey lines indicate cells that were stained only with the secondary antibody. (D) The scatter plot (mean standard deviation, n = 3) shows individual cell surface W6/32 measurements in STF1 and STF-TAP2 cells (black dots). In TAP2-deficient STF1 cells, surface expression of B*27:05-Y84C was about three times higher than for the wild type construct (left) whereas in TAP2-proficient cells, both constructs showed comparable cell surface expression (right). (TIF) pone.0200811.s001.tif (9.1M) GUID:?6E980085-415D-4082-8A6A-6696A74A95A1 S2 Fig: Surface Centrinone-B lifetimes of wild type and disulfide mutant of HLA B*27:05 can be rescued at the cell surface of TAP2-deficient cells at 25C. (A) Wild type B*27:05 reaches the cell surface of TAP2-deficient cells at 25C. Peptide-deficient STF1 cells expressing wild type B*27:05 were kept at 25 and 37C, respectively, stained with anti-HA and anti-mouse IgG conjugated with AlexaFluor-488, and subjected to flow cytometry. Wild type B*27:05 shows a much higher cell surface expression at 25 (blue line) than at 37C (orange line). The grey curve Centrinone-B in both histograms shows the background signal without primary antibody. Quantification of surface signals obtained at 25C (blue) and 37C (black, set to one) revealed a 4-fold increase in surface levels of wild type B*27:05 (scatter plot with mean standard deviation, right).(B) Averaged BFA decay from the cell surface at 25C. STF1 cells were kept at 25C and surface levels of B*27:05 and B*27:05-Y84C were detected by staining STF1 cells with anti-HA. Cells were harvested and stained at the times indicated representing the duration of treatment with Brefeldin A. The graph shows the cell surface levels normalized to the values detected at time point zero (SEM, n = 4), which was set to 100% with the following values depicted as its percentage. Both constructs show similar residence times at the cell surface when incubated at 25C. (C) B*27:05 free heavy chains on the surface of TAP-deficient cells. Scatter plot (mean standard deviation, n = 2,4,4) shows the levels of class I free heavy chains detected by HC-10 antibody at the surface of STF1, STF1-B*27:05 and STF1-B*27:05-Y84C cells at 37C, respectively. Acquired staining intensities from individual experiments were normalized to wild type B*27:05 levels. B*27:05-Y84C reveals approximately 4-fold higher more free heavy chains that this wild type protein. (D) Peptide binding to B*27:05 at the cell surface. STF1 cells expressing either B*27:05-WT or B*27:05-Y84C were Centrinone-B incubated with 20 M of the B*27:05-specific peptide IRAAPPPLF overnight (black bars). Amount of B*27:05 molecules were detected with anti-HA antibody and displayed in comparison with the samples without peptide addition (grey bars). IRAAPPPLF can bind and stabilize B*27:05-Y84C molecules that SERK1 have reached cell surface whereas surface levels of B*27:05-WT cannot be improved by the peptide. (TIF) pone.0200811.s002.tif (11M) GUID:?9AA1D3FF-D3BC-451F-8765-70E75CB30BC5 Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract HLA-B*27:05 is usually associated with the development of autoimmune spondyloarthropathies, but the precise causal relationship between the MHC haplotype and disease pathogenesis is usually yet to be elucidated. Studies focusing on the structure and cellular trafficking of HLA-B*27:05 implicate several links between the onset of inflammation and the unusual conformations of the molecule inside and at the surface of antigen presenting cells. Several lines of evidence emphasize the emergence of those unnatural protein conformations under conditions where peptide loading onto B*27:05 is usually impaired. To understand how cellular factors distinguish between poorly loaded molecules from the optimally loaded ones, we have Centrinone-B investigated the intracellular transport, folding, and cell surface expression of this particular B27 subtype. Our Centrinone-B findings show that B*27:05 is usually structurally unstable in the absence of peptide, and that an artificially introduced disulfide bond between residues 84 and 139 conferred enhanced conformational stability to the suboptimally loaded molecules. Empty or suboptimally loaded B*27:05 can escape intracellular retention and arrive at the cell surface leading to the appearance of increased number of analyses revealed increased molecular disorder in the alpha helices involved in the F pocket region in the 05 subtype compared to the 09, leading to a general instability of the heavy chains of B*27:05 . In order to test whether B*27:05 can be conformationally stabilized by limiting the mobility.