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Physiol. Genomics 28: 337-347, 2007. First published November 7, 2006; doi:10.1152/physiolgenomics.00087.2006
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Received 15 May 2006; accepted in final form 27 October 2006.
Physiological Genomics 28:337-347 (2007)
1094-8341/07 $8.00 © 2007 American Physiological Society

Genomic analysis of nucleoside transporters in Diptera and functional characterization of DmENT2, a Drosophila equilibrative nucleoside transporter

Jerry Machado1, Parween Abdulla1, W. J. Brad Hanna2, Arthur J. Hilliker1 and Imogen R. Coe1

1 Department of Biology, York University, Toronto, Ontario
2 Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, Canada


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The recent completion of genome sequencing projects in a number of eukaryotes allows comparative analysis of orthologs, which can aid in identifying evolutionary constraints on protein structure and function. Nucleoside transporters (NTs) are present in a diverse array of organisms and previous studies have suggested that there is low protein sequence similarity but conserved structure in invertebrate and vertebrate NT orthologs. In addition, most taxa possess multiple NT isoforms but their respective roles in the physiology of the organism are not clear. To investigate the evolution of the structure and function of NTs, we have extended our previous studies by identifying NT orthologs in the Dipteran Anopheles gambiae and comparing these proteins to human and Drosophila melanogaster (Dm) NTs. In addition, we have functionally characterized DmENT2, one of three putative D. melanogaster ENTs that we have previously described. DmENT2 has broad substrate specificity, is insensitive to standard nucleoside transport inhibitors and is expressed in the digestive tract of late stage embryos based on in situ hybridization. DmENT1 and DmENT2 are expressed in most stages during development with the exception of early embryogenesis suggesting specific physiological roles for each isoform. These data represent the first complete genomic analysis of Dipteran NTs and the first report of the functional characterization of any Dipteran NT.

concentrative nucleoside transporters; evolution; Anopheles


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
NUCLEOSIDE TRANSPORTERS (NTs) are integral membrane proteins responsible for movement of nucleosides across cell membranes (4, 13, 26). Nucleosides play key roles in eukaryote physiology, acting as signaling molecules, neuromodulators and in the regulation of cardiovascular activity (2, 4, 6). Nucleosides are also precursors of nucleic acids and are either synthesized de novo or salvaged from the extracellular environment via NTs. These salvage pathways are needed when de novo pathways are lacking, for example in protozoans, such as the malarial parasite Plasmodium, that cannot synthesize purines (20) and in specialized eukaryotic cells, such as in enterocytes, bone marrow, and certain brain cells (10).

NTs have been primarily studied in mammalian systems and are divided into two main categories based on their mechanism of transport. The equilibrative nucleoside transporters (ENTs) facilitate the movement of nucleosides down their concentration gradients, while the concentrative nucleoside transporters (CNTs) actively transport nucleosides against their concentration gradient by cotransport of a cation, usually Na+, down its gradient (13). In mammals, ENTs have broad permeant selectivity, whereas the CNTs tend to be selective of their substrate (4). ENTs have been further categorized by their sensitivity to nitrobenzylthioinosine (NBTI), a tight-binding and selective inhibitor of ENT1 in mammals. ENT1 is sensitive to NBTI at low concentrations (IC50 <5 nM), whereas ENT2 is inhibited at relatively high concentrations (IC50 >1 µM) (8). In humans, both ENT1 and ENT2 are sensitive to the coronary vasodilator drugs dipyridamole and dilazep, which inhibit influx of nucleosides.

ENTs are widely distributed in eukaryotes and additional ENT isoforms (ENT3, ENT4) have been identified by database mining (1). ENT3 has recently been characterized as an intracellular nucleoside transporter in mammals (5), while ENT4 has recently been characterized as a pH-dependent cardiac adenosine transporter (7). Although CNTs are functionally similar and found in many phyla, ENTs and CNTs are considered to be evolutionarily distinct (1, 10).

