Identification and characterization of purine nucleoside transporters from Crithidia fasciculata

doi:10.1016/j.molbiopara.2004.11.018

Molecular and Biochemical Parasitology

Volume 140, Issue 1, March 2005, Pages 1-12

https://doi.org/10.1016/j.molbiopara.2004.11.018 Get rights and content

Abstract

To initiate a molecular dissection into the mechanism by which purine transport is up-regulated in Crithidia, genes encoding nucleoside transporters from Crithidia fasciculata were cloned and functionally characterized. Sequence analysis revealed CfNT1 and CfNT2 to be members of the equilibrative nucleoside transporter family, and the genes isolated encompassed polypeptides of 497 and 502 amino acids, respectively, each with 11 predicted membrane-spanning domains. Heterologous expression of CfNT1 cRNA in Xenopus laevis oocytes or CfNT2 in nucleoside transport-deficient Leishmania donovani demonstrated that CfNT1 is a novel high affinity adenosine transporter that also recognizes inosine, hypoxanthine, and pyrimidine nucleosides, while CfNT2 is a high affinity permease specific for inosine and guanosine. Southern blot analysis revealed that CfNT2 is present as a single copy within the C. fasciculata genome. Starvation of parasites for purines increased CfNT2 transport activity by an order of magnitude, although Northern blot analysis indicated CfNT2 transcript levels increased by <2-fold. These data imply that this metabolic adaptation can mainly be ascribed to post-transcriptional events. Conversely, Southern analysis of CfNT1 suggests that it is a member of a highly homologous multi-copy gene family, indicating that adenosine transport by C. fasciculata is more complex than previously thought.

Introduction

Parasitic protozoa are incapable of synthesizing purine nucleotides de novo, and must, therefore, obligatorily depend upon environmental sources of preformed purines for their survival and growth [1]. Each genus of parasite has, therefore, evolved a unique purine salvage system that enables the organism to obtain nutrient purine. The purine salvage pathway in all protozoan parasites is initiated by the transport of host purines into the parasite. This translocation of purine bases and nucleosides in parasitic protozoa, by definition an indispensable nutritional process, is mediated by transport proteins of diverse ligand specificities and affinities [2].

A number of genes and cDNAs encoding parasite nucleoside transporters have been identified and functionally characterized in heterologous expression systems including Xenopus laevis oocytes and Saccharomyces cerevisiae, both of which are naturally deficient in purine nucleoside transport, as well as in mutationally derived nucleoside transport-deficient Leishmania donovani [3], [4], [5], [6], [7]. These parasite nucleoside transporters are members of the equilibrative nucleoside transporter (ENT) family whose members are characterized by a number of conserved amino acid residues and 11 predicted membrane-spanning domains. Crithidia fasciculata, a monogenetic parasite of the mosquito, has been previously reported to express two nucleoside transport activities of apparently non-overlapping ligand specificity; CfNT1, which recognizes adenosine and pyrimidine nucleosides, and CfNT2, which translocates inosine and guanosine [8]. Both CfNT1 and CfNT2 activities are increased 5–15-fold by incubation in purine-depleted medium [8]. Adenosine and guanosine, as well as adenine and hypoxanthine, transport activities are also markedly augmented by purine starvation of C. luciliae [9], [10], a trypanosomatid of the blowfly, and nucleoside transport into both Trypanosoma brucei [8] and L. donovani [11] is also modestly enhanced by purine limitation.

Because the adaptive response of transport to purine starvation in kinetoplastids has been examined most thoroughly in Crithidia [8], [9], [10], a molecular dissection of these biochemical alterations was initiated in C. fasciculata. Two distinct ENT genes were, therefore, cloned and functionally characterized from this species. The first, designated CfNT1, encodes a novel adenosine transport activity that also recognizes hypoxanthine and inosine, while the second, CfNT2, codes for a permease similar to the CfNT2 activity that was previously described in intact parasites [8]. Several lines of evidence suggest that CfNT1 is part of a family of adenosine transporter genes in C. fasciculata that may be individually regulated in response to exogenous purine levels. Up-regulation of CfNT2 transport activity observed after purine starvation does not appear to be controlled at the transcriptional level.

Section snippets

Cells and reagents

[α-³²P]dCTP (3000 Ci mmol⁻¹) and GeneScreen Plus membranes were purchased from NEN Life Sciences (Boston, MA). [2,8-³H]adenosine (40 Ci mmol⁻¹) was obtained from American Radiolabelled Chemicals Inc. (St. Louis, MO), while [8-³H]guanosine (5.5 Ci mmol⁻¹), [2,8-³H]inosine (17.3 Ci mmol⁻¹), [5,6-³H]uridine (44 Ci mmol⁻¹), [5-³H(N)]cytidine (18.1 Ci mmol⁻¹), and [2,8-³H]hypoxanthine (20 Ci mmol⁻¹) were procured from Moravek Biochemicals (Brea, CA). Synthetic oligonucleotides were acquired from lnvitrogen Corp.

