From the Departments of Obstetrics and Gynecology and
§ Biochemistry and Molecular Biology, Medical College of
Georgia, Augusta, Georgia 30912
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ABSTRACT |
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Previous studies have shown that a Na+-dependent transport system is responsible for the transplacental transfer of the vitamins pantothenate and biotin and the essential metabolite lipoate. We now report the isolation of a rat placental cDNA encoding a transport protein responsible for this function. The cloned cDNA, when expressed in HeLa cells, induces Na+-dependent pantothenate and biotin transport activities. The transporter is specific for pantothenate, biotin, and lipoate. The Michaelis-Menten constant (Kt) for the transport of pantothenate and biotin in cDNA-transfected cells is 4.9 ± 1.1 and 15.1 ± 1.2 µM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 µM. The nucleotide sequence of the cDNA (sodium-dependent multivitamin transporter (SMVT)) predicts a protein of 68.6 kDa with 634 amino acids and 12 potential transmembrane domains. Protein data base search indicates significant sequence similarity between SMVT and known members of the Na+-dependent glucose transporter family. Northern blot analysis shows that SMVT transcripts are present in all of the tissues that were tested. The size of the principal transcript is 3.2 kilobases. SMVT represents the first Na+-dependent vitamin transporter to be cloned from a mammalian tissue.
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INTRODUCTION |
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Vitamins are required for essential metabolic processes in all mammalian cells. Such cells have developed intrinsic mechanisms for active accumulation of essential vitamins. The small intestine and the kidney, tissues that are important for the absorption of vitamins, possess active transport mechanisms that mediate the transcellular transfer of these essential nutrients. The placenta also functions in the transcellular transfer of nutrients from mother to developing fetus. The syncytiotrophoblast, the absorptive epithelium of the placenta, possesses specific transport systems for several nutrients (1). Recent studies (2-4) have demonstrated the presence of a Na+-dependent vitamin transport system in the placenta that accepts as substrates the water-soluble vitamins pantothenate and biotin and the essential metabolite lipoate. The active transport system responsible for the uptake of these compounds is driven by a transmembrane Na+ gradient. The uptake of [14C]pantothenate into human placental brush border membrane vesicles (4) and JAR human placental choriocarcinoma cells (2) is mediated by a single saturable high affinity transport system with a Michaelis-Menten constant of 2-8 µM. The uptake of the radiolabeled pantothenate is inhibited by biotin and lipoate. The uptake of [3H]biotin into human placental brush border membrane vesicles is Na+-dependent and is inhibited by pantothenate and lipoate (3). Thus, a single transport system is responsible for the placental uptake of pantothenate, biotin, and lipoate. There is also evidence that a similar transport process for these vitamins operates in the small intestine and kidney in the absorption of these three essential vitamins (5, 6).
In this paper, we report on the cloning and functional expression of a rat placental cDNA that encodes a Na+-dependent vitamin transporter responsible for the placental uptake of pantothenate, biotin, and lipoate. This represents the first successful cloning of a Na+-dependent vitamin transporter from a mammalian tissue.
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EXPERIMENTAL PROCEDURES |
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Materials--
SuperScript Plasmid System for cDNA cloning
and Lipofectin were purchased from Life Technologies, Inc. Restriction
enzymes were obtained from Promega. Magna nylon transfer membranes were purchased from Micron Separations, Inc.
D-[14C]Pantothenate (51.5 mCi/mmol) and
[3H]biotin (58.2 Ci/mmol) were procured from NEN Life
Science Products. The rat chromaffin granule amine transporter (rCGAT)
(rVMAT II) clone used in the screening was kindly provided by Dr.
B. J. Hoffman (NIMH, Bethesda, MD). D-Pantothenate was
purchased from ICN Biochemicals, Inc., and D-biotin,
DL--lipoic acid, and DL-myo-inositol were bought from Sigma. HeLa cell line was obtained from ATCC (Rockville, MD) and routinely cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 µg/ml of penicillin, and 100 units/ml of streptomycin.