ENTs from diverse taxa generally have low sequence similarity based on their degree of amino acid identity but strikingly similar predicted topologies and functions (27). Therefore, comparative approaches can be useful in gaining insight into the cellular physiology of these important proteins while also providing information as to regions of the protein that have been conserved, presumably by structural or functional constraints, over evolutionary history. The completion of genome sequencing projects provides a unique opportunity to identify all NT family members within a single taxon using bioinformatic approaches. Although most databases still require significant manual curation in terms of correct annotation of sequences (3, 16, 22), it is possible to obtain enough sequence information to provide insight in the structure and distribution of NTs within a single taxon and to compare orthologs to other completed NT families (e.g., human, Caenorhabditis elegans). We have used this approach to identify new ENT family members (1) and to determine prototypic ENT structure across diverse phyla (27). Here we extend these studies to identify NTs in the recently completed Anopheles gambiae (Ag) genome, a comparator to the Dipteran, Drosophila melanogaster (Dm), and host to the malarial parasite, Plasmodium, a target of nucleoside analog chemotherapy (19, 20, 22). Moreover, the use of model systems allows whole animal approaches in studying the relevance of multiple NT members within a single taxon. D. melanogaster offers an excellent model to examine temporal and spatial expression of NTs during development and to begin to define roles of NTs in a multicellular context. Therefore, we have extended our previous studies (27) by functionally characterizing one of the Dipteran nucleoside transporters, DmENT2, to experimentally confirm our in silico analysis of a putative nucleoside transporter. In addition, we investigated spatial mRNA expression of DmENT2 by in situ hybridization in whole mount Drosophila embryos (28) to provide insight into the physiological expression of NTs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Drosophila and Anopheles NT genes.
Isolation and characterization of Drosophila ENT sequences have been previously described (27). Similar approaches were used to identify ENTs and CNTs in Anopheles and CNTs in Drosophila. Putative Anopheles transporters were also identified using Ensembl (http://www.ensembl.org/index.html).

Identification of gene and predicted protein structure.
The gene structure, exon, and intron splice sites of NTs were identified using reported mRNAs or predicted open reading frames and they were also checked with standard donor-acceptor consensus sites (GT/AG). The putative proteins were analyzed using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM/) to determine possible topology.

Sequence similarities and phylogenetic relationship between NTs.
Amino acid sequences of Drosophila, Anopheles, and human (h) NTs were obtained from GenBank and were aligned with their respective NT using ClustalW (http://www2.ebi.ac.uk/clustalw/). Human GenBank identifiers for hENT1, hENT2, hENT3, hENT4, hCNT1, hCNT2, and hCNT3 are gi:20136735, gi:2811137, gi:12656639, gi:25418480, gi:9296936, gi:9296940, and gi:10732815, respectively. The GenBank (http://www.ncbi.nlm.nih.gov) identifiers of DmENT1 (CG11907), DmENT2 (CG11045), and DmENT3 (CG11010) are gi:8132774, gi:7297138, and gi:23093598, respectively. NT sequences from Plasmodium were obtained from PlasmoDB (http://plasmodb.org/) using recently published data (23). The PlasmoDB identifiers for characterized and uncharacterized Plasmodium ENTs are PFA0160c, MAL8P1.32, PF13_0252, and PF14_0662. The resulting alignments were submitted through Boxshade 3.21 (http:www.ch.embnet.org/software/BOX_form.html) to produce amino acid identities and similarities. Percent identities of NTs were determined using the Network Protein Sequence Analysis Server (http://npsapbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NSPA/npsa_clustalw.html). To examine phylogenetic relationships between each respective NT family, a sequence alignment was performed using ClustalW. The aligned sequence was then converted to MEGA format using MEGA 3.1 (www.megasoftware.net) to produce a phylogenetic tree (18).

Isolation and cDNA cloning of DmENT1 and DmENT2.
All solutions and materials were purchased from Sigma-Aldrich (Oakville, ON, Canada), unless indicated, and restriction enzymes were purchased from New England Biolabs (Pickering, ON, Canada). Isolation and amplification of DmENT1 and DmENT2 were explained previously (27). DmENT1 and DmENT2 cDNAs were excised and purified using the QIAquick gel extraction kit protocol (Qiagen, Mississauga, ON, Canada) and cloned into pGEM-T (Promega, Mississauga, ON, Canada). DmENT2 was first cloned into pBlueScript (Stratagene, Cedar Creek, TX) and then into pcDNA3 (Invitrogen, Burlington, ON, Canada). DmENT2 was then removed from pcDNA3 using BamHI and XhoI and cloned into pCS2+ (generously provided by Dave Turner, University of Michigan). DmENT1 was amplified from pGEM-T using primers described previously (27) with the exception of the replacement of the 5'-end with a BamHI restriction site and the 3'-end with an EcoRI restriction site. The PCR product was amplified using the Tsg DNA polymerase (BioBasic, Markham, ON, Canada) with the following protocol: one cycle of 94°C for 2 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final cycle of 72°C for 10 min. The resulting PCR product was then cloned into the pCS2+ vector. hENT1 was amplified from a kidney cDNA library, using 5'-CCATCGATGGATGACAACCAGTCACCAGCCTCAGGACAGA-3' and 5'-GCTCTAGAGCTCACACAATTGCCCGGAACAGGAAGGAGAAAAC-3' for the forward and reverse primers, respectively. The PCR product was amplified using the Tsg DNA polymerase with the following protocol: one cycle of 94°C for 2 min; 30 cycles of 94°C for 30 s, 55°C for 30 s, 72°C for 30 s, and a final cycle of 72°C for 10 min. The 5'-end contained an engineered Cla1 restriction site and a 3'-end XbaI restriction site. The resulting PCR product was digested and then cloned into pCS2+. All cDNA sequences were sequenced at the Core Molecular Facility at York University (Toronto, ON, Canada).

