Pantothenate Uptake Research Articles

Pantothenate Auxotrophy in a Naturally Occurring Biocontrol Yeast.

The genus Hanseniaspora is characterized by some of the smallest genomes among budding yeasts. These fungi are primarily found on plant surfaces and in fermented products and represent promising biocontrol agents against notorious fungal plant pathogens. In this work, we identify pantothenate auxotrophy of a Hanseniaspora meyeri isolate that shows strong antagonism against the plant pathogen Fusarium oxysporum. Furthermore, strong biocontrol activity in vitro required both pantothenate and biotin in the growth medium. We show that the H. meyeri isolate APC 12.1 can obtain the vitamin from plants and other fungi. The underlying reason for the auxotrophy is the lack of two key pantothenate biosynthesis genes, but six genes encoding putative pantothenate transporters are present in the genome. By constructing and using a Saccharomyces cerevisiae reporter strain, we identified one Hanseniaspora transporter that conferred pantothenate uptake activity to S. cerevisiae. Pantothenate auxotrophy is rare and has been described in only a few bacteria and in S. cerevisiae strains that were isolated from sake. Such auxotrophic strains may seem an unexpected and unlikely choice as potential biocontrol agents, but they may be particularly competitive in their ecological niche and their specific growth requirements are an inherent biocontainment strategy preventing uncontrolled growth in the environment. Auxotrophic strains, such as the H. meyeri isolate APC 12.1, may thus represent a promising strategy for developing biocontrol agents that will be easier to register than prototrophic strains, which are normally used for such applications. IMPORTANCE As a precursor of the essential coenzyme A (CoA), pantothenate is present in all organisms. Plants, bacteria, and fungi are known to synthesize this vitamin, while animals must obtain it through their diet. Pantothenate auxotrophy has not been described in naturally occurring, environmental fungi and is an unexpected property for an antagonistic yeast. Here, we report that yeasts from the genus Hanseniaspora lack key enzymes for pantothenate biosynthesis and identify a transporter responsible for the acquisition of pantothenate from the environment. Hanseniaspora isolates are strong antagonists of fungal plant pathogens. Their pantothenate auxotrophy is a natural biocontainment feature that could make such isolates interesting candidates for new biocontrol approaches and allow easier registration as plant protection agents than prototrophic strains.

Pantothenic Acid Uptake and Metabolism by Red Blood Cells of Rats

Red blood cells (RBC) of rats and humans contain pantothenic acid (PA) derivatives, generally referred to as bound PA, which were never characterized or quantitated. This study was undertaken to define those factors that determine the rates of uptake and efflux of PA, and possibly PA derivatives, in RBC. Uptake of PA by RBC was studied by incubating RBC with different concentrations of PA (0.34–34.0 µmol/L), each containing [14C]PA (0.34 µmol/L) at 37°C for 3–360 min. The effects of Na+, energy and pH on the uptake of PA in RBC were determined in experiments varying media concentrations of Na+ (0–152 mmol/L), glucose (0 and 10 mmol/L) or pH (7.2–7.6). The release of PA from RBC was studied by incubating RBC containing [14C]PA (0.10–10.4 nmol/107 RBC, final concentration) in fresh media at 37°C for 10–240 min. Uptake and efflux of PA were found to be nonsaturable; uptake was not affected by Na+, energy or pH. Quantitative and qualitative determination of PA derivatives was by enzymatic hydrolysis of lysed RBC, followed by analysis of PA by RIA and paper chromatography. The RBC were found to contain PA, 4′-phosphopantothenic acid and pantetheine. We conclude that PA diffuses passively into and out of RBC and that RBC contain only PA, 4′-phosphopantothenic acid, pantetheine and no CoA.

Coenzyme A precursors flow from mother to zygote and from microbiome to host

Coenzyme A (CoA) is essential for metabolism and protein acetylation. Current knowledge holds that each cell obtains CoA exclusively through biosynthesis via the canonical five-step pathway, starting with pantothenate uptake. However, recent studies have suggested the presence of additional CoA-generating mechanisms, indicating a more complex system for CoA homeostasis. Here, we uncovered pathways for CoA generation through inter-organismal flows of CoA precursors. Using traceable compounds and fruit flies with a genetic block in CoA biosynthesis, we demonstrate that progeny survive embryonal and early larval development by obtaining CoA precursors from maternal sources. Later in life, the microbiome can provide the essential CoA building blocks to the host, enabling continuation of normal development. A flow of stable, long-lasting CoA precursors between living organisms is revealed. This indicates the presence of complex strategies to maintain CoA homeostasis.