Construction of cDNA Library from Rat Placental mRNA-- Rat placental mRNA was reverse transcribed using the SuperScript Plasmid System with a modified oligo(dT) primer adapter containing a NotI site. Following second strand synthesis and addition of SalI adapters, the cDNA was digested with NotI to generate cDNA with NotI and SalI "sticky" ends for unidirectional cloning. The cDNA was then size-fractionated by gel filtration in a Sephacryl column. cDNA larger than 1 kb1 was ligated to NotI-SalI-digested pSPORT vector and transformed into the competent DH10B strain of Escherichia coli by electroporation.
Screening of the cDNA Library--
This was done by colony
screening of the plasmid cDNA library grown on Magna nylon transfer
membranes as described by Vogeli and Kaytes (7). The cDNA probe
used for screening was a 1.45-kbp-long PstI fragment of
rCGAT cDNA (8) that consisted of 14 bases 5' to the translation
start site and bases coding for the entire protein except the last 45 amino acids. The probe was labeled with [-32P]dCTP
using the Ready-to-go oligolabeling kit from Pharmacia Biotech Inc.
Hybridization was carried out for 20 h at 60 °C in a solution
containing 5× SSPE (1× SSPE = 0.15 M NaCl, 10 mM NaH2PO4, and 1 mM
EDTA), 5× Denhardt's solution, 0.5% SDS, and 100 µg/ml of
denatured salmon sperm DNA. Post-hybridization washing was done at very
low stringency conditions, which involved extensive washes with 3×
SSPE/0.5% SDS at room temperature. Positive clones were identified,
and the colonies were purified by secondary screening.
DNA Sequencing-- Both sense and antisense strands of the cDNA were sequenced by primer walking. Sequencing by the dideoxynucleotide chain termination method was performed by Taq DyeDeoxy terminator cycle sequencing with an automated Perkin-Elmer Applied Biosystems 377 Prism DNA Sequencer. The sequence was analyzed using the GCG sequence analysis software package GCG version 7.B (Genetics Computer Group, Inc., Madison, WI). Data base searches were done using the GenBankTM Program BLAST (9).
Functional Expression of the cDNA-- The cDNA was functionally expressed in HeLa cells by vaccinia virus expression system (10) as described previously (11, 12). The cDNA was cloned in the plasmid pSPORT in such an orientation that the sense transcription of the cDNA was under the control of T7 promoter in the plasmid. Subconfluent HeLa cells grown in 24-well cell culture plates were first infected with a recombinant vaccinia virus (VTF7-3) encoding T7 RNA polymerase. This was followed by Lipofectin-mediated transfection of the plasmid cDNA into the cells. After 12 h post-transfection, transport measurements were made at room temperature using [14C]pantothenate and [3H]biotin. The transport buffer was composed of 25 mM Hepes/Tris (pH 7.5), supplemented with 140 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl2, 0.8 mM MgSO4, and 5 mM glucose. The incubation time for transport measurements was 30 min. Transport was terminated by aspiration of the uptake buffer followed by two washes with 2 ml of ice-cold transport buffer. Following this, the cells were solubilized with 0.5 ml of 1% SDS in 0.2 N NaOH and transferred to vials for quantitation of the radioactivity associated with the cells. The Na+ dependence of the transport process was examined by isoosmotically substituting NaCl in the transport buffer with choline chloride. HeLa cells transfected with empty vector under similar conditions served as control. In experiments dealing with saturation kinetics, data were analyzed by nonlinear regression and confirmed by linear regression.
Northern Blot Analysis--
Total RNA was isolated from various
rat tissues using TRIzol reagent. Poly(A)+ RNA was isolated
from these total RNAs by oligo(dT)-cellulose affinity chromatography. 6 µg of poly(A)+ RNA isolated from different tissues was
separated on a 1% formaldehyde-agarose gel and blotted onto a Hybond-N
transfer membrane by capillary blotting. The
SalI-XbaI fragment consisting of the complete
cDNA was labeled with [-32P]dCTP using a
Ready-to-go oligolabeling kit and used as the probe. Hybridizations
were performed at 42 °C in 6× SSPE, 50% formamide, 10×
Denhardt's, 2% SDS, and 100 µg/ml salmon sperm DNA. The
post-hybridization wash was done at high stringency conditions that
involved a final wash with 0.5× SSPE, 0.5% SDS at 60 °C for 30 min.
