Supplementary Table 1

Summary of SPHK1 Mutations, Predictions, and Conservation. It outlines the properties of four SPHK1 mutations observed in prostate cancer samples, including their position, amino acid change, and functional predictions from SIFT and PolyPhen. It also incorporates conservation information from multiple sequence alignment (MSA). Mutations A186T and G369S are both predicted to be damaging and occur in conserved regions, indicating high potential impact. In contrast, P89S is less conserved and borderline tolerated, suggesting moderate functional relevance. R47 is a synonymous variant with no predicted effect.

Supplementary Figure 1

Orthologous SPHK1 Protein Sequences Used in Conservation Analysis. It lists the UniProt accession numbers for SPHK1 orthologs retrieved from human, mouse, rat, and chicken. These sequences were used to perform multiple sequence alignment and assess evolutionary conservation across species at mutation sites of interest.

Supplementary Figure 2

3D Structure of SPHK1 with Labeled Mutation Sites. AlphaFold-predicted structure of the human SPHK1 protein, visualized in PyMOL and colored by residue position from N-terminus (blue) to C-terminus (red). The positions of four recurrent mutations observed in prostate cancer: S47, P89, A186, and G369 are labeled. These residues are distributed across structured and flexible regions, suggesting possible variation in their structural and functional impact.

Supplementary Figure 3

Sequence Coverage of SPHK1 Predictions. A) Normal (wild-type) SPHK1 and B) Mutated SPHK1 sequence coverage as computed during AlphaFold prediction. The x-axis represents residue positions, and the y-axis shows the number of aligned sequences. The color gradient indicates sequence identity to the query, with higher identity (blue) reflecting more reliable evolutionary alignment.

Supplementary Figure 4

Multiple sequence alignment of SPHK1 orthologs across species. It shows aligned SPHK1 protein sequences from human, mouse, rat, and chicken, focusing on regions surrounding mutation hotspots (e.g., P89S, A186T, G369S). The alignment is color-coded using the Clustal2 scheme, which highlights residue type conservation. The top sequence in each panel corresponds to the human SPHK1 protein (Q9NYA1). All mutation sites of interest are highly conserved across species, especially residues A186 and G369, supporting their likely functional importance. This comparative analysis reinforces the hypothesis that mutations in these conserved positions may disrupt SPHK1 structure or function.

Supplementary Figure 5

Cross-species multiple sequence alignment centered on SPHK1 G369 position. This figure illustrates the conservation of SPHK1 residues across five species (human, mouse, rat, chicken, and a UniProt reference entry), focusing on the region around position 369. Each panel shows the same region with a different sequence placed on top for comparison. The G369 residue is highly conserved across all species shown, reinforcing its likely functional significance. The alignment uses the Clustal2 coloring scheme to highlight conserved and variable residues. This supports the interpretation that the G369S mutation occurs in an evolutionarily constrained region, where substitution may influence protein structure or function.

Supplementary Figure 6

Variant Effect Predictor (VEP) results for SPHK1 mutations. It displays the output from the Ensembl Variant Effect Predictor (VEP) used to assess the potential functional consequences of SPHK1 mutations found in prostate cancer samples. Pie charts summarize the types of variants identified, with missense variants being the most common. The detailed variant table lists amino acid changes, codons, SIFT and PolyPhen scores, and conservation annotations. Notably, the mutations A186T and G369S are predicted as “damaging” by both SIFT and PolyPhen, with high somatic occurrence, supporting their potential functional relevance.

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