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. 2020 Jan 17;10:3088. doi: 10.3389/fimmu.2019.03088

Figure 5.

Figure 5

HA peptide prevents loading of MBP146−170 onto the HLA-DRB1*01:01. (A) Recombinant HLA-DRB1*01:01 (150 nM) and HLA-DM (150 nM) were incubated with biotinylated thioredoxin-fused HA306−318 (Trx-[HA]bio) (150 nM) in the presence of increasing concentrations (0-1 μM) of thioredoxin-fused MBP151−164 (Trx-[MBP]), HA306−311-MBP157−164 (Trx-[HA-MBP]), MBP151−156-HA312−318 (Trx-[MBP-HA]) and HA306−318 (Trx-[HA]). Thioredoxin (Trx) without any peptide was used as a control. At indicated timepoints 8, 6, 4, 2, and 0 h, the amount of bound Trx-[HA]bio was determined by addition of streptavidin-HRP. (B) Binding of thioredoxin fusions containing HA306−318 (red) and chimeric peptides assembled from N- and C-terminal parts of HA306−318 and MBP151−164 (Trx-[MBP-HA] and Trx-[HA-MBP]) in different concentrations (3 nM−10 μM) with recombinant HLA-DRB1*01:01 (5 nM) measured by DELFIA with time-resolved fluorescence detection. (C) Recombinant HLA-DRB1*01:01 (150 nM) was incubated with biotinylated thioredoxin-fused HA306−318 (Trx-[HA]bio) (150 nM) in the presence of increasing concentrations (7.8 nM−1 μM) of thioredoxin-fused HA306−318 (Trx-[HA]) and chimeric peptides assembled from N- and C-terminal parts of HA306−318 and MBP151−164 (Trx-[MBP-HA] and Trx-[HA-MBP]). After 18 h of incubation, the amount of bound Trx-[HA]bio was determined by addition of streptavidin-HRP. (D) Recombinant HLA-DRB1*15:01 (150 nM) and HLA-DM (150 nM) were incubated with biotinylated thioredoxin-fused pp65109−123 (Trx-[pp65]bio) (150 nM) in the presence of increasing concentrations (0-1 μM) of thioredoxin-fused MBP85−97 (Trx-[MBP]), pp65109−114-MBP91−97 (Trx-[pp65-MBP]), and pp65109−123 (Trx-[pp65]). Thioredoxin (Trx) without any peptide was used as a control. At indicated timepoints 5.5, 3.5, 1.5, and 0.5 h, the amount of bound Trx-[pp65]bio was determined by addition of streptavidin-HRP. (E) Recombinant HLA-DRB1*15:01 (150 nM) was incubated with biotinylated thioredoxin-fused pp65109−123 (Trx-[pp65]bio) (150 nM) in the presence of increasing concentrations (7.8 nM−1 μM) of thioredoxin-fused MBP85−97 (Trx-[MBP]), pp65109−114-MBP91−97 (Trx-[pp65-MBP]), and pp65109−123 (Trx-[pp65]). After 18 h of incubation, the amount of bound Trx-[pp65]bio was determined by addition of streptavidin-HRP.