Metabolomics Profiling Reveals Markers for Chemosensitivity and Clinical Outcomes in Pediatric AML Patients

Acute myeloid leukemia (AML) is a clinically challenging disease with high interpatient variability in response to chemotherapy. Despite continuing advances in treatment options, current 5-year survival rates for pediatric AML are suboptimal at ~60%. The heterogeneous nature of AML contributes significantly to the variability in treatment response and survival outcomes. Several known genetic lesions and cytogenetic features contribute to disease progression. However, our understanding of how molecular mechanisms contribute to variation in treatment outcomes is still limited. Previous metabolomics studies have successfully identified significant metabolic alterations in hematological malignancies, but very few metabolomics studies have been conducted for the pediatric AML patient population. In this study, we used global and targeted metabolomics to identify differential metabolite abundance associated with chemosensitivity and treatment outcomes in pediatric AML patients.Serum metabolomics profiles were generated with serum samples obtained at diagnosis from patients treated in the multicenter AML02 study (n=94, NCT00136084). Clinical outcomes tested for association included half-maximal inhibitory concentration (IC50) of cytarabine, minimal residual disease (MRD), relapse free survival (RFS), and overall survival (OS). Global metabolomics profiling was performed using liquid chromatography/mass spectrometry (LC/MS). Targeted metabolomics profiling was generated for a select group of organic acids and acylcarnitines. The organic acid panel included eight metabolites related to the tricarboxylic acid cycle and glycolysis. The acylcarnitine panel featured 20 varieties of acylcarnitines detectable in human serum. Statistical analyses were performed using MetaboAnalyst and various R packages.A total of 3205 features were detected in the global metabolome, with 124 known metabolites and 3081 unknown features. All metabolites were used for association analysis, while annotated metabolites were used in pathway analyses. Association analysis of clinical endpoints vs. metabolome identified 10 known metabolites significantly associated with IC50 values, 17 associated with MRD, 7 associated with RFS, and 7 associated with OS (p<0.05). Targeted metabolomics generated the absolute abundance profile of 8 organic acid metabolites and 20 acylcarnitine metabolites in patient samples. Spearman correlation analysis identified five acylcarnitines significantly correlated with IC50 values. Among the significant metabolites, the most interesting is pantothenic acid, showing higher serum abundance associated with poorer IC50, MRD, and RFS outcomes. Pantothenic acid is an essential component for Coenzyme A synthesis, leading into energy production through the tricarboxylic acid cycle. A previous study has shown a reduced capacity for pantothenic acid uptake in leukemia cells resistant to daunorubicin. Our results suggest a similar relationship for pantothenic acid uptake and cytarabine resistance.Pathway enrichment analysis identified 11 metabolic pathways showing significant association with IC50 values and 12 pathways associated with MRD (FDR<0.05). Some of the most significantly associated pathways included alanine, aspartate and glutamate metabolism, arginine and proline metabolism, and pantothenic acid based CoA biosynthesis. Overall, differences in chemosensitivity and clinical outcomes appear to be most closely related to amino acid synthesis and energy production.This study identifies several metabolites and metabolic pathways significantly associated with chemosensitivity and clinical endpoints in pediatric AML patients. These results help expand on previously conducted AML pilot studies, and metabolomics studies on other cancer types, to further clarify the metabolic differences associated with interpatient variability in chemotherapy response for AML patients. Continued metabolic profiling of AML patient populations can help establish targetable pathways that can be used to improve treatment efficiency for AML. In addition, in vitro functional modeling to validate results of the metabolomics study are currently underway. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Rewired Metabolism in Drug-resistant Leukemia Cells: A METABOLIC SWITCH HALLMARKED BY REDUCED DEPENDENCE ON EXOGENOUS GLUTAMINE

Cancer cells that escape induction therapy are a major cause of relapse. Understanding metabolic alterations associated with drug resistance opens up unexplored opportunities for the development of new therapeutic strategies. Here, we applied a broad spectrum of technologies including RNA sequencing, global untargeted metabolomics, and stable isotope labeling mass spectrometry to identify metabolic changes in P-glycoprotein overexpressing T-cell acute lymphoblastic leukemia (ALL) cells, which escaped a therapeutically relevant daunorubicin treatment. We show that compared with sensitive ALL cells, resistant leukemia cells possess a fundamentally rewired central metabolism characterized by reduced dependence on glutamine despite a lack of expression of glutamate-ammonia ligase (GLUL), a higher demand for glucose and an altered rate of fatty acid β-oxidation, accompanied by a decreased pantothenic acid uptake capacity. We experimentally validate our findings by selectively targeting components of this metabolic switch, using approved drugs and starvation approaches followed by cell viability analyses in both the ALL cells and in an acute myeloid leukemia (AML) sensitive/resistant cell line pair. We demonstrate how comparative metabolomics and RNA expression profiling of drug-sensitive and -resistant cells expose targetable metabolic changes and potential resistance markers. Our results show that drug resistance is associated with significant metabolic costs in cancer cells, which could be exploited using new therapeutic strategies.

