Fig. 2: Ttyh1 mediates endolysosomal clearance of ceramide 1-phosphate (C1P).
a, b Simulated 3-dimensional docking between a human TTYH1 monomer and ceramide 1-phosphate (CerP(d18:1/22:0)). TMD transmembrane domain. Constituent residues of the docking cavity are shown in surface representation. Residues in green were predicted to form hydrophobic interactions with the docked C1P. E210, R213, and Y217 are highlighted in orange. Magnification of the docking region is shown in (b). c Peptide sequence alignment between TTYH1 orthologs of the indicated species. Shown are the partial sequences of the third transmembrane helix. Positions of E210, R213, and Y217 of human TTYH1 are indicated by orange dots. The evolutionarily conserved E210 and R213 are colored in red. d Quantifications of NBD-C1P pulse-chase assay. Ttyh1 WT and Ttyh1 KO astrocytes were pulsed with 2 µM NBD-C1P for 15 min, before chasing in ACSF for 0, 15, 30, and 60 min. NBD fluorescence intensities for each time-point were normalized to the mean of Ttyh1 WT values. Box plots show the median, the bounds of the box (25th and 75th percentiles) and whiskers representing minimum and maximum values. Number of astrocytes (n) from two independent experiments is shown in bracket underneath each box plot. Mann–Whitney test. n.s. not significant, P = 0.379. e E210 and R213 confer C1P clearance function. The two residues in human TTYH1 were substituted with nonpolar alanine to create the TTYH1ER/AA mutant. Astrocytes expressing either wild-type TTYH1 or TTYH1ER/AA mutant were subject to the NBD-C1P pulse-chase assay. Astrocytes were pulsed with 2 µM NBD-C1P for 15 min, followed by chasing for 60 min. Shown are quantifications of NBD fluorescence before and after the chase. Fluorescence intensities were normalized to the mean of the TTYH1-expressing Ttyh1 WT astrocytes values at 0-min chase. Box plots show the median, the bounds of the box (25th and 75th percentiles) and whiskers representing minimum and maximum values. Number of astrocytes (n) from two independent experiments is shown in brackets underneath each box plot. One-way ANOVA (F = 19.3, df = 188) followed by Bonferroni’s multiple comparisons post hoc test. n.s. not significant, P > 0.9999. Source data are provided as a Source Data file.
