Figure 2. TAPBPR functions as a peptide loading catalyst and peptide editor for HLA-A2.
(A,B) Dissociation, (C) association and (D) peptide competition of fluorescent peptide FLPSDC*FPSV on HLA-A*02:01 in the absence or presence of TAPBPR or TAPBPRTN5 as measured by fluorescence polarisation. (A) 0.15 µM HLA-A*02:01fos molecules were mixed with 1.2 µM human β2m and loaded with 0.1 μM FLPSDC*FPSV and (B) 0.5 µM HLA-A*02:01 molecules were loaded with 0.125 μM FLPSDC*FPSV. The complexes were then split and incubated with 1000 fold molar excess of (A) FLPSDCFPSV or (B) NLVPMVATV with either A) buffer (No protein) or supplemented with 0.75 μM TAPBPR, or (B) buffer (No protein) or supplemented with 0.25 μM TAPBPR or TAPBPRTN5. Note, the slight difference in dissociation rate observed in A & B is likely to be related to the concentration of TAPBPR used in each experiment and not to the sequence of the competing peptide used. The data shown in Figure 2A,B is representative of 13 independent experiments, which all produced similar results. (C) 0.5 µM HLA-A*02:01 molecules were made peptide receptive and then the binding of 0.125 µM FLPSDC*FPSV was followed in the presence or absence of 0.05 µM TAPBPR or TAPBPRTN5. One representative association experiment of 13 experiments is shown. (D) 0.5 µM HLA-A*02:01 molecules were made peptide-receptive and incubated with 0.125 μM high affinity peptide FLPSDC*FPSV and various concentrations of the lower affinity competing peptide NLVPMVATV (0–20 μM) in presence or absence of 0.25 μM TAPBPR or TAPBPRTN5. One of two experiments is shown. (E–G) Comparison of the dissociation of seven peptides from HLA-A*02:01fos in the presence or absence of TAPBPR or tapasin-Jun as performed in Figure 2—figure supplement 1. The results of three (E) and four (F,G) independent experiments were combined and are shown. Data from each experiment was processed in GraphPad Prism using one-phase exponential decay non-linear regression. The half-lives that were calculated for the dissociation of the indicated peptide in the presence of either (E) Competitor only, (F) Competitor and Tapasin-Jun or (G) Competitor and TAPBPR were plotted as a percentage of the half-life calculated for the dissociation of KLWEAESK*L in the equivalent condition. Error bars show the standard deviation of the relative half-lives measured in the different experiments. While there were slight modifications of experimental conditions between replicate experiments, results consistent with the presented results were observed.
Figure 2—figure supplement 1. Dissociation of seven different peptides from HLA-A*02:01fos in the presence or absence of TAPBPR or Tapasin-Jun.