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RESULTS AND DISCUSSION |
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Screening of the Placental cDNA Library-- Previous studies from our laboratory have provided evidence for the expression of an organic cation/H+ antiporter in the placenta (13, 14). With an intent to clone this transporter from the rat placenta, we screened a rat placental cDNA library with rCGAT as the probe. rCGAT transports monoamines, which are cationic at physiological pH, in exchange for protons. Thus there is a functional similarity between the organic cation/H+ antiporter and rCGAT. We therefore thought that the two proteins may have significant sequence homology and that the rCGAT cDNA probe may lead to the identification of the organic cation/H+ antiporter cDNA clone by cross-hybridization. When the rat placental cDNA library was screened under low stringency conditions with the rCGAT cDNA probe, several positive clones were identified. Plasmid DNA was isolated from these clones, and the cloned inserts were released by restriction digestion with XbaI-SalI enzymes. The sizes of the inserts were analyzed on agarose gels, and clones larger than 2.0 kbp in size were sequenced at the 5' end. Based on the nucleotide sequence so obtained, one clone was identical to the cloned rCGAT, another turned out to be rVMAT I (rSVAT) (8, 15), and several other clones did not have any homology to known cloned transporters. One of these clones, approximately 2.0 kbp in size, showed significant homology with known members of Na+-dependent glucose transporter family (Na+-dependent glucose transporters, Na+-dependent iodide transporter, Na+-dependent nucleoside transporters, and Na+-dependent myo-inositol transporter). This clone was partial and lacked the 5' end of the cDNA including the translation start site. Therefore, a second screening of the same placental cDNA library was done using this partial cDNA as the probe. Three clones larger than 3.0 kbp were obtained, two of which turned out to be identical to the partial clone used as the probe except that they contained a ~1.0-kbp-long 5' extension. One of these was arbitrarily chosen for further characterization by complete sequence analysis and functional expression.
Structural Features of the cDNA-- The cloned cDNA is 3091 bp long and has an open reading frame of 1905 bp including the termination codon. The open reading frame is flanked by a 412-bp-long 5' noncoding sequence and 774-bp-long 3' noncoding sequence. The putative initiation codon is preceded by a Kozak consensus sequence (GTG AGG) (16). At the 3' end, the cDNA has a polyadenylation signal (AATAAA) followed by a poly(A)+ tail. The open reading frame encodes a protein of 634 amino acids. The primary structure of this protein is shown in Fig. 1. The protein has an estimated core molecular mass of 68.6 kDa and a pI of 7.86. Hydropathy analysis of the primary amino acid sequence using the Kyte-Doolittle method (17) with a window size of 20 amino acids shows that the protein is highly hydrophobic with 12 putative transmembrane domains (Fig. 1). When modeled as most other cloned transporters, both the N-terminal and C-terminal ends of the protein face the cytoplasm. Three putative N-glycosylation sites are present in the extracellular loop between transmembrane domains 11 and 12. The C-terminal end of the protein following the last transmembrane domain is about 84 amino acids long and is highly hydrophilic.
