HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 275, Issue 27, 20935-20941, July 7, 2000

This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20935    most recent M002418200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carter, N. S. Articles by Ullman, B. Search for Related Content PubMed PubMed Citation Articles by Carter, N. S. Articles by Ullman, B. Social Bookmarking                      What's this?

Cloning of a Novel Inosine-Guanosine Transporter Gene from Leishmania donovani by Functional Rescue of a Transport-deficient Mutant^*

Nicola S. Carter, Mark E. Drew^§, Marco Sanchez^§, Gayatri Vasudevan^§, Scott M. Landfear^§¶, and Buddy Ullman^¶

From the  Department of Biochemistry and Molecular Biology and the ^§ Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland, Oregon 97201

Received for publication, March 21, 2000, and in revised form, April 25, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purine transport is an indispensable nutritional function for protozoan parasites, since they are incapable of purine biosynthesis and must, therefore, acquire purines from the host milieu. Exploiting a mutant cell line (FBD5) of Leishmania donovani deficient in inosine and guanosine transport activity, the gene encoding this transporter (LdNT2) has been cloned by functional rescue of the mutant phenotype. LdNT2 encodes a polypeptide of 499 amino acids that shows substantial homology to other members of the equilibrative nucleoside transporter family. Molecular analysis revealed that LdNT2 is present as a single gene copy within the leishmanial genome and encodes a single transcript of 3 kilobase pairs. Transfection of FBD5 parasites with LdNT2 re-established their ability to take up inosine and guanosine with a concurrent restoration of sensitivity to the inosine analog formycin B. Kinetic analyses reveal that LdNT2 is highly specific for inosine (K_m = 0.3 µM) and guanosine (K_m = 1.7 µM) and does not recognize other naturally occurring nucleosides. Expression of LdNT2 cRNA in Xenopus oocytes significantly augmented their ability to take up inosine and guanosine, establishing that LdNT2 by itself suffices to mediate nucleoside transport. These results authenticate genetically and biochemically that LdNT2 is a novel nucleoside transporter with an unusual and strict specificity for inosine and guanosine.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Leishmania donovani is a protozoan parasite and the etiologic agent of visceral leishmaniasis, a devastating and invariably fatal disease if untreated. The parasite exhibits an intricate life cycle in which the extracellular, flagellated promastigote exists in the phlebotomine sandfly vector, and the intracellular amastigote resides in the phagolysosome of macrophages and other reticuloendothelial cells of the mammalian host. Drugs are the only defense against visceral leishmaniasis, but the efficacy of these empirically derived agents is compromised both by drug toxicity and resistance (1). Thus, it is increasingly imperative to identify new and unique biochemical targets in the parasite for potential therapeutic exploitation.

Among the most conspicuous metabolic differences between parasites and their mammalian hosts is the purine pathway. Whereas animal cells synthesize purine nucleotides de novo, all protozoan parasites are incapable of synthesizing purines and depend upon purine acquisition from their hosts to survive and proliferate (2). Hence, each genus of parasite has evolved a unique complement of purine salvage enzymes in order to scavenge purines from the host milieu (2). The first step in this salvage process involves the translocation of purines across the parasite plasma membrane, a process mediated by membrane permeases. These permeases also initiate the uptake of pyrazolopyrimidine nucleobase and nucleoside analogs of hypoxanthine and inosine that are selectively toxic to Leishmania spp. (3, 4). Thus, purine transporters play vital roles in both purine nutrition and antiparasitic drug targeting intimating that these membrane proteins could be targets for either inhibitor or cytotoxic ligand development.

Genetic and biochemical investigations have demonstrated that L. donovani promastigotes express two nucleoside transport activities of nonoverlapping ligand specificity (5). The first, LdNT1, transports adenosine, pyrimidine nucleosides, and the cytotoxic adenosine analog tubercidin, and the second, LdNT2, recognizes inosine, guanosine, and the cytotoxic inosine isomer formycin B (FoB)¹ (5, 6). Mutant L. donovani clones deficient in LdNT1 or LdNT2 activity have been isolated by virtue of their resistance to either tubercidin or FoB, respectively (5). The availability of these transport-deficient mutants and the ability to transfect Leishmania with cosmid expression libraries (7) provided an avenue for cloning the genes encoding nucleoside transporter proteins by selecting for functional recovery of the wild type drug sensitivity phenotype and, thus, nucleoside transport capability. This functional rescue scheme was previously employed to clone the LdNT1 locus (8).