In vitro RNA transcription.
cRNA was transcribed using the mMessage mMachine protocol (Ambion, Austin, TX). DmENT2 that was cloned into pCS2+ was linearized with SacII; hENT1 and DmENT1 that were cloned into pCS2+ were linearized with NotI and all were transcribed using the SP6 RNA polymerase. DmENT3 was available commercially and was cloned into pFLC-I (Invitrogen), linearized with ApaI, and transcribed using the T7 RNA polymerase. cRNA was analyzed with a spectrophotometer and by agarose formaldehyde gel electrophoresis to determine concentration and integrity, respectively.

Expression and transport assays in Xenopus laevis oocytes.
Oocytes were harvested during surgery and placed in ND96 medium (96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, and 2.5 mM pyruvate, pH 7.5) supplemented with gentamicin sulphate (50 µg/ml) and bovine serum albumin (0.1% wt/vol). Follicular cells from the oocytes were removed by treatment with collagenase (1 mg/ml, Sigma-Aldrich) for 30–60 min. DmENT1, DmENT2, DmENT3, and hENT1 cRNA (18 ng) were individually injected into defolliculated Xenopus oocytes, and transport characteristics were studied by standard methods (e.g., 5, 14, 17). Oocytes were incubated at 16–18°C for 72 h in ND96 buffer, and uptake assays were performed. Assays were performed on groups of 10–12 oocytes at 25°C. Oocytes were incubated in 200 µl of NaCl transport buffer (100 mM CaCl2, 2 mM KCl, 1 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES, pH 7.5) for 15 min before the start of each experiment. The buffer was removed, and 200 µl of NaCl transport buffer containing [14C]uridine (0.1 mCi/ml; Moravek, Brea, CA) and unlabeled uridine at a concentration of 100 µM were added. Incubation periods that were used were 5, 10, 30, 60, 90, and 120 min. Uridine concentrations of 10, 50, 100, 250, 500, 1,000 µM were used to determine kinetic parameters at 30 min. For inhibition assays, unlabeled nucleosides were used at a concentration of 2 mM for a single 30-min uptake assay. For experiments involving NBTI, dilazep, and dipyridamole, oocytes were treated with an inhibitor (1 µM) for 1 h prior to each assay. Nonlinear curve fitting was performed using the Prism (v. 3.02) software (GraphPAD). Each experiment was performed at least twice using different batches of oocytes in triplicate.

Developmental profiling.
For analysis of NT expression in D. melanogaster, the Drosophila Rapid-Scan Gene Expression Panel (Origene Technologies, Rockville, MD) was used. This product contains first-strand cDNAs made from 12 Drosophila tissues and developmental stages. Primers designed for DmENT1, DmENT2, and DmENT3 RT-PCR were used to investigate developmental expression patterns. Each panel contained serially diluted rows and was performed in duplicate. At each developmental stage, expression of the mRNA was noted as positive expression, some expression occurred in serially diluted rows or in a duplicate experiment, and these stages were seen as positive NT expression.

Digoxigenin probe labeling for DmENT2 and whole mount in situ hybridization.
Digoxigenin (DIG) probe labeling and whole mount in situ hybridization were performed as outlined in the nonradioactive in situ hybridization manual using 3 µg of template DNA (Roche Molecular Biochemicals, Mannheim, Germany) with modifications. Embryos were collected on grape juice agar plates. Grape juice agar plates consisted of 350 ml distilled H20, 150 ml grape juice, 5 ml 100% (vol/vol) ethanol, and 15 g agar, which were then autoclaved, and to it 2.5 ml of glacial acetic acid was added. Embryos at the age of 12–24 h were transferred to Eppendorf tubes and were fixed. After prehybridization, embryos were hybridized with the DmENT2 DIG probe and added to 10 µg of sonicated salmon sperm DNA, which was denatured at 100°C for 3 min and cooled on ice briefly. The probe was added to the hybridization solution (0.5 µg/ml) containing the embryos and then incubated overnight at 45°C.

