Cloning, Heterologous Expression, and in Situ Characterization of the First High Affinity Nucleobase Transporter from a Protozoan

Burchmore, Richard; Wallace, Lynsey J. M.; Candlish, Denise; Al-Salabi, Mohammed I.; Beal, Paul R.; Barrett, Michael P.; Baldwin, Stephen A.; Koning, Harry P. de

doi:10.1074/jbc.m301252200

Cited by 46 publications

(48 citation statements)

References 43 publications

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“…Several ENT homologues were identified from TBLASTN searches of the Wellcome Trust Sanger Institute sequence databases (http://www.sanger-.ac.uk/projects/T_brucei). The cloning and characterization of one of these was previously reported as TbNBT1 (Burchmore et al, 2003). Two others were designated AT-B and AT-D and the following PCR primers from regions flanking the ORF were designed: AT-B: forward, 5Ј-CTTCACCATCTAGCTGAGTCC-3Ј; reverse, 5Ј-TTACCTC-CTCTTAGGGACAG-3Ј; AT-D: forward 5Ј-GATGCTCGGTTTTTAT-TCAG-3Ј; reverse, 5Ј-ATGTCACACTGGTTATTTCAGC-3Ј.…”

Section: Methodsmentioning

confidence: 99%

Molecular Interactions Underlying the Unusually High Adenosine Affinity of a NovelTrypanosoma bruceiNucleoside Transporter

Al-Salabi¹,

Wallace²,

Lüscher³

et al. 2006

Mol Pharmacol

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Trypanosoma brucei encodes a relatively high number of genes of the equilibrative nucleoside transporter (ENT) family. We report here the cloning and in-depth characterization of one T. brucei brucei ENT member, TbNT9/AT-D. This transporter was expressed in Saccharomyces cerevisiae and displayed a uniquely high affinity for adenosine (K m ϭ 0.068 Ϯ 0.013 M), as well as broader selectivity for other purine nucleosides in the low micromolar range, but was not inhibited by nucleobases or pyrimidines. This selectivity profile is consistent with the P1 transport activity observed previously in procyclic and longslender bloodstream T. brucei, apart from the 40-fold higher affinity for adenosine than for inosine. We found that, like the previously investigated P1 activity of long/slender bloodstream trypanosomes, the 3Ј-hydroxy, 5Ј-hydroxy, N3, and N7 functional groups contribute to transporter binding. In addition, we show that the 6-position amine group of adenosine, but not the inosine 6-keto group, makes a major contribution to binding (⌬G 0 ϭ 12 kJ/mol), explaining the different K m values of the purine nucleosides. We further found that P1 activity in procyclic and long-slender trypanosomes is pharmacologically distinct, and we identified the main gene encoding this activity in procyclic cells as NT10/AT-B. The presence of multiple P1-type nucleoside transport activities in T. brucei brucei facilitates the development of nucleoside-based treatments for African trypanosomiasis and would delay the onset of uptake-related drug resistance to such therapy. We show that both TbNT9/ AT-D and NT10/AT-B transport a range of potentially therapeutic nucleoside analogs.

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Section: Methodsmentioning

confidence: 99%

Molecular Interactions Underlying the Unusually High Adenosine Affinity of a NovelTrypanosoma bruceiNucleoside Transporter

Al-Salabi¹,

Wallace²,

Lüscher³

et al. 2006

Mol Pharmacol

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“…TbNT8 mRNAs are expressed in both PF parasites and BF parasites but are more abundant in PF parasites. An independent study (8) identified what is apparently another member of the TbNT8 family that was designated TbNBT1. Its expression in yeast revealed high-affinity transport activities for hypoxanthine, xanthine, adenine, guanine, guanosine, and allopurinol and a moderate-affinity activity for inosine.…”