Functional rescue has now been exploited to isolate LdNT2. LdNT2 is present as a single copy gene within the leishmanial genome and encodes a transcript of ~3 kb. Functional expression of LdNT2 in nucleoside transport-deficient L. donovani and in Xenopus laevis oocytes revealed LdNT2 to be a novel high affinity inosine-guanosine transporter with a singular predicted membrane topology.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals, Materials, and Reagents-- Restriction endonucleases and DNA-modifying enzymes were obtained from New England Biolabs, Inc. (Beverly, MA), Roche Molecular Biochemicals, or Life Technologies, Inc. Radiolabeled D-[2,8-³H]inosine (31.3 Ci mmol¹) and D-[8-³H]guanosine (5.0 Ci mmol¹) were purchased from Moravek (Brea, CA). -[³²P]dCTP (3000 Ci mmol¹) was procured from ICN Biomedicals (Costa Mesa, CA). Hygromycin B was purchased from Roche Molecular Biochemicals and G418 from BioWhittaker (Walkersville, MD). All other chemicals, materials, and reagents were of the highest grade commercially available and bought from either Aldrich or Sigma.

Parasite Cell Culture-- L. donovani wild type (DI700) and LdNT2-deficient (FBD5) parasites (5) were propagated at 26 °C in Dulbecco's modified Eagle's-Leishmania (DME-L) medium (9) containing 100 µM xanthine as a purine source. The FBD5 cells were maintained continuously in DME-L supplemented with 1 µM FoB to ensure that the population did not harbor any wild type revertants. Transfectants were propagated in DME-L supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 50 µg ml¹ of hygromycin B (DME-L/FBS/HYG).

Transfection and Isolation of Cosmids-- To isolate cosmids containing LdNT2, 30 independent transfections were performed, using previously reported parameters (10), on exponentially growing FBD5 cells resuspended at 1 × 10⁸ cells ml¹ in electroporation buffer (10). FBD5 promastigotes were transfected with 10 µg of DNA prepared from a cosmid library of L. donovani Ld4 strain DNA in the shuttle vector cLHYG, which encompasses the hygromycin phosphotransferase gene (7). Transfectants were maintained in the absence of hygromycin B for 24 h post-transfection, after which time they were plated on semi-solid (1% Noble agar, Difco) DME-L/FBS/HYG and incubated at 26 °C in 5% CO₂. After 2-3 weeks, isolated colonies were picked and inoculated into individual wells of 96-well microtiter plates containing 200 µl of DME-L/FBS/HYG and proliferated at 26 °C in 5% CO₂ until individual wells contained at least 5 × 10⁶ cells ml¹. To screen for FoB-sensitive FBD5 transfectants, aliquots of 10 µl from individual wells of the master microtiter plate were transferred into wells of two replica 96-well microtiter plates, one plate containing DME-L/FBS/HYG supplemented with 1 µM FoB and the other containing DME-L/FBS/HYG alone. Clones that were determined to be susceptible to 1 µM FoB were expanded from the appropriate wells in the master microtiter plates, and the cosmids were rescued from these transfectants by alkaline lysis (10).

Isolation of LdNT2-- Cosmids F30F12 and F44H9 were subjected to restriction digestion with BamHI, BglII, EcoRI, EcoRV, HindIII, NotI, SacII, and XbaI. To localize the LdNT2 gene within the F30F12 cosmid, various restriction fragments were subcloned into the leishmanial shuttle vector pSNAR, encompassing the neomycin phosphotransferase gene (11), and tested for restoration of FoB sensitivity after transfection into FBD5 cells and selection in 20 µg ml¹ G418. An ~5-kb EcoRV fragment that conferred the appropriate phenotype was sequenced in its entirety on a Perkin-Elmer Applied Biosystems 377 DNA automated sequencer using dye-terminator cycle methodology by the Core Facility located within the Department of Molecular Microbiology and Immunology at the Oregon Health Sciences University. The LdNT2 open reading frame (ORF) was identified by BLAST searching of available data bases (12) and subsequently sequenced in both directions. Pairwise alignments with other members of the equilibrative nucleoside transporter (ENT) family (8, 13-16) were performed using the algorithm of Needleman and Wunsch (17). Multisequence alignments were conducted using the Feng-Doolittle algorithm (18). Membrane spanning domains were deduced from hydropathy plots constructed from three independent algorithms (19-21).