Washing and detection.
Hybridized embryos were washed twice for 30 min in 500 µl of hybridization solution at 45°C. They were then washed in serial dilutions of hybridization solution and PBT (phosphate-buffered saline plus Tween 20) [130 mM NaCl; 10 mM sodium phosphate, pH 7.2; 0.1% vol/vol Tween 20] (4:1, 3:2, 2:3, and 1:4) for 10 min each at room temperature. Embryos were preabsorbed with an anti-DIG antibody conjugate (1:200) for 1 h to reduce nonspecific binding. Following this incubation, the solution was diluted to a concentration of 1:2,000 and incubated for an additional hour. The embryos were washed three times for 20 min each in PBT and then three times for 5 min in staining buffer (100 mM NaCl, 50 mM MgCl2, 100 mM Tris·HCl pH 9.5). The embryos were stained with 1 ml of staining buffer containing 4.5 µl of nitro blue tetrazolium solution and 3.5 µl 5-bromo-4-chloro-3-indoyl phosphate solution. Color was allowed to develop for 45 min, and the reaction was stopped with five rapid washes in PBT. Embryos were then dehydrated in a series of dilutions of ethanol (50, 70, 95, and 100%) for 1 min each and mounted in glycerol-based mounting medium [10 mg/ml 1,4-phenylenediamine in 10x phosphate-buffered saline (stock) diluted 1/10th in glycerol].


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Drosophila and Anopheles NT genes.
The identification of putative Drosophila NTs was previously described (27). In addition to the three putative Drosophila ENTs (DmENT1, DmENT2, and DmENT3), two CNTs were found based on sequence similarity to known NTs and are designated as DmCNT1 (CG11778, gi:7303952) and DmCNT2 (CG8083, gi:7303953). For putative Anopheles NTs, similar approaches previously described (27) were performed. Three putative ENTs were found and are designated as AgENT1, AgENT2, and AgENT3 and two CNTs designated as AgCNT1 and AgCNT2. The GenBank identifiers of AgENT1, AgENT2, and AgENT3 are gi:55235449, gi:55246144, and gi:55234886, respectively. Considering that the AgENT amino acid lengths were found to be incomplete, the sequence was extended to a terminating stop codon and the 5'-prime ends were extended to a putative initiating methionine by aligning the amino acid sequences with DmENTs. The GenBank identifiers of AgCNT1 and AgCNT2 are gi:58375104 and gi:58388093, respectively.

Chromosomal location of Drosophila and Anopheles NTs.
Drosophila has four sets of chromosomes: a sex chromosome and chromosomes 2, 3, and 4. DmENT1 and DmENT2 are both located on chromosome 2L (2-21C1 and 2-26E3, respectively) and are oriented in opposite directions on the chromosome. DmENT3 is found on chromosome 3L (3-69E6). DmCNT1 and DmCNT2 are both adjacent to one another on chromosome 2R (2-45A1 and 2-45A1, respectively). Anopheles has three sets of chromosomes: a sex chromosome and chromosomes 2 and 3. AgENT1, AgENT2, and AgENT3 are found on chromosomes 3R, 2R, and 3L respectively. Both AgCNT1 and AgCNT2 are found on chromosome 2L.

Gene and protein structure of Drosophila and Anopheles NTs.
The gene structure, exon, and intron splice sites of each NT were identified using reported mRNAs or predicted open reading frames and were checked at donor acceptor consensus sites (GT/AG) (Figs. 1 and 2). Drosophila ENTs genes are simpler than human ENT genes with fewer exons and relatively small or nonexistent introns (26). DmCNT1 consists of four exons and codes for a predicted protein of 528 amino acids, while DmCNT2 has eight exons and encodes a predicted protein of 603 amino acids. Like Drosophila ENTs and CNTs, the Anopheles NT gene structure is less complex than the hNT gene family. The AgENT1 gene structure contains only three exons, while AgENT2 and AgENT3 contain six exons, with amino acids lengths of 476, 473, and 683, respectively. These genes generally have small introns with the exception of a large intron in AgENT2 and AgENT3 that is not found in the DmENT family. AgCNT1 has two exons encoding for a protein of 397 amino acids, although the predicted intron does not conform to the general splice donor-acceptor site rule, and further analysis is required for the actual gene structure. AgCNT2 has four exons encoding for a 521-amino acid protein when extended to a terminating stop codon.


Figure 1
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  		Fig. 1. Gene organization of Anopheles gambiae (Ag) equilibrative nucleoside transporters (ENTs). Exon and intron sizes were obtained from GenBank and Ensembl, and splice donor/acceptance sites were confirmed using consensus sequences (GT/AG) from reported mRNAs or predicted open reading frames. The predicted gene structure shown includes a putative 5'-initiating methionine and 3'-terminating codon, respectively. aa, Amino acid.

 

Figure 2
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  		Fig. 2. Gene organization of Drosophila and Anopheles concentrative nucleoside transporters (CNT). Exon and intron sizes were obtained from GenBank and Ensembl, and splice donor/acceptance sites were confirmed using consensus sequences (GT/AG) from reported mRNAs or predicted open reading frames. Drosophila melanogaster (Dm) CNT terminal ends include putative untranslated regions. Anopheles CNT gene structure and 5'- and 3'-ends are undefined due to conceptual translation of the genes.

