Functional expression and characterization of a purine nucleobase transporter gene from Leishmania major

Abstract

Leishmania major, like all the other kinetoplastid protozoa, are unable to synthesize purines and rely on purine nucleobase and nucleoside acquisition across the parasite plasma membrane by specific permeases. Although, several genes have been cloned that encode nucleoside transporters in Leishmania and Trypanosoma brucei, much less progress has been made on nucleobase transporters, especially at the molecular level. The studies reported here have cloned and expressed the first gene for a L. major nucleobase transporter, designated LmaNT3. The LmaNT3 permease shows 33% identity to L. donovani nucleoside transporter 1.1 (LdNT1.1) and is, thus, a member of the equilibrative nucleoside transporter (ENT) family. ENT family members identified to date are nucleoside transporters, some of which also transport one or several nucleobases. Functional expression studies in Xenopus laevis oocytes revealed that LmaNT3 mediates high levels of uptake of hypoxanthine, xanthine, adenine and guanine. Moreover, LmaNT3 is an high affinity transporter with K_m values for hypoxanthine, xanthine, adenine and guanine of 16.5±1.5, 8.5±0.6, 8.5±1.1 and 8.8±4.0 μM, respectively. LmaNT3 is, thus, the first member of the ENT family identified in any organism that functions as a nucleobase rather than nucleoside or nucleoside/nucleobase transporter.

• Leishmania
• purines
• kinetoplastida
• nucleobase transporter

LmaNT3, Leishmania major nucleobase transporter 3; LmaNT3, the gene encoding LmaNT3; ORF, open reading frame; S.D., standard deviation; bp, base pairs; ENT, equilibrative nucleoside transporter; TMD, transmembrane domain; PAC, P1 artificial chromosome; NAT, nucleobase-ascorbate transporter; PRT, purine-related transporter; PCR, polymerase chain reaction

LmaNT3, Leishmania major nucleobase transporter 3; LmaNT3, the gene encoding LmaNT3; ORF, open reading frame; S.D., standard deviation; bp, base pairs; ENT, equilibrative nucleoside transporter; TMD, transmembrane domain; PAC, P1 artificial chromosome; NAT, nucleobase-ascorbate transporter; PRT, purine-related transporter; PCR, polymerase chain reaction