Southern Analysis-- Genomic DNA was isolated from L. donovani promastigotes according to standard procedures (22). Restriction enzyme-digested DNA was blotted onto GeneScreen Plus® hybridization transfer membrane (NEN Life Science Products) and hybridized to a 2.2-kb HindIII fragment derived from the 5-kb EcoRV fragment in pSNAR that contains the LdNT2 ORF.

RNA Extraction and Northern Blotting-- Total cellular RNA was isolated from ~5 × 10⁸ exponentially growing L. donovani promastigotes using the RNeasy Midi kit (Qiagen Inc., Valencia, CA). Poly(A)⁺ RNA, prepared from total RNA using the Oligotex mRNA mini kit (Qiagen Inc., Valencia, CA), was subjected to denaturing agarose electrophoresis, transferred to a GeneScreen Plus® hybridization transfer membrane (NEN Life Science Products), and probed with the 2.2-kb HindIII fragment described above. Signals were normalized by hybridization to probes corresponding to Leishmania enriettii -tubulin gene (23).

Vectors for Transport Assays-- LdNT2-mediated transport was measured both in FBD5 L. donovani and X. laevis oocytes. For expression in oocytes, a 2.2-kb HindIII fragment encompassing the entire LdNT2 ORF, ~100 bp of 5'-untranslated region, and 600 bp of 3'-untranslated region was excised from the 5-kb EcoRV fragment and subcloned into the pOG-1 oocyte expression vector (24), creating pLdNT2-OG-1. For expression in L. donovani, an ~2.5-kb BamHI-NotI fragment from pLdNT2-OG-1 encompassing the 2.2-kb HindIII LdNT2 fragment and ~300 bp of pOG-1 vector-derived sequence encoding the 3'-untranslated region of the X. laevis -globin gene was subcloned into the appropriate sites within the leishmanial expression vector pALTNEO (25) generating pLdNT2-ALTNEO.

Nucleoside Uptake into FBD5 Cells Expressing LdNT2-- FBD5 promastigotes were transfected with either pLdNT2-ALTNEO or pALTNEO, and the resulting transfectants were maintained continuously in DME-L/FBS supplemented with 25 µg ml¹ G418. Uptake of [³H]inosine (0.31 Ci mmol¹) and [³H]guanosine (0.05 Ci mmol¹) was measured in FBD5 cells harboring either pLdNT2-ALTNEO or pALTNEO as described previously (6). Briefly, FBD5 promastigotes (4 × 10⁸ cells ml¹), resuspended in phosphate-buffered saline, pH 7.4, and supplemented with 10 mM D-glucose, were mixed with radiolabel for various times. Uptake was terminated by a modified oil-stop technique using a dibutyl phthalate cushion (6). All transport measurements on the LdNT2 transfectants were performed on stationary phase parasites, since inosine transport into exponentially growing parasites was nonlinear at concentrations proximal to the K_m value even after a few seconds. Technical limitations of the transport assay precluded the use of fewer cells. Initial rates for each nucleoside concentration were determined by linear regression analysis over the linear portions of the assay, and K_m values were determined by Hanes analysis. Competition experiments were performed in the same buffer containing 1 µM [³H]inosine (0.31 Ci mmol¹) and 100 µM unlabeled inhibitor.