 
Sequence similarities and phylogeny between NTs.
Amino acid sequences of Drosophila and Anopheles predicted NT proteins were aligned to determine amino acid similarities and identities (Figs. 3 and 4). Percent identities of predicted and known NT proteins from Drosophila, Anopheles, and humans were examined to investigate sequence similarity and help elucidate evolutionary histories of NT protein families. The predicted DmENTs and AgENTs have similar, relatively low levels of familial identity (14–22% within DmENTs, 13–24% within AgENTs) compared with hENTs (21–47%). However, both Dipteran ENT families show comparable levels of similarity to hENTs (DmENTs to hENTs, 15–29%; AgENTs to hENTs; 14–31%), while DmENTs and AgENTs were generally more similar to each other (13–56% identity). From our analysis, AgENTs were identified as orthologs to DmENTs based on percent identity and named accordingly (i.e., AgENT1 is most similar to DmENT1, etc.). In the CNT family, the predicted DmCNT1 and DmCNT2 are more alike (38% identity) compared with the AgCNTs (27% identity), which contrasts with the hCNTs (40–62% identity). Again, the Dipteran families were relatively more similar to each other (DmCNTs vs. AgCNTs 16–48%) than to hCNTs (DmCNTs vs. hCNTs 24–33%; AgCNTs vs. hCNTs 19–32%). Phylogenetic analysis of each putative NT helped to name putative orthologs (Fig. 5), and it appears that hENT4 should be considered the ortholog of Dm/AgENT3, while hENT3 is derived from a similar lineage to Dm/AgENT2. In contrast, the CNT family shows a distinct split into the hCNT family and Dipteran family members, implying a different evolutionary history for CNTs compared with ENTs. Interestingly, AgCNT1 appears to be evolutionarily distinct from all other CNT members analyzed and may have diverged earlier in the history of CNT development.


Figure 3
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  		Fig. 3. Multiple sequence alignment of ENTs from Drosophila and Anopheles. Sequence alignments were performed for ENTs using ClustalW. Black boxes denote aa identity, and grey boxes signify similarity. Putative transmembrane domains (TMDs) are shown by black bars and are numbered.

 

Figure 4
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  		Fig. 4. Multiple sequence alignment of CNT aa’s from Drosophila and Anopheles. Sequence alignments were performed for CNTs using ClustalW. Black boxes denote aa identity, and grey boxes signify similarity. Putative TMDs are shown by black bars labeled with each CNT. AgCNT1 = A1, AgCNT2 = A2, DmCNT1 = D1, DmCNT2 = D2. Aa sequence between the 2 arrows denotes sequence used for phylogenetic analysis.

 

Figure 5
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  		Fig. 5. Phylogenetic relationship between Drosophila, Anopheles, and human nucleoside transporters (NTs). The protein sequences were aligned using ClustalW and subject to phylogenetic analysis with 500 bootstrap replications using MEGA 3.1. The numbers at each node represent the percentage of times that node was found in the bootstrap analysis.

 
Predicted transmembrane domains.
Since the percent identities observed were often low between ENT families in different taxa, predicted transmembrane domains (TMDs) of Drosophila, Anopheles, and humans were compared using TMHMM (data not shown). Remarkably, similar hydropathy profiles have been previously observed for DmENTs and hENTs (27), and we observed similar results for the AgENTs. AgENT1 and AgENT2 are predicted to have a large extracellular loop between TMD 1 and 2, similar to the hENT (1–3) and DmENT family. They also share a putative prototypic ENT feature of a large intracellular loop between TMD 6 and 7 and an internal NH2-terminal tail and a short COOH-terminal tail, with the exception of AgENT3’s long COOH-terminal tail. Notably, hENT4, DmENT3, and AgENT3s have similar topologies to each other than to the other ENTs. Most Dipteran ENTs had between 10 and 11 TMDs. AgENT1 was found to be missing TMD 8 and AgENT3 missing TMD 5 using TMHMM (Fig. 3). Furthermore, DmENT3 was only found to have eight putative TMDs, although further analysis would be required considering it is similar to AgENT3, which has 10 putative TMDs. TMHMM analysis for the CNTs was less clear with variations between 7 and 12 TMDs in DmCNTs and AgCNTs compared with the predicted 13 in the hCNT family, and thus further analysis of CNT family topology would be required. Putative TMDs have been denoted in Figs. 3 and 4.

