title: Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2.
abstract: Protein glycosylation is important for nucleoside transport, and this has been demonstrated for the human equilibrative nucleoside transporter-1 (hENT1). It is not known whether glycosylation affects the functions of hENT2 or where hENT2 is glycosylated. We address these questions using N-glycosylation mutants (N48D, N57D, and N48/57D) and demonstrate that hENT2 is glycosylated at Asn(48) and Asn(57). Our results show that although the apparent affinities for [3H]uridine and [3H]cytidine of the mutants were indistinguishable from those of the wild-type protein, N-glycosylation was required for efficient targeting of hENT2 to the plasma membrane. All mutants had a two- to threefold increase in IC(50) for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC(50) for NBMPR. We conclude that the relative insensitivity of hENT2 to inhibitors is primarily due to its primary structure and not to glycosylation. Glycosylation modulates hENT1 function, but is not required for hENT2.
authors.affiliation: Department of Medicine, Division of Gastroenterology, The Johns Hopkins University School of Medicine, Ross 925, 720 Rutland Avenune, Baltimore, Maryland 21205, USA.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Ward
authors.forename: George P H
authors.initials: GP
authors.lastname: Leung
authors.forename: Shuy-Vang
authors.initials: SV
authors.lastname: Toan
authors.forename: Chung-Ming
authors.initials: CM
authors.lastname: Tse
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D001216:Asparagine; D001692:Biological Transport; D002460:Cell Line; D003001:Cloning, Molecular; D033722:Equilibrative-Nucleoside Transporter 2 / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D007668:Kidney; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013552:Swine; D014162:Transfection; D014529:Uridine / Q000378:metabolism
chemical_list: D033722:Equilibrative-Nucleoside Transporter 2; D011994:Recombinant Proteins; D001216:Asparagine; D014529:Uridine