Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analog drugs across cellular membranes and is necessary for the uptake of many anti-cancer, anti-parasitic, and anti-viral drugs. Previous work, and in silico prediction, suggests that hENT1 is glycosylated at Asn48 in the first extracellular loop of the protein and that glycosylation plays a role in correct localization and function of hENT1. Site-directed mutagenesis of wild-type (wt) hENT1 removed potential glycosylation sites. Constructs (wt 3xFLAG-hENT1, N48Q-3xFLAG-hENT1 or N288Q-3xFLAG-hENT2) were transiently transfected into HEK293 cells and cell lysates were treated with or without PNGase-F, followed by immunoblotting analysis. Substitution of N48 prevents hENT1 glycosylation, confirming a single N -linked glycosylation site. N48Q-hENT1 protein is found at the plasma membrane in HEK293 cells but at lower levels compared to wt hENT1 based on NBTI binding analysis (wt 3xFLAG-ENT1 Bmax, 41.5±2.9 pmol/mg protein; N48Q-3xFLAG-ENT1 Bmax, 13.5±0.45 pmol/mg protein) and immunofluorescence microscopy. Although present at the membrane, chloroadenosine transport assays suggest that N48Q-hENT1 is non-functional (wt 3xFLAG-ENT1, 170.80±44.01pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06pmol/mg protein; mock-transfected 74.31±19.65pmol/mg protein). Co-immunoprecipitation analyses suggest that N48Q ENT1 is unable to interact with self or with wt hENT1. Based on these data we propose that glycosylation at N48 is critical for the localization, function, and oligomerization of hENT1.
- Equilibrative Nucleoside Transporter 1
- N-linked glycosylation
- ©2016 The Author(s)
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