Functional expression of DmENT1, DmENT2, and DmENT3.
To functionally characterize DmENT1, DmENT2, and DmENT3, their proteins were expressed in Xenopus oocytes. Time course analysis of uridine transport with each putative ENT was compared against hENT1 and water-injected oocytes (Fig. 6A). As expected, hENT1-injected oocytes exhibited higher transport kinetics than water-injected control oocytes. The Km of uridine uptake in hENT1-injected oocytes was 0.5 ± 0.2 mM, with a Vmax estimate of 24 ± 4 pmol/oocyte/30 min (mean ± SE, n = 3). These data are comparable to previously published values (14); therefore, we continued with analysis of the DmENTs. DmENT1- and DmENT3-injected oocytes showed no significant uptake compared with controls up to 60 min (data not shown). We confirmed that the cRNA of both DmENT1 and DmENT3 was not degraded by formaldehyde agarose gel analysis; therefore, the reasons for lack of uptake by these proteins are not clear and will be further discussed. In contrast DmENT2-injected oocytes (Fig. 6A) had significantly greater uptake of [14C]uridine (9.4 ± 0.7 pmol/30 min/oocyte) compared with the water-injected control (3.8 ± 0.9 pmol/30 min/oocyte). The Km of DmENT2-dependent uridine uptake was 0.6 ± 0.1 mM with a Vmax estimate of 62.8 ± 6.2 pmol/oocyte/30 min (mean ± SE, n = 3). We therefore determined that DmENT2 was an NT and continued analysis of its transport characteristics.


Figure 6
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  		Fig. 6. Transport of nucleosides using Xenopus oocytes. A: oocytes were injected with in vitro transcribed RNA from each respective NT, and a transport assay using 100 µM radiolabeled uridine was performed. Transport by each transporter was compared with water-injected oocytes (negative control). B: uridine (100 µM) uptake was measured in the presence of nonradioactive nucleosides or nucleobases (2 mM). C: uridine (100 µM) uptake was measured in the presence of transport inhibitors (1 µM). Each inhibitor was added to the oocytes 1 h prior to each transport assay. Each value for each experiment is the mean ± SE or SD of at least 2 experiments, each using triplicate batches of 10–12 oocytes.

 
To gain insight in the substrate specificity of DmENT2, a broad range of purine and pyrimidine nucleosides and nucleobases were tested for their ability to inhibit uridine uptake in DmENT2-injected oocytes. Data show that uridine uptake was differentially inhibited by nucleosides and nucleobases, 30–80% by other nucleosides and ~50% by the nucleobases (Fig. 6B). Nucleosides lacking a hydroxyl group at the 2'-position almost completely abolished uridine transport. In addition, NBTI, dipyridamole, and dilazep, which are inhibitors of mammalian ENT1 and ENT2, were used to determine if uridine transport via DmENT2 was sensitive to any of these compounds. Our data show that DmENT2 was resistant to inhibition by all three compounds (Fig. 6C), while transport via hENT1 was almost completely abolished. Residual apparent uptake was not above the signal obtained in water-injected oocytes. These results suggest that DmENT2 is a broad-substrate, inhibitor-insensitive (ei) ENT (4).

Developmental profiles of DmENTs.
Data from PCR analysis of the Drosophila Rapid-Scan Gene Expression Panel suggested DmENT1 and DmENT2 are expressed at every stage of development except during early embryogenesis (Fig. 7). DmENT1 was expressed in 2nd instar larvae in a serially diluted row (data not shown). Strongest signals for DmENT1 were observed in both male and female heads, and there was a lack of expression in the female body. DmENT2 developmental expression occurs during most stages although apparently at differing levels. Analysis of the developmental profile of DmENT3 expression by this approach was inconclusive. The primers for DmENT3 successfully produced a PCR product from the DmENT3 cDNA clone, suggesting they can successfully amplify DmENT3 if present; therefore, it is possible that DmENT3 is expressed at very low levels and is difficult to detect using this technique.


Figure 7
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  		Fig. 7. Developmental regulation of DmENT expression. Primers for DmENTs were used to amplify PCR products at various developmental stages in the D. melanogaster life cycle. Developmental stages are denoted below panel; + signifies expression, and (+) denotes expression in a serially diluted or repeated expression panel. Representative experiment shown.

 
In situ hybridization of DmENT2 in whole mount embryos.
To observe if we could correlate the expression pattern of DmENT2 with physiology, we looked at spatial expression of DmENT2 in late-stage embryos. Comparing embryos that had been treated with no probe (Fig. 8A) with those that had been treated with labeled DmENT2 probe (Fig. 8B) showed a clear difference in expression. In the control embryos no patterning or labeling was present, but in embryos treated with a DmENT2-labeled probed, localization of the DmENT2 protein in a late stage of the embryo was found to be in the digestive tract (purple color). For comparison, embryonic expression of the gene CG31259 (stages 13–16) from the Berkeley Drosophila Genome Project (BDGP) (http://www.fruitfly.org/cgi-bin/ex/basic.pl) was localized to the midgut and is comparable to DmENT2 expression (Fig. 8C).


