Fig. 6

A GSEA analysis of transcriptome displayed significant change of sphingolipid metabolism and related pathway in aldh3b1^-/- and 2-HD treated aldh3b1^+/+larvae. Baldh3b1 deficiency and 2-HD treatment decreased S1pr5 protein in zebrafish larvae (n = 3 for each group). Caldh3b1 deficiency decreased S1pr5 protein in the zebrafish eye (n = 4/4). D, E Reduced S1pr5 expression by s1pr5a/b morpholinos (MO) injection increased hyaloid sprouts (D) and branchpoints (E) of 5dpf zebrafish larvae (n = 14 for larvae injected with s1pr5b MO, and n = 13 for all other groups). F S1pr5 morpholinos injection decreased S1pr5 and Fsp1 proteins, but Gpx4 remained unaltered (n = 4 for each group). G, Haldh3b1^-/- and 2-HD induced hyaloid vascular alterations can be rescued by the S1PR5 agonist A-971432 (n = 14 for each group). I SPR assay revealed 2-HD bind to S1PR5 in a concentration-dependent manner. J Flow cytometry analysis showed 100 and 200 μmol 2-HD significantly decreased surface S1PR5 signal of NK cells (n = 5). K Computational model demonstrated interaction modes between 2-HD and S1PR5 with ONO-5430608-S1PR5 as controls. The red surface highlights 2-HD, the purple surface denotes ONO-5430608 (ONO), yellow sticks represent consensus residue shared by two modulators, and green sticks indicate 2-HD unique binding sites. (L) Schematic diagram of the ligand binding pocket and interactions between 2-HD and S1PR5 compared to interactions of ONO-S1PR5 and S1P-S1PR3. Residues are color-coded according to their interaction specificity. Yellow for consensus residues between 2-HD and ONO, green for 2-HD’s unique binding sites, purple for consensus residues shared between 2-HD and S1P, and pink for consensus residues shared among ONO, 2-HD, and S1P. ECM, extracellular matrix; S1PR5, Sphingosine-1-phosphate receptor 5; MO, morpholino; A-971, A-971432; SPR, Surface Plasmon Resonance. Statistical analysis was performed using one-way ANOVA for panels (B, D–H, and J) two-tailed Student’s t test for C. The bars indicate mean ± SD values. For Fig. 6A, GSEA was performed using a permutation-based statistical test. Significance was assessed using the Normalized Enrichment Score (NES) and the False Discovery Rate (FDR) q-value to account for multiple hypothesis testing. Source data are provided as a Source Data file.