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Abstract
GM3 Synthase Deficiency (GM3SD) is a rare neurological disorder typically characterized by seizures, severe intellectual disability, choreoathetosis, dysmorphic facial features, and altered skin pigmentation. GM3SD results from variants in the ST3GAL5 gene that encodes GM3 synthase, a glycosphingolipid (GSL)-specific sialyltransferase. GM3 Synthase (ST3GAL5) adds sialic acid to lactosylceramide (LacCer) to synthesize GM3 ganglioside, which in turn, can be extended by other glycosyltransferases to generate the majority of complex gangliosides. Both ST3GAL5 and gangliosides are highly enriched in neural tissues. Patient fibroblasts from two individuals with different variants in ST3GAL5 were reprogrammed to pluripotency; one patient line was derived from an African-American cohort (Salt & Pepper Syndrome, SP-ST3) and has a missense variant in the S-motif of the sialyltransferase domain, while the other was derived from an Amish cohort (A-ST3) and has a variant that truncates the polypeptide between the L- and S-motifs of the catalytic domain. The resulting induced pluripotent stem cells (iPSCs) were differentiated to neural crest cells (NCCs) to investigate the impact of loss of GM3 on neural-specific glycosylation and cell signaling during differentiation. Consistent with our understanding of the variants and with previous research, GM3 and GM3-derived gangliosides are undetectable in iPSCs and NCCs derived from both cohorts. However, GSL profiles exhibited different compensatory responses in relation to how LacCer is utilized and in GSL ceramide composition. Since GSLs are known to be important for membrane organization and cell signaling, we investigated the cell surface proteome and expression levels of receptor tyrosine kinases across NCC differentiation. In SP-ST3 cells, we detected altered relative abundances of signaling receptors, adhesion molecules, and membrane trafficking proteins at the cell surface compared to WT. For both GM3SD variants, we discovered altered ErbB3 and EGFR abundance compared to the differentiation of WT cells. However, the magnitude and timing of altered EGFR and ErbB3 protein expression was different in the two alleles compared to WT. Thus, altered GSL biosynthesis impacts the cell surface proteome and signaling events that accompany differentiation and survival of neural precursors. Additionally, GM3SD cells provide a novel platform to investigate structure/function relationships that connect GSL diversity to cell signaling.