RF catabolites refold with MR1 and modulate cell surface expression. (A and B) Shown is the gel filtration (S200 10/300 GL; GE Healthcare) purification (left) and SDS-PAGE analysis (right) of MR1-β2m binary complexes loaded with (A) 5-OP-RU, non-exposed RF, and photodegraded RF, as well as the RF catabolites (B) FMF, lumichrome, lumiflavin, and alloxazine. Absorption at 280 nm and volume (ml) are shown on the y and x axis, respectively. (C) Crystallographic omit maps of UV-treated RF are presented as a Fobserved – Fcalculated omit map (green mesh) contoured at 2σ showing the position of the RF degradation product(s) within MR1 cleft connected with Lys-43. The superimposed green and yellow sticks represent lumichrome and carboxymethylflavin, respectively, which have been suggested by mass spectrometry to be the main RF photodegradation catabolites in the refold sample. (D) Bar graph shows the frequency of viable cells in single lymphocytes gated for C1R.MR1 cells after 16-h treatment of the indicated ligands. (E–G) C1R cells (E) overexpressing HLA-A*02:01, (F) expressing wild-type level of MR1*01, or (G) overexpressing MR1.K43A mutant were incubated for the indicated periods with titrated quantities of ligand followed by flow cytometry. The C1R and C1R.A2 cells were stained with biotinylated 8F2F9 and W6/32, respectively, followed by PE-labeled streptavidin. Data in D–G are depicted as a fold change from basal surface expression upon incubation with the vehicle (1 mM NaOH). Each column represents the average of at least three independent experiments performed in duplicate with standard error (SEM) represented by error bars. One-way ANOVA statistical analysis was performed for all samples followed by Dunnett’s multiple comparison test using NaOH as a control (ns; not significant, ****P < 0.0001). Source data are available for this figure: SourceData FS1.
Figure S1.

RF catabolites refold with MR1 and modulate cell surface expression. (A and B) Shown is the gel filtration (S200 10/300 GL; GE Healthcare) purification (left) and SDS-PAGE analysis (right) of MR1-β2m binary complexes loaded with (A) 5-OP-RU, non-exposed RF, and photodegraded RF, as well as the RF catabolites (B) FMF, lumichrome, lumiflavin, and alloxazine. Absorption at 280 nm and volume (ml) are shown on the y and x axis, respectively. (C) Crystallographic omit maps of UV-treated RF are presented as a Fobserved – Fcalculated omit map (green mesh) contoured at 2σ showing the position of the RF degradation product(s) within MR1 cleft connected with Lys-43. The superimposed green and yellow sticks represent lumichrome and carboxymethylflavin, respectively, which have been suggested by mass spectrometry to be the main RF photodegradation catabolites in the refold sample. (D) Bar graph shows the frequency of viable cells in single lymphocytes gated for C1R.MR1 cells after 16-h treatment of the indicated ligands. (E–G) C1R cells (E) overexpressing HLA-A*02:01, (F) expressing wild-type level of MR1*01, or (G) overexpressing MR1.K43A mutant were incubated for the indicated periods with titrated quantities of ligand followed by flow cytometry. The C1R and C1R.A2 cells were stained with biotinylated 8F2F9 and W6/32, respectively, followed by PE-labeled streptavidin. Data in D–G are depicted as a fold change from basal surface expression upon incubation with the vehicle (1 mM NaOH). Each column represents the average of at least three independent experiments performed in duplicate with standard error (SEM) represented by error bars. One-way ANOVA statistical analysis was performed for all samples followed by Dunnett’s multiple comparison test using NaOH as a control (ns; not significant, ****P < 0.0001). Source data are available for this figure: SourceData FS1.

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