Nucleoside Uptake into X. laevis Oocytes-- Oocytes were dissected and defolliculated as described previously (26, 27) and maintained at 16 °C in frog Ringer's solution supplemented with 2.5 mM pyruvate, 0.5 mM theophylline, and 50 µg/ml gentamycin (Life Technologies, Inc.). The pLdNT2-OG-1 plasmid was linearized with NotI and capped cRNA synthesized in the presence of the cap analog m⁷GpppG by T7 polymerase (28). Stage V-VI oocytes were microinjected with 5-20 ng of cRNA 1 day after defolliculation. Control oocytes were injected with equivalent volumes of water. [³H]Inosine (31.3 Ci mmol¹) and [³H]guanosine (5.0 Ci mmol¹) uptake was assayed after 3 days of cRNA expression. Each oocyte was dissolved in 0.25 ml of 5% sodium dodecyl sulfate, and oocyte-associated radiolabel incorporation was quantitated by liquid scintillation spectrometry.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cloning of LdNT2 by Functional Rescue of the FoB Sensitivity Phenotype-- The cloning of LdNT2 was predicated on the functional restoration of a wild type phenotype (FoB-sensitive, nucleoside transport-competent) in a FoB-resistant, nucleoside transport-deficient (FBD5) background (5). Six thousand independent hygromycin B-resistant colonies representing more than five genome equivalents of leishmanial DNA (7) were picked after transfection with a leishmanial cosmid library and tested for sensitivity to 1 µM FoB. Of these, only two colonies exhibited a wild type FoB-sensitive phenotype, and both were subsequently determined to be inosine transport-proficient (data not shown). The cosmids, designated F30F12 and F44H9, were recovered from the two FoB-sensitive transfectants and subjected to restriction endonuclease analysis with the enzymes EcoRI, NotI, HindIII, XbaI, SacII, EcoRV, BglII, and BamHI. Restriction mapping revealed that both cosmids were distinct, but both contained a common ~11-kb EcoRI fragment. The cosmid F30F12 (Fig. 1A) was selected for further analysis. The location of the LdNT2 gene was determined by subcloning fragments of F30F12 DNA into the leishmanial transfection vector pSNAR (11), transfecting them back into FBD5 cells, and testing for concomitant restoration of FoB sensitivity and inosine transport capability (Fig. 1). A 5-kb EcoRV fragment conferring the appropriate phenotype was sequenced in its entirety, and an ORF (LdNT2) was identified that upon conceptual translation encoded a hydrophobic polypeptide with significant homology to other parasite and mammalian equilibrative nucleoside transporters (8, 13-16, 26, 29).

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Restriction map of the F30F12 cosmid. A, partial map of the insert within the F30F12 cosmid. B, map of an 11-kb EcoRI fragment derived from the F30F12 cosmid. Fragments subcloned into the pSNAR transfection vector (11) and retransfected into FBD5 cells are indicated by the arrows. Whether fragments conferred FoB sensitivity is indicated by either a +, positive, or a , negative. The location of the LdNT2 ORF is indicated by a solid gray bar. Restriction sites are as follows: for panel A, R, EcoRI; N, NotI; X, XbaI; for panel B, R, EcoRI; S, SalI; B, BglII; RV, EcoRV; H, HindIII; P, PstI; N, NotI.

Sequence Analysis of LdNT2-- The LdNT2 ORF comprises 1,497 bp and predicts a polypeptide of 499 amino acids (Fig. 2). A multisequence alignment of LdNT2 with the human equilibrative nucleoside transporters, hENT1(13) and hENT2 (14, 15), the L. donovani LdNT1.1 transporter (8), and the Trypanosoma brucei TbNT2 transporter (16) is depicted in Fig. 2. Pairwise alignments between LdNT2 and each of the other nucleoside transporters in Fig. 2 showed amino acid identities between 25 and 44%. Hydropathy predictions by three independent algorithms (19-21) suggest that LdNT2 accommodates nine transmembrane domains (TMs) with a large hydrophilic loop between TMs 5 and 6 (Figs. 2 and 3). LdNT2 also has two potential Asn-linked glycosylation sites, Asn³²⁶ and Asn⁴⁹⁰ (Fig. 3), although only Asn³²⁶ is predicted to be within an exposed loop.

View larger version (111K): [in this window] [in a new window]   Fig. 2.   Protein sequence alignments with LdNT2. The deduced protein sequence of LdNT2 was aligned with hENT1 (13), hENT2 (14, 15), TbNT2 (16), and LdNT1 (8), by the method of Feng and Doolittle (18). Amino acids identical among all five transporters are shaded black, and conserved amino acids are shaded gray. The predicted membrane-spanning domains for hENT1 and LdNT2 are indicated by the solid lines above and below the aligned proteins and are numbered sequentially.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Predicted topology of LdNT2. LdNT2 topology was predicted by three hydropathy algorithms (19-21), which predict nine membrane-spanning domains and an NH2 terminus on the opposite side of the membrane to the large hydrophilic loop between membrane-spanning domains 5 and 6. Open circles indicate both hydrophobic and polar residues; open circles with either + or  indicate charged residues; gray circles with the letter N indicate potential N-linked glycosylation sites; and black circles with white letters indicate residues that are invariant between LdNT2 and the four other transporters aligned in Fig. 3.