Figure 8
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  		Fig. 8. In situ hybridization of DmENT2 using whole mount Drosophila embryos. Embryos were collected on grape juice agar plates and were incubated (25°C) for 12–24 h. A: control embryo (x10 magnification) showing no hybridization signal. B: digoxigenin (DIG)-labeled DmENT2 hybridized embryo showing signal localized to internal structures (x40 magnification). Representative experiment shown. Assay was performed twice with similar results. C: expression of CG31259 in the embryonic midgut is shown for comparison (Berkeley Drosophila Genome Project).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Complete genome sequencing of both D. melanogaster and A. gambiae allowed the identification and analysis of NTs in two different species of Diptera, which are thought to have diverged ~250 million yr ago (9). About half of the genes in these arthropods are thought to be orthologs with an average of ~56% sequence identity (32). DmENT1, DmENT2, and DmENT3 were found in our analysis to share an average of 50% amino acid sequence identity with their respective orthologs AgENT1, AgENT2, and AgENT3. DmCNT1 and AgCNT1 were found to have a significantly lower average amino acid sequence identity (16%) compared with DmCNT2 and AgCNT2, which share a higher similarity (48%). Interestingly, AgCNT2 shares a higher homology than AgCNT1 to both putative DmCNTs, implying that AgCNT1 diverged earlier in CNT evolution.

The generation of gene families encoding similar proteins may be the result of duplication of the entire genome (polyploidization) and/or small regional duplications (27). Interestingly, duplication of genes allows the preservation of an ancestral function while producing related genes with similar functional characteristics or substrate preferences. Most of these paralogous gene duplications occur on the same chromosome and this appears to be true for DmENT1 and DmENT2, DmCNT1 and DmCNT2, and AgCNT1 and AgCNT2. Comparison of the general genomic architecture of Drosophila and Anopheles showed that significant homologies are found between chromosomal arms in these two species. The homologies for each chromosomal arm previously described (9) are as follows: XAg/XDm, 2RAg/3RDm, 2LAg/3LDm, 3RAg/2LDm, and 3LAg/2RDm, suggesting large chromosomal rearrangements have occurred since their divergence. In our analysis this chromosomal arm homology does not hold true for the NTs analyzed (ENTs and CNTs), since each respective transporter is maintained on the same chromosome number with the exception of DmENT1 and AgENT1.

Orthologs in Anopheles and Drosophila have been found to have an equivalent number of introns, equivalent exon length, and shorter intron lengths in Drosophila (on average half the length). This intron gain or loss equates to a rate of approximately one per gene per 125 million yr (32). Comparison of the AgENT and DmENT gene structure showed similar exon lengths, a slight increase in intron number for AgENTs, and similar intron lengths excluding AgENT2 and AgENT3’s relatively large intron. For the CNT family, the DmCNTs have about twice the number of introns, similar exon lengths, and similar intron lengths with the AgCNT family with the exception of a relatively larger intron for DmCNT2. Comparison of the NTs of these two species supports the existence of orthologs containing similar exon lengths but variable numbers of introns and similar intron lengths.

There is relatively low amino acid sequence identity between AgNTs, DmNTs, and hNTs, although hydropathic analysis suggests conservation of topology among ENTs (27), implying similar function. Notably, comparison of the AgENTs with the putative ENTs identified in the malarial genome (22) suggests very low sequence similarity (data not shown). Comparison of the one functionally characterized Plasmodium NT, PfENT1/PfNT1 (24), with each AgENT shows only 12–14% identity (and ~20% similarity). This is not substantially different from the levels of similarity of the hENTs to PfENT1 (13–15% identity, 19–22% similar). Whether the similarities and differences in these proteins can be exploited for improved drug treatments for malaria remains to be determined, but the identification of putative NTs in both Anopheles and Plasmodium is an important first step.

AgCNT2 was found to be more similar than AgCNT1 compared with the hCNT family. Whether they share the same substrate translocational domain still needs to be examined, since conservation of the number of TMDs suggests that transportation of substrates and/or interaction with the cell membrane may be essential (21). DmCNT2 shares a close similarity with the hCNT family with the exception of a TMD between domains 8 and 9. AgCNT1 is significantly different in its topology compared with the hCNT and DmCNT family, although AgCNT1 appears to contain the core region of an NT, and further analysis would be required to fully annotate its full sequence.

