Unveiling the biochemical secrets of ST3GAL3: a multi-approach characterization of the glycosphingolipid pathway

ST3GAL3 is a sialyltransferase that transfer sialic acid to the C-3 position of galactose of glycoproteins and gangliosides. Patients carrying inactive ST3GAL3 variants present neurological symptoms and express normal levels of circulating CA19.9. Enzymatic studies showed that ST3GAL3 uses both Galb1,3GlcNAc (lacto-series) and the Galb1,3GalNAc (ganglio-series) glycosphingolipids, suggesting that it generates of a definite pool of minor but essential brain gangliosides. Cloned ST3GAL3 cDNA variants were transfected into HEK-293T cells to measure sialyltransferase activity through LC-MS/MS. ST3GAL3-KO and b4GALNT1-KO HEK-293T and HCT-15 cells were also obtained through CRISPR-Cas9 genome editing and characterized by end-point RT-PCR. Plasma samples from patients were analyzed by ultra-sensitive HR-LC-MS/MS . Enzyme activity assays indicated that almost all pathogenic ST3GAL3 variants completely lack activity. cDNAs encompassing the whole coding sequence of ST3GAL3 and B4GALNT1, respectively, were detected upon reverse transcription of RNA extracted from parental HEK-293T and HCT-15 cells but not by the respective CRISPR-Cas9 clones, indicating actual gene KO. Semi-quantitative LC-MS/MS analysis detected lower levels of sLc4 (Siaa2,3Galb1,3GlcNAcb1,4Galb1,4Glc-Cer)/GM1b (Siaa2,3Galb1,3GalNAcb1,4Galb1,4Glc-Cer), in plasma patients when compared to controls. We are going to determine by LC-MS/MS the glycosphingolipid profile of the obtained KO cells in steady-state and upon metabolic flux performed after seeding cells with deuterated precursors, and to study variants directly in fibroblasts and plasma samples from two other patients. Together with recent literature data showing no relevant alteration of sialylated glycoproteins in neurons differentiated from patient induced-pluripotent stem cells, our data corroborate the hypothesis that ST3GAL3 plays a crucial role in ganglioside biosynthesis.