Section: Nucleoside and Nucleobase Permeases In T Bruceimentioning

confidence: 99%

Nucleoside and Nucleobase Transporters in Parasitic Protozoa

Landfear¹,

et al. 2004

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PURINE TRANSPORT AND SALVAGE IN PARASITIC PROTOZOAOne distinctive feature of the biochemistry of parasitic protozoa is their absolute reliance upon the salvage of preformed purines from their vertebrate and invertebrate hosts. While many mammalian cells possess the innate ability to synthesize purines de novo, all protozoa so far examined that exhibit a parasitic life style lack these biosynthetic pathways and have therefore elaborated a variety of salvage pathways ( Fig. 1) that enable them to acquire preformed purines from their hosts (14). These nutrients are imported from the host as either nucleosides or nucleobases, either of which can serve as a purine source for the parasite. The first step in these salvage pathways entails the uptake of the purine nucleosides or nucleobases from the host milieu and is mediated by various nucleoside or nucleobase transporters, located in the plasma membrane of the parasite, that provide substrate-specific permeation routes. While the enzymes of purine salvage have been studied in considerable molecular detail (7), the identification of individual purine transporters at the molecular level is a more recent development. However, over the past several years a plethora of genes encoding nucleoside or nucleobase permeases from parasitic protozoa have been identified and functionally expressed. The increasing understanding of these carriers at the molecular level provides an appropriate setting for a timely review of these important transporters and a comparison of their properties to related and distinct purine permeases of other organisms. This article will concentrate primarily on purine transporters from the Kinetoplastid parasites Leishmania and Trypanosoma brucei, since these are the protozoa with which the majority of the studies have been accomplished. However, we will also briefly discuss related work on transporters from the Apicomplexa Plasmodium falciparum and Toxoplasma gondii. This review does not attempt to be exhaustive but focuses rather upon recent results utilizing molecular genetic approaches.A principal reason for the interest in purine transport in these protozoa is the essential nature of purine salvage for this large family of parasites. Thus, while some permeases may provide nutrients that are nonessential, albeit advantageous, to the fitness of the organism in its natural environment, the purine transporters as a group are likely to be required for parasite viability in all life cycle stages. A second compelling reason to study these transporters is that they also mediate the uptake of a variety of cytotoxic drugs, many, but not all, of which are purine homologs (11,13,49). Consequently, the import of subversive substrates that are metabolized, often uniquely, by parasite purine salvage enzymes is absolutely dependent upon the purine permeases. Thus, this family of permeases plays an important role in the delivery of drugs or experimental therapeutic compounds, and mutations in these carriers can cause drug resistance (5, 13, 36). STUDIES OF PURINE TR...

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“…None of the 12 identified Arabidopsis NAT loci are yet functionally characterized. Equilibrative nucleoside transporters (ENT) found in plants, protozoans and mammals, act primarily as nucleoside transporters but can also transport nucleobases adenine, guanine, xanthine and hypoxanthine or cytokinin [7][8][9][10][11][12][13]. Eight identified ENT loci are present in the Arabidopsis genome.…”

Section: Introductionmentioning

confidence: 99%

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

2008

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a b s t r a c tIn plants, nucleobase biochemistry is highly compartmented relying upon a well-regulated and selective membrane transport system. In Arabidopsis two proteins, AtAzg1 and AtAzg2, show substantial amino acid sequence similarity to the adenine-guanine-hypoxanthine transporter AzgA of Aspergillus nidulans. Analysis of single and double mutant lines harboring T-DNA insertion alleles AtAzg1-1 and AtAzg2-1 reveal a marked resistance to growth in the presence of 8-azaadenine and 8-azaguanine but not to other toxic nucleobase analogues. Conversely, yeast strains expressing AtAzg1 and AtAzg2 gain heightened sensitivity to growth on 8-azaadenine and 8-azaguanine. Radio-labeled purine uptake experiments in yeast and in planta confirm the function of AtAzg1 and AtAzg2 as plant adenine-guanine transporters.

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Cloning, Heterologous Expression, and in Situ Characterization of the First High Affinity Nucleobase Transporter from a Protozoan

Cited by 46 publications

References 43 publications

Molecular Interactions Underlying the Unusually High Adenosine Affinity of a NovelTrypanosoma bruceiNucleoside Transporter

Molecular Interactions Underlying the Unusually High Adenosine Affinity of a NovelTrypanosoma bruceiNucleoside Transporter

Nucleoside and Nucleobase Transporters in Parasitic Protozoa

AtAzg1 and AtAzg2 comprise a novel family of purine transporters in Arabidopsis

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