The functional properties of DmENT1, DmENT2, and DmENT3 were examined in a heterologous expression assay. DmENT1 and DmENT3 produced similar nucleoside uptake compared with water-injected controls, although DmENT2 was found to have significantly greater uridine influx than the water-injected control during the uptake assays, and saturation was reached upon 1,000 µM of uridine. Therefore, DmENT2 was further characterized. Possible reasons for the inability of DmENT1- and DmENT3-injected oocytes to transport uridine using this assay may be due to their inability to transport uridine as efficiently as other nucleosides, although uridine is often considered to be a universal permeant transported by all mammalian NTs and is therefore typically used as a standard in these types of assays (11). Alternatively, or in addition, DmENT1 and DmENT3 may not be plasma membrane proteins but rather may be proteins found in membranes of intracellular compartments as has been observed in human fibroblast lysosomes (25) and mitochondrial membranes (15). Similarly, when hENT3 and mENT3 transcripts were injected into Xenopus oocytes, nucleoside uptake was not observed, indicating that they may function as organellar transporters (10). It may also be possible that DmENT1 and DmENT3 are proton dependent as in the Arabidopsis thaliana NT, AtENT1 (23) and the Leishmania donovani NT, LdNT1 (30). However, we note that, based on these studies and our previous work, DmENT3 is a member of the ENT4 lineage (1), (not an ortholog of hENT3). DmENT1 is a member of the ENT1/2/3 lineage but not a direct ortholog of any specific mammalian isoform.

Inhibitory studies using high concentrations of nucleosides showed that DmENT2 exhibits a broad specificity for nucleosides, a common characteristic of transport by ENT family members (17). While we cannot confirm that inhibition is competitive, this approach has been routinely used to provide insight into potential competitive inhibitors. It is interesting that DmENT2 appears to transport nucleobases, suggesting that DmENT2 may be derived from an ancestral NT that was able to transport many substrates, and additional transporters (e.g., DmENT1 and DmENT3) may possibly specialize in specific nucleoside and/or nucleobase uptake. This is in contrast to humans where hENT1 transports only nucleosides while hENT2 is able to transport both nucleosides and nucleobases (31).

From the developmental profile of Drosophila NTs analyzed by the Drosophila Rapid-Scan Gene Expression Panel, DmENT1 and DmENT2 were found to be expressed at all stages except during early embryological stages, but generally embryos that are 0–4 h of age have an underrepresented transporter expression relative to the genome. The fact that early Drosophila blastoderm is a syncitium may result in the underrepresentation of transporter expression. The delay in transcription until cellularization reflects an efficient utilization of limited metabolic resources (28). Given that DmENT2 appears to have broad substrate specificity, it would make sense that it would be expressed at the majority of stages to handle the uptake and release of the broad spectrum of nucleosides for growth and physiological processes. DmENT3 expression was not detected using this technique and may be the result of a low level of expression. These data suggest that NTs are important during the life cycle of an organism, considering nucleosides are important in many cell types, and they require NTs to perform many physiological processes (6).

To study gene expression patterns during Drosophila embryogenesis, DmENT2 was examined by in situ hybridization in whole mount Drosophila embryos. Many genes are often expressed in dynamic patterns throughout early development and several staining patterns are often observed for individual genes (28). DmENT2 was observed to be expressed primarily in the digestive tract of D. melanogaster. This is similar to the tissue distribution expression of DmCNT2, which has been localized to the amnioserosa, embryonic midgut, and embryonic hindgut during stages 13–16 as determined by the BDGP. Similarly, members of the CNT family in other species can be found in gastrointestinal structures and in specialized cell types and are found to coexist with ENTs (12).

Upon analyzing genes used for in situ hybridization of whole mount embryos in the BDGP, we found a putative Drosophila nucleobase transporter, CG14767. This is the first published report to our knowledge of a putative nucleobase transporter in Drosophila. When the predicted amino acid sequence was analyzed by BLASTP in NCBI, a high amino acid sequence identity (43%) with a lysosomal-associated transmembrane protein from the silkworm Bombyx mori (gi:14582691) was found. This protein was localized by BDGP to embryonic/larval garland cells. Perhaps the gene may be a Drosophila lysosomal nucleobase transporter, although further studies would be required.

The analysis of putative Drosophila and Anopheles NTs by in silico analysis in our report showed that during evolutionary history, many characteristics of putative NTs have been conserved. This research allowed us to compare two Dipteran species that have diverged millions of years ago but maintained several genomic and putative topological architecture. Furthermore, the identification and comparison of similar characteristics between putative NTs and known NTs were used for functional characterization of the first Dipteran NT, DmENT2. Also, the NT expression examined will be important in determining its physiological relevance in developmental and spatial regulation. The characterization of Drosophila and Anopheles NTs using comparative analysis of Dipteran NTs will allow for future characterization of other Dipteran NTs.


   GRANTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
Funding for these studies was provided by the Natural Sciences and Engineering Research Council of Canada (I. R. Coe) and the Premier’s Research Excellence Awards (I. R. Coe).


   ACKNOWLEDGMENTS
 
We thank John Burger (Department of Biomedical Sciences, University of Guelph) for expertise and isolation of X. laevis oocytes. We also thank Dave Turner (University of Michigan) for the gift of the pCS2+ vector.


   FOOTNOTES
 
Address for reprint requests and other correspondence: I. Coe, Dept. of Biology, York Univ., 4700 Keele St., Toronto M3J 1P3, Canada (e-mail: coe{at}yorku.ca).

Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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