Molecular Characterization of LdNT2 in Wild Type and FBD5 Cells-- Southern blot analysis of L. donovani genomic DNA digested with a battery of restriction enzymes that cut either within (PstI, PvuI, and SalI) or outside (EcoRI and HindIII) the ORF indicated that LdNT2 was a single copy gene (Fig. 4A). This result is compatible with the nucleotide sequence of the 5-kb EcoRV fragment that encompasses LdNT2. The Southern blot of FBD5 genomic DNA hybridized with the LdNT2 ORF was identical to that of wild type DNA (Fig. 4A), demonstrating that neither a gross deletion nor rearrangement of the LdNT2 locus confers the nucleoside transport-deficient phenotype on FBD5 cells.

View larger version (37K): [in this window] [in a new window]   Fig. 4.   Southern and Northern analysis of LdNT2. A, Southern analysis of the LdNT2 locus in wild type (WT) and FBD5 cells. Genomic DNA (~5 µg) isolated from either wild type or FBD5 cells (22) was digested with PstI, SalI, HindIII, PvuI, or EcoRI and probed with the entire LdNT2 ORF. B, Northern analysis was performed, as described under "Experimental Procedures," on polyadenylated RNA (1 µg) from wild type (lane I), FBD5 (lane II), FBD5 transfected with pLdNT2-ALTNEO (lane III), and FBD5 transfected with pALTNEO (lane IV) and probed with either the LdNT2 ORF or the L. enriettii -tubulin gene (23).

Northern analysis of wild type poly(A)⁺ RNA revealed a major LdNT2 transcript of ~3-kb (Fig. 4B). A fainter hybridizing band at ~5 kb was also observed, which could conceivably be an unprocessed mRNA. FBD5 cells, as well as FBD5 cells transfected with either pALTNEO or pLdNT2-ALTNEO, also expressed both the major and minor transcripts, indicating that loss of LdNT2 function in FBD5 cells cannot be attributed to a lack of LdNT2 transcription (Fig. 4B). As expected, LdNT2 is overexpressed in the FBD5 strain transfected with pLdNT2-ALTNEO. The size of this transcript, which arises from splice acceptors within the vector and insert, is also ~3 kb. Levels of poly(A)⁺ RNA were normalized for each cell line using the L. enriettii -tubulin gene (23). There is an additional band that hybridizes to the -tubulin probe in the pLdNT2-ALTNEO lane, suggestive of an alternatively spliced transcript, the reason for which is unclear.

Functional Characterization of LdNT2 in L. donovani-- To establish that LdNT2 is a functional nucleoside transporter, LdNT2 was subcloned into the pALTNEO leishmanial expression vector and transfected into FBD5 cells. Uptake assays using 5 µM [³H]inosine confirmed that transfection with LdNT2 bestows a robust inosine transport phenotype on FBD5 cells (71 pmol/s/10⁸ cells) (Fig. 5). FBD5 cells transfected with pALTNEO alone displayed only minimal inosine transport capability (1 pmol/s/10⁸ cells) (Fig. 5). Substrate saturation curves with FBD5 pLdNT2-ALTNEO cells revealed that [³H]inosine and [³H]guanosine transport displayed Michaelis-Menten kinetics with an apparent K_m value of 0.3 ± 0.1 (n = 4) and 1.7 ± 0.5 µM (n = 3), respectively. Representative experiments for both inosine and guanosine are displayed in Fig. 6. These values are comparable to those determined for wild type parasites (data not shown and Ref. 6).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Functional expression of LdNT2 in FBD5 cells. Uptake of 5 µM D-[2,8-3H]inosine (specific activity 0.31 Ci mmol1) by FBD5 cells transfected with either pLdNT2-ALTNEO () or pALTNEO () was assayed as described under "Experimental Procedures" over a 7-s period. Each time point is the mean ± S.D. of duplicate experiments.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   LdNT2-mediated transport kinetics. A and B, uptake of D-[2,8-3H]inosine (specific activity 0.31 Ci mmol1) or D-[8-3H]guanosine (specific activity 0.05 Ci mmol1) by FBD5 cells transfected with pLdNT2-ALTNEO was determined over a 7-s period for a range of substrate concentrations (either 0.25-5 µM for inosine or 0.5-10 µM for guanosine). The rate of uptake was determined at each substrate concentration by linear regression analysis. The results represented as a Hanes analysis are expressed as either inosine (µM) versus inosine (µM)/rate of uptake (pmol/s/108 cells) or guanosine (µM) versus guanosine (µM)/rate of uptake (pmol/s/108 cells).

