BPS2026 – Structural and functional characterization of the human vitamin C tranporter 2 (hSVCT2)

Vitamin C plays vital roles in redox balance and enzymatic processes. Humans rely on the sodium-dependent vitamin C transporters, hSVCT1 and hSVCT2, for efficient acquisition of dietary vitamin C to support health. These transporters differ in tissue distribution and transport kinetics with hSVCT1 responsible for dietary absorption and systemic distribution, while hSVCT2 concentrates vitamin C in metabolically active tissues and unusually acts as a transceptor. To date, structural studies have focused mainly on hSVCT1, with little known about hSVCT2. In addition, virtually nothing is known about the influence of membrane lipids on either transporter. My research investigates the structure and lipid-interactions of hSVCT2. Wild-type (WT) hSVCT2 was successfully expressed in Expi293 cells and purified to high homogeneity suitable for cryo-EM studies. Single particle analysis revealed that WT hSVCT2 tends to dissociate with mainly monomeric particles obtained in 2D class averages. The hSVCT2 mutant G473P is more stable and yields a greater proportion of dimeric views. We are currently optimizing sample and grid preparation for data collection on protein at higher concentrations in thinner ice regions to facilitate high-resolution structural characterization. In addition, I developed a proteoliposome-based uptake assay to investigate the impact of specific lipids on hSVCT2 activity. Cholesterol (CHL) is key for hSVCT2 function. Phospholipids (PLs), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and 1-stearoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (SOPS), further substantially enhance ascorbic acid uptake, demonstrating additive effects in combination with CHL, suggesting that the PLs and CHL have different effects on the transporter. POPE + SOPS has no further effect than the individual PLs alone and sphingomyelin has no detectable effect when added with CHL. More lipid compositions will be explored, and molecular dynamic simulations carried out to gain greater insights into the molecular mechanism of hSVCT2.