N-linked glycosylation regulates SNAT2 trafficking and stability in pancreatic ductal adenocarcinoma cells

The sodium-dependent neutral amino acid transporter 2 (SNAT2) is a plasma membrane transporter that facilitates the uptake of small, aliphatic amino acids. Restricting SNAT2-mediated alanine uptake may be a promising therapeutic strategy for various diseases, including pancreatic ductal adenocarcinoma (PDAC). The post-translational mechanisms regulating the distribution of SNAT2 to intracellular membranes and its turnover remain uncharacterized in the context of PDAC and may be useful to devise future strategies to inhibit SNAT2 function. Human SNAT2 contains three conserved extracellular N-linked glycosylation moieties at N254, N258, and N274. A screen of SNAT2 expression across several human and mouse PDAC cell lines revealed that plasma membrane SNAT2 is exclusively modified by N-linked glycosylation. Preventing SNAT2 N-glycosylation using pharmacological inhibitors or mutagenesis abolished its glycosylation and attenuated its plasma membrane, but not lysosomal, localization. Further, overexpressing glycosylation-deficient SNAT2 in knockout cell lines fails to restore cell proliferative capacity relative to wild-type SNAT2, despite partially rescuing the metabolomic defect associated with SNAT2-deficiency. Using an inducible expression system, we also demonstrate that N-linked glycosylation-deficient SNAT2 mutants exhibit altered degradation kinetics but use similar pathways as wild-type SNAT2 to coordinate its turnover. Our results highlight the importance of N-linked glycosylation for regulating the stability and cell surface expression of nascent SNAT2 in PDAC cells.