Plasmodium yoelii vitamin B5 pantothenate transporter candidate is essential for parasite transmission to the mosquito.

In nearly all non-photosynthetic cells, pantothenate (vitamin B5) transport and utilization are prerequisites for the synthesis of the universal essential cofactor Coenzyme A (CoA). Early studies showed that human malaria parasites rely on the uptake of pantothenate across the parasite plasma membrane for survival within erythrocytes. Recently, a P. falciparum candidate pantothenate transporter (PAT) was characterized by functional complementation in yeast. These studies revealed that PfPAT mediated survival of yeast cells in low pantothenate concentrations and restored sensitivity of yeast cells lacking pantothenate uptake to fenpropimorph. In addition, PfPAT was refractory to deletion in P. falciparum in vitro, but nothing is known about the in vivo functions of PAT in Plasmodium life cycle stages. Herein, we used gene-targeting techniques to delete PAT in Plasmodium yoelii. Parasites lacking PAT displayed normal asexual and sexual blood stage development compared to wild-type (WT) and WT-like p230p(-) parasites. However, progression from the ookinete to the oocyst stage and sporozoite formation were completely abolished in pat(-) parasites. These studies provide the first evidence for an essential role of a candidate pantothenate transport in malaria transmission to Anopheles mosquitoes. This will set the stage for the development of PAT inhibitors against multiple parasite life cycle stages.

Pantothenate Utilization by Plasmodium as a Target for Antimalarial Chemotherapy

In the absence of an effective vaccine against malaria suitable for widespread deployment, the control of this lethal infectious disease relies heavily on antimalarial chemotherapies. The most virulent of the parasites that cause malaria (Plasmodium falciparum) has, however, developed resistance to all antimalarial agents in clinical use, and there is a desperate need for new antimalarial agents that target previously unexploited parasite processes. P. falciparum requires an extracellular supply of pantothenate to support its proliferation during the erythrocytic stage of its development in humans. This requirement highlights the mechanisms involved in the utilization (uptake and metabolism) of pantothenate as potential targets for chemotherapeutic attack. Here we review the evidence demonstrating pantothenate to be an essential nutrient for P. falciparum and data from studies investigating whether this parasite has the capacity to utilize exogenous supplies of the cofactor (coenzyme A; CoA) for which pantothenate serves as a precursor. The results of recent studies aimed at characterising the mechanisms by which pantothenate is taken up by the P. falciparum-infected erythrocyte and intracellular parasite, and metabolised to CoA, are described. The unique properties that may be exploited to develop selective inhibitors of pantothenate utilization by P. falciparum-infected erythrocytes are highlighted. The molecular identity of P. falciparum pantothenate transporters and CoA biosynthesis enzymes remain unconfirmed. We consider the possible identities, and emphasize the importance of generating these proteins in pure, functionally active form. The tools currently available for identifying inhibitors of pantothenate utilization that may be potent antiplasmodial agents are also discussed.

Biosynthesis of Pantothenic Acid and Coenzyme A.

Pantothenate is vitamin B5 and is the key precursor for the biosynthesis of coenzyme A (CoA), a universal and essential cofactor involved in a myriad of metabolic reactions, including the synthesis of phospholipids, the synthesis and degradation of fatty acids, and the operation of the tricarboxylic acid cycle. CoA is also the only source of the phosphopantetheine prosthetic group for enzymes that shuttle intermediates between the active sites of enzymes involved in fatty acid, nonribosomal peptide, and polyketide synthesis. Pantothenate can be synthesized de novo and/or transported into the cell through a pantothenatepermease. Pantothenate uptake is essential for those organisms that lack the genes to synthesize this vitamin. The intracellular levels of CoA are controlled by the balance between synthesis and degradation. In particular, CoA is assembled in five enzymatic steps, starting from the phosphorylation of pantothenate to phosphopantothenatecatalyzed by pantothenate kinase, the product of the coaA gene. In some bacteria, the production of phosphopantothenate by pantothenate kinase is the rate limiting and most regulated step in the biosynthetic pathway. CoA synthesis additionally networks with other vitamin-associated pathways, such as thiamine and folic acid.