Isolation of CfNT1 and CfNT2

Both CfNT1 and CfNT2 were cloned from a C. fasciculata genomic library by hybridization to probes generated to portions of LdNT1 and LdNT2, their well-characterized counterparts from L. donovani, respectively [3], [4]. Nucleotide sequence analysis of the two ORFs revealed that CfNT1 and CfNT2 encode polypeptides of 497 and 502 amino acids, respectively (Fig. 1). Each protein bears significant homology with other members of the ENT family. CfNT1 shares 72% identity with the adenosine/pyrimidine

Discussion

To initiate a molecular dissection of the regulatory mechanisms that trigger the augmentation of purine transport activities in Crithidia cultured in the absence of purines, genes encoding nucleoside transporters of C. fasciculata were cloned by cross-hybridization to their counterparts from L. donovani [3], [4]. Sequence analysis revealed that CfNT1 and CfNT2 were >70% identical to LdNTl and LdNT2, respectively, a level of homology that is considerably greater than that observed among ENTs

Acknowledgements

This work was supported in part by grants RO1 AI 23682, RO1 AI 44138, and RO1 51507 from the National Institutes of Health (BU) and a grant 0360022Z from the American Heart Association (NSC). CSA was supported by postdoctoral fellowship grant PF-02-097-01-CSM from the American Cancer Society, and an institutional Molecular Hematology Training Grant (T32 HL007781).

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Most eukaryotic organisms including protozoans like Crithidia, Leishmania, and Plasmodium encode a repertoire of equilibrative nucleoside transporters (ENTs). Using genomic sequencing data from Crithidia fasciculata, we discovered that this organism contains multiple ENT genes of highly similar sequence to the previously cloned and characterized adenosine transporter CfNT1: CfAT1 and CfNT3, and an allele of CfAT1, named CfAT1.2. Characterization of CfAT1 shows that it is an adenosine-only transporter, 87% identical to CfNT1 in protein sequence, with a 50-fold lower K_m for adenosine. Site directed mutation of a key residue in transmembrane domain 4 (TM4) in both CfNT1 and CfAT1 shows that lysine at this position results in a high affinity phenotype, while threonine decreases adenosine affinity in both transporters. These results show that C. fasciculata has at least two adenosine transporters, and that as in other protozoan ENTs, a lysine residue in TM4 plays a key role in ligand affinity.
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Citation Excerpt :
The genetic selection was performed using the yeast S. cerevisiae, an organism that lacks endogenous purine nucleoside transport activity (6, 39), has an available mutant strain that relies on exogenous purines for growth (ade2), and has genetic tools that facilitate rapid and efficient screening of plasmid libraries. Expression of CfNT2 conferred upon yeast the ability to grow on medium containing inosine as the sole purine source (Fig. 2A) (17, 27) but not on adenosine (Fig. 2B), which is not a ligand of the wild-type transporter (compare with robust growth of cells expressing CfNT1, a C. fasciculata adenosine transporter (17) (Fig. 2B)). Following PCR mutagenesis of the CfNT2 ORF, transformants were selected directly on medium containing 100 μm adenosine as the sole purine source to identify cfnt2 mutants that had gained the ability to transport adenosine.
Equilibrative nucleoside transporters play essential roles in nutrient uptake, cardiovascular and renal function, and purine analog drug chemotherapies. Limited structural information is available for this family of transporters; however, residues in transmembrane domains 1, 2, 4, and 5 appear to be important for ligand and inhibitor binding. In order to identify regions of the transporter that are important for ligand specificity, a genetic selection for mutants of the inosine-guanosine-specific Crithidia fasciculata nucleoside transporter 2 (CfNT2) that had gained the ability to transport adenosine was carried out in the yeast Saccharomyces cerevisiae. Nearly all positive clones from the genetic selection carried mutations at lysine 155 in transmembrane domain 4, highlighting lysine 155 as a pivotal residue governing the ligand specificity of CfNT2. Mutation of lysine 155 to asparagine conferred affinity for adenosine on the mutant transporter at the expense of inosine and guanosine affinity due to weakened contacts to the purine ring of the ligand. Following systematic cysteine-scanning mutagenesis, thiol-specific modification of several positions within transmembrane domain 4 was found to interfere with inosine transport capability, indicating that this helix lines the water-filled ligand translocation channel. Additionally, the pattern of modification of transmembrane domain 4 suggested that it may deviate from helicity in the vicinity of residue 155. Position 155 was also protected from modification in the presence of ligand, suggesting that lysine 155 is in or near the ligand binding site. Transmembrane domain 4 and particularly lysine 155 appear to play key roles in ligand discrimination and translocation by CfNT2.
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All expression of CfNT2 derivatives in yeast was driven from the high-copy pRS426-Cu yeast–E. coli shuttle vector [22], which allows copper-inducible expression of the inserted gene and contains a URA3 selectable marker. The CfNT2 gene from the protozoan parasite C. fasciculata encodes a 502-amino acid inosine/guanosine equilibrative nucleoside transporter [23]. Of 10 cysteine residues within the CfNT2 open reading frame, 7 were mutated to other amino acids by the Quikchange method (Stratagene, La Jolla, CA, USA).
We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2 to facilitate biochemical studies using thiol-specific modifying reagents. Of 10 endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single- and double-mutant libraries were produced by combining a previously reported site saturation mutagenesis scheme based on the Stratagene Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in Saccharomyces cerevisiae cells auxotrophic for purines, several highly functional noncysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position.
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: Note: Nucleotide sequences for the Crithidia fasciculata CfNT1 and CfNT2 genes have been deposited in GenBank under accession numbers 10764225 and 10764227, respectively.

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Identification and characterization of purine nucleoside transporters from Crithidia fasciculata

Abstract

Introduction

Section snippets

Cells and reagents

Isolation of CfNT1 and CfNT2

Discussion

Acknowledgements

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