An inhibition profile determined for LdNT2-mediated transport into pLdNT2-ALTNEO FBD5 transfectants revealed that 1 µM [³H]inosine transport was inhibited by a 100-fold excess of nonradiolabeled inosine (96%), guanosine (96%), and several analogs, including 8-aminoguanosine (95%), 6-thioguanosine (94%), FoB (74%), 4-thiopurinol riboside (54%), and allopurinol riboside (44%). No inhibitory effects were observed with 9-deazainosine (5%) and 2-aminopurine riboside (<1%) (Fig. 7). By contrast a 100-fold excess of the naturally occurring nucleosides, adenosine (<1%), xanthosine (35%), uridine (33%), cytidine (<1%), and thymidine (14%), as well as the nucleobases, adenine (6%), hypoxanthine (24%), guanine (26%), xanthine (12%), thymine (<1%), and uracil (1%), only inhibited 1 µM [³H]D-inosine marginally (Fig. 7). LdNT2-mediated inosine transport was also not impacted by 100 µM 4-nitrobenzyl-6-thioinosine (NBMPR) (<1%) (Fig. 7), a potent inhibitor of mammalian nucleoside transport at nanomolar concentrations (30).

View larger version (32K): [in this window] [in a new window]   Fig. 7.   Substrate specificity profile of LdNT2-mediated transport. Uptake of 1 µM D-[2,8-3H]inosine (specific activity 0.31Ci mmol1) by FBD5 cells transfected with pLdNT2-ALTNEO was determined over a 7-s period in the presence or absence of various competitors (100 µM). Results are expressed as percent uptake compared with non-competed 1 µM D-[2,8-3H]inosine uptake and each value is the mean rate of uptake ± S.D. (n = 3).

The phenotypic consequence of restored nucleoside transport proficiency in FBD5 cells was also assessed by growth phenotype in FoB. The effective concentration of FoB that inhibited growth of the LdNT2 transfectants by 50% (EC₅₀ value) was 4.5 ± 3.9 nM (n = 4), a value similar to that determined for wild type promatigotes (5.1 ± 3.5 nM) (n = 4) (5). In contrast, FBD5 cells transfected with the empty pALTNEO vector exhibited an EC₅₀ value of 5.7 ± 0.9 µM (n = 4).

Functional Expression of LdNT2 in X. laevis Oocytes-- To confirm that LdNT2 by itself encodes a functional inosine-guanosine transporter, LdNT2 cRNA was expressed in Xenopus oocytes. Oocytes injected with the LdNT2 cRNA transported [³H]inosine and [³H]guanosine 10-20-fold more efficiently than water-injected control oocytes (Fig. 8). Rates of uptake were 0.36 and 0.32 pmol min¹ (oocyte)¹ for inosine and guanosine, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 8.   Functional expression of LdNT2 in Xenopus oocytes. Uptake of 5 µCi ml1 of either D-[2,8-3H]inosine (specific activity 31.3 Ci mmol1) or D-[8-3H]guanosine (specific activity 5 Ci mmol1) by either LdNT2-injected (10 ng/oocyte) (black bars) or water-injected oocytes (white bars) was assayed for 60 min. Each time point is the mean ± S.D. of 5 individual oocytes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

LdNT2 encoding the L. donovani inosine-guanosine transporter was identified and cloned after screening for restoration of a wild type phenotype in inosine-guanosine transport-deficient FBD5 cells following transfection with an LdNT2-containing cosmid. A similar functional rescue strategy has also been exploited to pinpoint and isolate the LdNT1 locus encoding the L. donovani adenosine-pyrimidine nucleoside transporters (8) and offers a powerful genetic approach toward the isolation of any gene for which a selection or screen can be devised. Other leishmanial genes that have been isolated by complementation of mutant phenotypes include several involved in the biosynthesis of lipophosphoglycan (31, 32), an important cell surface glycoconjugate and one crucial for biogenesis of the glycosome (33), a unique kinetoplastid peroxisomal-like microbody that accommodates glycolytic and other nutritional enzymes (34).