Feedback Inhibition of Pantothenate Kinase Regulates Pantothenol Uptake by the Malaria Parasite

To survive, the human malaria parasite Plasmodium falciparum must acquire pantothenate (vitamin B5) from the external medium. Pantothenol (provitamin B5) inhibits parasite growth by competing with pantothenate for pantothenate kinase, the first enzyme in the coenzyme A biosynthesis pathway. In this study we investigated pantothenol uptake by P. falciparum and in doing so gained insights into the regulation of the parasite's coenzyme A biosynthesis pathway. Pantothenol was shown to enter P. falciparum-infected erythrocytes via two routes, the furosemide-inhibited "new permeation pathways" induced by the parasite in the infected erythrocyte membrane (the sole access route for pantothenate) and a second, furosemide-insensitive pathway. Having entered the erythrocyte, pantothenol is taken up by the intracellular parasite via a mechanism showing functional characteristics distinct from those of the parasite's pantothenate uptake mechanism. On reaching the parasite cytosol, pantothenol is phosphorylated and thereby trapped by pantothenate kinase, shown here to be under feedback inhibition control by coenzyme A. Furosemide reduced this inherent feedback inhibition by competing with coenzyme A for binding to pantothenate kinase, thereby increasing pantothenol uptake.

Cell division defects of Schizosaccharomyces pombe liz1- mutants are caused by defects in pantothenate uptake.

The liz1+ gene of the fission yeast Schizosaccharomyces pombe was previously identified by complementation of a mutation that causes abnormal mitosis when ribonucleotide reductase is inhibited. Liz1 has similarity to transport proteins from Saccharomyces cerevisiae, but the potential substrate and its connection to the cell division cycle remain elusive. We report here that liz1+ encodes a plasma membrane-localized active transport protein for the vitamin pantothenate, the precursor of coenzyme A (CoA). Liz1 is required for pantothenate uptake at low extracellular concentrations. A lack of pantothenate uptake results in three phenotypes: (i) slow growth, (ii) delayed septation, and (iii) aberrant mitosis in the presence of hydroxyurea (HU). All three phenotypes are suppressed by high extracellular concentrations of pantothenate, where pantothenate uptake occurs by passive diffusion. liz1Delta mutants are viable because they can synthesize pantothenate from uracil as an endogenous source. The use of uracil for both pantothenate biosynthesis and deoxyribonucleotide generation provides an explanation for the aberrant mitosis in the presence of HU. HU blocks ribonucleotide reductase, and we propose that the accumulation of ribonucleotides reduces uracil biosynthesis by feedback inhibition of aspartate transcarbamoylase. Thus, the addition of HU to liz1Delta mutants results in a shortage of pantothenate. Because liz1Delta mutants show striking similarities to mutants with defects in fatty acid biosynthesis, we propose that the shortage of pantothenate compromises fatty acid synthesis, resulting in slow growth and mitotic defects.

Keratinocytes Join Forces with Immune Cells in the Prosecution of SMVT as a “False” Biotin Transporter

SMVT, a sodium-dependent multivitamin transporter, was cloned in our laboratory first from rat placenta (Prasad et al, 1998) and subsequently from human placenta (Wang et al, 1999) and rabbit intestine (Prasad et al, 1999). This transporter mediates the active transport of three different water-soluble vitamins, namely biotin, pantothenate, and lipoate and the transport process is energized by a transmembrane electrochemical Na+ gradient (Prasad and Ganapathy, 2000). The Kt (Michaelis-Menten constant) values for biotin and pantothenate for transport via human SMVT are 5 and 2M, respectively. While the Kt value for pantothenate is only about 20 times higher than the plasma levels of this vitamin (0.1 M), the Kt value for biotin is four orders of magnitude higher than the plasma levels of biotin. The concentration of biotin in human blood is in the range of 0.5–2 nM (Mock et al, 1995). This raises the question as to whether SMVT can mediate the cellular uptake of biotin from the circulation efficiently at normal physiological concentrations of biotin. It was purely coincidental that in the same year that we cloned SMVT,Zempleni and Mock (1998) reported on the characteristics of biotin uptake in peripheral blood mononuclear cells which indicated the presence of a biotin transporter different from SMVT. The transporter in immune cells is Nadependent as is SMVT, but the Kt value for biotin in the case of the new transporter is 2.5 nM. Moreover, the transporter in immune cells does not interact with pantothenate or lipoate. Thus, the newly identified transporter is specific for biotin and operates with an affinity close to the plasma levels of biotin. This transporter is induced in immune cells during mitogen-induced proliferation (Zempleni and Mock, 2000). Interestingly, SMVT is also expressed in immune cells. Mock and his coworkers have hypothesized that while SMVT is needed in these cells for the cellular uptake of pantothenate, the newly identified transporter is predominantly responsible for biotin uptake under normal conditions (Zempleni and Mock, 2000). That the biotin-specific transporter identified in immune cells is distinct from SMVT is further substantiated by a recent report (Mardach et al, 2002) in which a biotin transport defect was seen in peripheral blood mononuclear cells in a patient associated with biotin dependency. The patient had no noticeable defect in the functional activity of SMVT.

