title: N-linked glycosylation of N48 is required for equilibrative nucleoside transporter 1 (ENT1) function.
abstract: Human equilibrative nucleoside transporter 1 (hENT1) transports nucleosides and nucleoside analogue 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, suggest that hENT1 is glycosylated at Asn(48) 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 peptide-N-glycosidase F (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 with wt hENT1 based on S-(4-nitrobenzyl)-6-thioinosine (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±44.01 pmol/mg protein; N48Q-3xFLAG-ENT1, 57.91±17.06 pmol/mg protein; mock-transfected 74.31±19.65 pmol/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.
authors.affiliation: Department of Biology, York University, Toronto, Canada M3J 1P3.
authors.forename: Alex
authors.initials: A
authors.lastname: Bicket
authors.affiliation: Department of Biology, York University, Toronto, Canada M3J 1P3 Department of Chemistry and Biology, Ryerson University, Toronto, Canada M5B 2K3 imogen.coe@ryerson.ca.
authors.forename: Imogen R
authors.initials: IR
authors.lastname: Coe
mesh_terms: D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D033721:Equilibrative Nucleoside Transporter 1 / Q000378:metabolism*; D006031:Glycosylation; D057809:HEK293 Cells; D006801:Humans; D016297:Mutagenesis, Site-Directed / Q000379:methods; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism
chemical_list: D033721:Equilibrative Nucleoside Transporter 1; C438649:SLC29A1 protein, human; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine
keywords: N-linked glycosylation; equilibrative nucleoside transporter 1; function; immunofluorescence; oligomerization; trafficking