The predicted amino acid sequence indicates that LdNT2 is a member of the ENT family. This family includes transporters from mammalian cells (13-15), a variety of protozoan parasites (8, 16, 26, 29, 35), and as yet many functionally uncharacterized ORFs from eukaryotic cells uncovered among a variety of genome sequencing projects (13). ENTs are distinct from bacterial nucleoside transporters (36, 37), as well as from mammalian concentrative nucleoside transporters (38, 39) both in their primary sequences and topological profiles. Multisequence alignment of LdNT2 with other members of this family reveal that LdNT2 shares a number of common residues, most of which reside in predicted membrane-spanning domains, including the charged residues aspartate 389 and arginine 393 within predicted TM 7 (Fig. 2). Hydropathy predictions suggest that LdNT2 exhibits an unusual nine membrane-spanning topology with a large hydrophilic loop between TMs 5 and 6, whereas other members of the ENT family are conjectured to possess 11 membrane-spanning domains. The inferred model for LdNT2 lacks a membrane-spanning domain equivalent to TM 2 of other members of the ENT family and predicts that TM 8 encompasses two of the carboxyl-terminal membrane-spanning domains of the other transporters. If this model is correct, it suggests that the large hydrophilic loop would be on the opposite side of the membrane from the amino terminus, unlike the other ENTs. Whether this loop is exo- or endofacial in any of these transporters, however, is unknown, since these topological predictions have yet to be confirmed experimentally for any ENT member. The topological predictions for LdNT2 were made by three independent algorithms (19-21) in which individual parameters for length of membrane-spanning regions, hydrophobicity, and surface probability were considered. These same algorithms predict 11 membrane-spanning regions for other ENT members.

Functional characterization of LdNT2 indicates that it is a novel high affinity transport system for both inosine and guanosine (Fig. 6) that excludes other purine and pyrimidine nucleosides and bases (Fig. 7). This unusual ligand specificity and affinity are distinct from all previously characterized ENTs from both mammalian cells and parasites. For instance, hENT1 and hENT2 both exhibit a broad ligand specificity for all naturally occurring purine and pyrimidine D-nucleosides (30), but the affinities of the human transporters for these nucleoside ligands are much lower than for either LdNT1 (8) or LdNT2. It is worth noting, however, a sodium-dependent nucleoside transport activity that appears to be specific for guanosine has been described in human acute promyelocytic leukemia cells (40).

LdNT2 also recognizes a variety of cytotoxic inosine and guanosine analogs, although large excesses of 2-aminopurine riboside and 9-deazainosine failed to impede inosine entry (Fig. 7). The inability of LdNT2 to recognize efficiently either 2-aminopurine riboside or adenosine implies that the exocyclic oxygen on C-6 of the purine ring is a critical determinant for ligand recognition by LdNT2. The inosine analog NBMPR, in which the C-6 hydroxyl is replaced by an S-nitrobenzyl moiety, does not inhibit LdNT2 at 100 µM, a concentration 3 orders of magnitude greater than that required to inhibit hENT1 (30).

Southern analysis revealed that LdNT2 is present as a single copy within the leishmanial genome and that there are no gross anomalies in the LdNT2 locus of the mutant FBD5 line. Moreover, LdNT2-specific transcripts of a comparable size and intensity were observed in both wild type and mutant parasites. These results indicate that loss of LdNT2 function in FBD5 cells is most likely due to minor deletions or point mutations within the LdNT2 ORF. Whether this loss of function is due to mutations within both LdNT2 alleles or to mutations within one allele with a concomitant loss of heterozygosity, as has been previously observed for Leishmania (41, 42), is unknown. Isolation and sequence analysis of the mutant LdNT2 locus from FBD5 cells should differentiate between these two models.

The availability of a molecular clone encoding LdNT2, a mutant strain deficient in LdNT2 activity, and both homologous and heterologous expression systems in which to assess LdNT2 function provide a foundation for a thorough analysis of nucleoside transport activity in Leishmania. LdNT2-deficient mutants, the ability to create further mutants, and the identification of conserved residues among nucleoside transporters provide an avenue for the genetic dissection of nucleoside transport both by forward and reverse genetic techniques and suggest LdNT2 as a paradigm for the study of nucleoside transport in higher eukaryotes. Particularly noteworthy is the ability to generate nonlethal loss-of-function mutants with facility. Finally, it is worth noting that functional differences between parasite and mammalian transporters might be exploited therapeutically. Indeed, the selective toxicity of the drugs melarsoprol and pentamidine, both currently employed in the treatment of African trypanosomiasis, is mediated by their uptake on a novel adenine-adenosine transporter (P2) (43, 44). Since LdNT2 differs from its human counterparts in terms of ligand specificity, ligand affinities, and inhibitor profiles, these discrepancies might ultimately be availed of pharmacologically.

	ACKNOWLEDGEMENT

We thank Stephen M. Beverley for the generous gift of the leishmanial cosmid library and for critically reading the manuscript.