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Functional Characterization-- The functional expression of the clone was done in HeLa cells by transient transfection followed by vaccinia virus-induced expression of the cDNA. The function was monitored by the transport of radioactive substrates. Cells transfected with the empty vector were used to measure the endogenous transport activity. The ability of the expressed transporter to transport several substrates known to have sodium-dependent uptake mechanisms like ascorbate, carnitine, myoinositol, pantothenate, and biotin was investigated. Of these substrates tested, the uptake of only pantothenate and biotin was significantly increased in the cells transfected with the cDNA in comparison with the vector-transfected cells. The uptake of [14C]pantothenate was typically 4-fold higher in the cDNA-transfected cells compared with the endogenous transport measured in the vector-transfected cells (Fig. 2A) but varied between 3- and 8-fold between experiments. Replacement of Na+ with choline in the transport buffer resulted in the complete loss of the uptake activity, demonstrating the Na+ dependence of the transport process. Significant uptake of Na+-dependent pantothenate is also seen in HeLa cells transfected with empty vector, indicating that there is endogenous expression of this transporter in HeLa cells. The substrate specificity of the cloned transporter was analyzed by competition studies. Unlabeled pantothenate, biotin, and lipoate significantly inhibited the uptake of [14C]pantothenate into the cDNA-transfected cells, indicating that all three essential nutrients are substrates for the carrier. Myo-inositol, which is not a substrate of the carrier, failed to inhibit the uptake of the radiolabeled pantothenate.
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Sequence Homology of SMVT with Other Transporters-- Sequence comparison with the sequences in the SwissProt data base shows that SMVT exhibits significant homology with some of the other known sodium-dependent nutrient transporters. Of these, the pantothenate permease from E. coli (20) and Hemophilus influenzae (21) and the mammalian iodide transporter, glucose transporters, and myo-inositol transporter show maximal homology with SMVT. The percentage of similarity and identity values obtained from such a comparison are presented in Table I. Interestingly, the pantothenate permeases from E. coli and H. influenzae are functionally similar to SMVT. The pantothenate permease (panF) gene of E. coli has been functionally expressed following cloning (20). The gene codes for a protein that catalyzes the sodium-dependent uptake of pantothenate into the bacterium. The permease gene of H. influenza was identified by homology search. Similar to the mammalian SMVT, the bacterial and viral pantothenate permeases are integral membrane proteins with 12 putative transmembrane domains. The bacterial protein is specific to pantothenate with a Kt of 0.4 µM (22), which is lower in comparison with the mammalian counterpart, which has a Kt of 2-8 µM. However, it is not known whether biotin and lipoate are also transported by the bacterial and viral pantothenate permeases. It is interesting to note that rCGAT, which does not belong to the Na+-dependent glucose transporter family, has 46% similarity and 20% identity with rSMVT at the protein level. This probably explains why the rCGAT cDNA probe hybridized to the rSMVT cDNA during the library screening.
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Tissue Distribution of SMVT Transcripts-- Poly(A)+ RNA isolated from several tissues of rat was analyzed by Northern blot hybridization for the presence of mRNA transcripts of SMVT (Fig. 4). Transcripts were detected in all of the tissues analyzed. The size of the primary transcript was 3.2 kb. An additional minor hybridizing transcript of 6.5 kb was also seen in all tissues. Quantitatively, the absorptive tissues like the intestinal mucosa, kidney, and placenta have very high amounts of the SMVT-specific mRNA. Significant amounts of SMVT transcripts are seen in other tissues such as the liver, brain, lung, heart, and skeletal muscle. This is not surprising because SMVT transports three different vitamins that are obligatory for the proper metabolic functioning of every cell.
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ACKNOWLEDGEMENTS |
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We thank Dr. Beth J. Hoffman (NIMH, Bethesda, MD) for providing the rat CGAT clone. We also thank Dr. Matthias Brandsch and Dr. Reinhard Paschke (Biocentre, Martin-Luther-Universität, Halle-Wittenberg, Germany) for the analysis and comparison of the three-dimensional structures of pantothenate, biotin, and lipoate.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants HD 24451 and HD 33347.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 GenBankTM/EMBL Data Bank with accession number(s) AF026554.
¶ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Medical College of Georgia, Augusta, GA 30912-2100. Tel.: 706-721-1761; Fax: 706-721-6608; E-mail: pprasad{at}mail.mcg.edu.
1 The abbreviations used are: kb, kilobase(s); kbp, kilobase pair(s); bp, base pair(s); rCGAT, rat chromaffin granule amine transporter; SMVT, sodium-dependent multivitamin transporter; rSMVT, rat SMVT.
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REFERENCES |
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