Proliferation of peripheral blood mononuclear cells causes increased expression of the sodium-dependent multivitamin transporter gene and increased uptake of pantothenic acid

Antigenic or mitogenic stimulation of peripheral blood mononuclear cells (PBMC) causes rapid cell proliferation. PBMC proliferation is associated with increased activities of pantothenic acid-dependent metabolic pathways, suggesting increased demand for pantothenic acid. We sought to determine whether PBMC respond to proliferation by increased cellular uptake of pantothenic acid and, if so, by what mechanism(s) the increased uptake is mediated. Uptake of pantothenic acid into PBMC was mediated by the sodium-dependent multivitamin transporter, SMVT, as judged by sodium dependency of uptake, substrate affinity and specificity, and RT-PCR of PBMC RNA. Proliferating PBMC accumulated two times more [3H]pantothenic acid than quiescent PBMC. Rates of [3H]pantothenic acid uptake paralleled rates of PBMC proliferation, as judged by uptake of [3H]thymidine. The increased uptake of [3H]pantothenic acid into proliferating PBMC was mediated by increased expression of SMVT (as judged by RT-PCR using total RNA from PBMC), leading to an increased number of transporters on the cell surface (as judged by maximal transport rates for pantothenic acid). We conclude that proliferating PBMC increase expression of the gene encoding SMVT to increase uptake of pantothenic acid.

H+-coupled Pantothenate Transport in the Intracellular Malaria Parasite

Pantothenate, the precursor of coenzyme A, is an essential nutrient for the intraerythrocytic stage of the malaria parasite Plasmodium falciparum. Pantothenate enters the malaria-infected erythrocyte via new permeation pathways induced by the parasite in the host cell membrane (Saliba, K. J., Horner, H. A., and Kirk, K. (1998) J. Biol. Chem. 273, 10190-10195). We show here that pantothenate is taken up by the intracellular parasite via a novel H(+)-coupled transporter, quite different from the Na(+)-coupled transporters that mediate pantothenate uptake into mammalian cells. The plasmodial H(+):pantothenate transporter has a low affinity for pantothenate (K(m) approximately 23 mm) and a stoichiometry of 1 H(+):1 pantothenate. It is inhibited by low concentrations of the bioflavonoid phloretin and the thiol-modifying agent p-chloromercuribenzene sulfonate. On entering the parasite, pantothenate is phosphorylated (and thereby trapped) by an unusually high affinity pantothenate kinase (K(m) approximately 300 nm). The combination of H(+)-coupled transporter and kinase provides the parasite with an efficient, high affinity pantothenate uptake system, which is distinct from that of the host and is therefore an attractive target for antimalarial chemotherapy.

Electrogenic Nature of Rat Sodium-Dependent Multivitamin Transport

We report on the electrogenic nature of the transport process mediated by the rat sodium-dependent multivitamin transporter. In Cos-7 cells, the relationship of Na+ concentration versus biotin and pantothenate uptake rate was sigmoidal with a Na+:substrate stoichiometry of 2:1. In Cos-7 cells expressing rat SMVT biotin transport was significantly higher when the membrane was hyperpolarized and considerably reduced when the membrane was depolarized. Similarly, biotin uptake in X. laevis oocytes expressing rat SMVT was inhibited with depolarized oocyte membrane by altering the K+ permeability across the membrane. It is concluded that the transport of biotin and pantothenate mediated by rat SMVT is electrogenic with a Na+:substrate coupling ratio of 2:1 and that the transport process is associated with the transfer of one net positive charge across the membrane per transport cycle.