	FOOTNOTES

^* This work was supported by Grants AI23682 (to B. U.) and AI44138 (to S. M. L.) from the National Institutes of Health and in part by a grant from the Burroughs Wellcome Fund.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AF245276.

^¶ Scholar in Molecular Parasitology.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Oregon Health Sciences University, Portland, OR 97201. Tel.: 503-494-8437; Fax: 503-494-8393; E-mail: ullmanb@ohsu.edu.

Published, JBC Papers in Press, April 26, 2000, DOI 10.1074/jbc.M002418200

	ABBREVIATIONS

The abbreviations used are: FoB, formycin B; DME-L, Dulbecco's modified Eagle's-Leishmania; FBS, fetal bovine serum; HYG, hygromycin B; ORF, open reading frame; ENT, equilibrative nucleoside transporter; TM, transmembrane domain; NBMPR, 4-nitrobenzyl-6thioinosine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

This article has been cited by other articles:

				R. Valdes, W. Liu, B. Ullman, and S. M. Landfear Comprehensive Examination of Charged Intramembrane Residues in a Nucleoside Transporter J. Biol. Chem., August 11, 2006; 281(32): 22647 - 22655. [Abstract] [Full Text] [PDF]

				S. Arastu-Kapur, C. S. Arendt, T. Purnat, N. S. Carter, and B. Ullman Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter J. Biol. Chem., January 21, 2005; 280(3): 2213 - 2219. [Abstract] [Full Text] [PDF]

				S. M. Landfear, B. Ullman, N. S. Carter, and M. A. Sanchez Nucleoside and Nucleobase Transporters in Parasitic Protozoa Eukaryot. Cell, April 1, 2004; 3(2): 245 - 254. [Full Text] [PDF]

				A. Stein, G. Vaseduvan, N. S. Carter, B. Ullman, S. M. Landfear, and M. P. Kavanaugh Equilibrative Nucleoside Transporter Family Members from Leishmania donovani Are Electrogenic Proton Symporters J. Biol. Chem., September 12, 2003; 278(37): 35127 - 35134. [Abstract] [Full Text] [PDF]

				S. Arastu-Kapur, E. Ford, B. Ullman, and N. S. Carter Functional Analysis of an Inosine-Guanosine Transporter from Leishmania donovani: THE ROLE OF CONSERVED RESIDUES, ASPARTATE 389 AND ARGININE 393 J. Biol. Chem., August 29, 2003; 278(35): 33327 - 33333. [Abstract] [Full Text] [PDF]

				R. J. S. Burchmore, L. J. M. Wallace, D. Candlish, M. I. Al-Salabi, P. R. Beal, M. P. Barrett, S. A. Baldwin, and H. P. de Koning Cloning, Heterologous Expression, and in Situ Characterization of the First High Affinity Nucleobase Transporter from a Protozoan J. Biol. Chem., June 20, 2003; 278(26): 23502 - 23507. [Abstract] [Full Text] [PDF]

				M. I. Al-Salabi, L. J. M. Wallace, and H. P. de Koning A Leishmania major Nucleobase Transporter Responsible for Allopurinol Uptake Is a Functional Homolog of the Trypanosoma brucei H2 Transporter. Mol. Pharmacol., April 1, 2003; 63(4): 814 - 820. [Abstract] [Full Text] [PDF]

				J. Martinussen, S. L. L. Wadskov-Hansen, and K. Hammer Two Nucleoside Uptake Systems in Lactococcus lactis: Competition between Purine Nucleosides and Cytidine Allows for Modulation of Intracellular Nucleotide Pools J. Bacteriol., March 1, 2003; 185(5): 1503 - 1508. [Abstract] [Full Text] [PDF]

				Y. Acimovic and I. R. Coe Molecular Evolution of the Equilibrative Nucleoside Transporter Family: Identification of Novel Family Members in Prokaryotes and Eukaryotes Mol. Biol. Evol., December 1, 2002; 19(12): 2199 - 2210. [Abstract] [Full Text] [PDF]

				G. Vasudevan, B. Ullman, and S. M. Landfear Point mutations in a nucleoside transporter gene from Leishmania donovani confer drug resistance and alter substrate selectivity PNAS, May 22, 2001; 98(11): 6092 - 6097. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: 275/27/20935    most recent M002418200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Carter, N. S. Articles by Ullman, B. Search for Related Content PubMed