Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2

Jeffrey L. Ward, George P H Leung, Shuy Vang Toan, Chung Ming Tse

Research output: Contribution to journalArticle

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 Asn48 and Asn57. 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 IC50 for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC50 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.

Original languageEnglish (US)
Pages (from-to)19-26
Number of pages8
JournalArchives of Biochemistry and Biophysics
Volume411
Issue number1
DOIs
StatePublished - Mar 1 2003

Fingerprint

Glycosylation
Functional analysis
Inhibitory Concentration 50
Cytidine
Dipyridamole
Uridine
Cell membranes
Nucleosides
human SLC29A2 protein
Proteins
Cell Membrane

Keywords

  • Mutagenesis
  • N-linked glycosylation
  • Nucleoside transport
  • Nucleosides

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Functional analysis of site-directed glycosylation mutants of the human equilibrative nucleoside transporter-2. / Ward, Jeffrey L.; Leung, George P H; Toan, Shuy Vang; Tse, Chung Ming.

In: Archives of Biochemistry and Biophysics, Vol. 411, No. 1, 01.03.2003, p. 19-26.

Research output: Contribution to journalArticle

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AB - 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 Asn48 and Asn57. 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 IC50 for dipyridamole. N57D and N48/57D, but not N48D, also had a twofold increase in IC50 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.

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