Molecular and Functional Characterization of the Intestinal Na+-Dependent Multivitamin Transporter

We have cloned a Na+-dependent multivitamin transporter from rabbit intestine (riSMVT). The cDNA codes for a protein of 636 amino acids with 12 putative transmembrane domains. When expressed in mammalian cells, the cDNA induces Na+-dependent uptake of the vitamins pantothenate and biotin. Lipoate is also a substrate for the cDNA-induced uptake process. The affinity constant for the cDNA-specific transport of pantothenate and biotin is ∼2 and ∼8 μM, respectively. The Na+:vitamin stoichiometry is greater than 1, indicating that the transport process is electrogenic. The SMVT-specific transcripts of 3.2 kbp are equally distributed throughout the small intestine. We have also cloned SMVT from the human intestinal cell line Caco-2. The Caco-2 SMVT cDNA codes for a protein of 635 amino acids which is homologous to riSMVT and is identical to the SMVT expressed in the human choriocarcinoma cell line JAR. Caco-2 SMVT also catalyzes Na+-dependent uptake of pantothenate, biotin, and lipoate. In oocytes expressing Caco-2 SMVT, all three vitamins evoke inward currents, confirming the electrogenicity of the transport process.

Cloning of a cDNA encoding the human sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate

Cloning of a cDNA encoding the human sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate

Cloning and Functional Expression of a cDNA Encoding a Mammalian Sodium-dependent Vitamin Transporter Mediating the Uptake of Pantothenate, Biotin, and Lipoate

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 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. 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.

Cloning and functional expression of a cDNA encoding a mammalian sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin, and lipoate

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 microM, respectively. The transport of both vitamins in cDNA-transfected cells is inhibited by lipoate with an inhibition constant (Ki) of approximately 5 microM. 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.

Characterization of a sodium-dependent vitamin transporter mediating the uptake of pantothenate, biotin and lipoate in human placental choriocarcinoma cells

The characteristics of the uptake of the vitamin pantothenate into JAr human placental choriocarcinoma cells were investigated and these cells were found to accumulate the vitamin against a concentration gradient by a Na +-dependent process. Substitution of Na + with other monovalent cations abolished the uptake completely. The transport process showed no preference for any particular anion. Kinetic analysis indicated the presence of a single saturable transport system with a Michaelis-Menten constant of 2.1 ± 0.2 μ and a maximal velocity of 341 ± 12 pmol/mg of protein per 10 min. The dependence of the uptake rate of pantothenate on Na + concentration exhibited sigmoidal kinetics, indicating interaction of more than one Na + ion with the transporter. The Hill coefficient for this process was calculated to be 1.6. The Na +/pantothenate coupling ratio being greater than unity suggests that the transport process is electrogenic, resulting in net transfer of positive charge across the membrane. This was confirmed in plasma membrane vesicles prepared from JAr cells where the uptake of pantothenate was found to be significantly stimulated by valinomycin-induced inside-negative K +-diffusion potential. Substrate specificity studies showed that, in addition to pantothenate, the transporter interacts with two other vitamins, namely biotin and lipoate. The characteristics of pantothenate uptake in the placental cell line BeWo was also investigated. These cells were also found to express a pantothenate transport system similar to that expressed in the JAr cells.

Human placental brush-border membrane Na(+)-pantothenate cotransport

Membrane transport pathways for transplacental transfer of the water-soluble vitamin pantothenate were investigated by assessing the possible presence of a Na(+)-pantothenate cotransport mechanism in the maternal facing membrane of human placental epithelial cells. The presence of Na(+)-pantothenate cotransport was determined from radiolabeled tracer flux measurements of pantothenate uptake using preparations of purified brush-border membrane vesicles. Compared with other cations the imposition of an inward Na+ gradient stimulated vesicle uptake of pantothenate to levels approximately 40-fold greater than those observed at equilibrium. The observed stimulation of pantothenate uptake was not the result of indirect electrostatic coupling to an inside positive Na+ diffusion potential. In the absence of Na+ and pantothenate concentration gradients an inside negative voltage difference induced a Na(+)-dependent net influx of pantothenate, suggesting the presence of an electrogenic Na(+)-pantothenate cotransport mechanism. The effect of biotin on the kinetics of Na(+)-dependent pantothenate uptake and the effect of pantothenate on the kinetics of Na(+)-dependent biotin uptake suggested that placental absorption of biotin and pantothenate from the maternal circulation occurs by a common Na+ cotransport mechanism in apical brush-border membrane.