Effect of genetic polymorphisms involved in folate metabolism on the concentration of serum folate and plasma total homocysteine (p-tHcy) in healthy subjects after short-term folic acid supplementation: a randomized,double blind,crossover study

Data on the effect of combined genetic polymorphisms, involved in folate metabolism, on the concentration of serum folate after folic acid supplementation are scarce. Therefore, we investigated the impact of seven gene polymorphisms on the concentration of serum folate and p-tHcy in healthy subjects after short-term folic acid supplementation. In a randomized, double blind, crossover study, apparently healthy subjects were given either 0.8 mg folic acid per day (n = 46) or placebo (n = 45) for 14 days. The washout period was 14 days. Fasting blood samples were collected on day 1, 15, 30 and 45. Data on subjects on folic acid supplementation (n = 91) and on placebo (n = 45) were used for the statistical analysis. The concentration of serum folate increased higher in subjects with higher age (53.5 ± 7.0 years) than in subjects with lower age (24.3 ± 3.2 years) after folic acid supplementation (p = 0.006). The baseline concentration of serum folate in subjects with polymorphism combination, reduced folate carrier protein, RFC1-80 GA and methylenetetrahydrofolate reductase, MTHFR677 CT+TT, was lower than RFC1-80 AA and MTHFR677 CT+TT (p = 0.002). After folic acid supplementation, a higher increase in the concentration of serum folate was detected in subjects with polymorphism combination RFC1-80 GA and MTHFR677 CC than RFC1-80 GG and MTHFR CT+TT combination (p < 0.0001). The baseline concentration of plasma total homocysteine (p-tHcy) was altered by combined polymorphisms in genes associated with folate metabolism. After folic acid supplementation, in subjects with combined polymorphisms in methylenetetrahydrofolate dehydrogenase, MTHFD1-1958 and MTHFR-677 genes, the concentration of p-tHcy was changed (p = 0.002). The combination of RFC1-80 and MTHFR-677 polymorphisms had a profound affect on the concentration of serum folate in healthy subjects before and after folic acid supplementation.     

Identification of the Minimal Functional Unit of the Homo-oligomeric Human Reduced Folate Carrier

The reduced folate carrier (RFC) is the major transport system for folates in mammals. We previously demonstrated the existence of human RFC (hRFC) homo-oligomers and established the importance of these higher order structures to intracellular trafficking and carrier function. In this report, we examined the operational significance of hRFC oligomerization and the minimal functional unit for transport. In negative dominance experiments, multimeric transporters composed of different ratios of active (either wild type (WT) or cysteine-less (CLFL)) and inactive (either inherently inactive (Y281L and R373A) due to mutation, or resulting from inactivation of the Y126C mutant by (2-sulfonatoethyl) methanethiosulfonate (MTSES)) hRFC monomers were expressed in hRFC-null HeLa (R5) cells, and residual WT or CLFL activity was measured. In either case, residual transport activity with increasing levels of inactive mutant correlated linearly with the fraction of WT or CLFL hRFC in plasma membranes. When active covalent hRFC dimers, generated by fusing CLFL and Y126C monomers, were expressed in R5 cells and treated with MTSES, transport activity of the CLFL-CLFL dimer was unaffected, whereas Y126C-Y126C was potently (64%) inhibited; heterodimeric CLFL-Y126C and Y126C-CLFL were only partly (27 and 23%, respectively) inhibited by MTSES. In contrast to Y126C-Y126C, trans-stimulation of methotrexate uptake by intracellular folates for Y126C-CLFL and CLFL-Y126C was nominally affected by MTSES. Collectively, these results strongly support the notion that each hRFC monomer comprises a single translocation pathway for anionic folate substrates and functions independently of other monomers (i.e. despite an oligomeric structure, hRFC functions as a monomer).     

Simultaneous activity of two different mechanisms of folate transport in ovarian carcinoma cell lines

We investigated whether the folate receptor α-isoform (FRα), which is overexpressed on ovarian carcinoma cells, is functionally active in internalizing the physiological form of folate, 5-methyl tetrahydrofolate (THF). Six ovarian tumor cell lines, expressing different levels of FRα (COR ≫ OVCAR3 > IGROV1 > OVCAR4 > SKOV3 > OVCAR5), were maintained in folate-depleted medium and internalization of 10 nM evaluated as acid-resistant radioactivity at 0° and 37°C. The amount of 5-methyl[³H]THF present in this fraction was not strictly related to the number of membrane receptors, since even cell lines with low FRα expression, e.g., OVCAR4, showed efficient internalization. Time-course studies indicated that, whereas no uptake was detected at 0°C, at 37°C the internalized fraction showed a slow and constant increase, until 4 h. At this time, the internalized radioactivity represented <50% of the total bound in COR, OVCAR3 and IGROV1 cells, whereas the other cell lines tested internalized fourfold more folate than their surface binding capacity. The incubation in the presence of a concentration (50 nM) of 5-methyl[³H]THF, which best ensures receptors saturation on cells with highest FR levels (COR and OVCAR3), had slight effect on surface binding of all the tested cell lines, including IGROV1 and SKOV3. In contrast, the increase of the uptake was more pronounced, particularly in SKOV3 cells. These results, together with the accumulation curves of folic acid (FA) and 5-methylTHF at 37°C, suggested the presence of a molecule on ovarian carcinoma cells with high affinity for reduced folates, possibly a reduced folate carrier (RFC). Measurement of radioactivity present in the supernatant of IGROV1 and SKOV3 cells, subjected to hypotonic lysis and cell fractionation, further indicated that 5-methyl[³H]THF was translocated to the cytosol and, despite differences in membrane levels of FRα expression this internalized fraction was similar in both cell lines. Inhibition experiments to selectively block FRα or RFC activity showed a differential sensitivity of the two pathways depending on the cell line examined. Internalization was more consistently inhibited on IGROV1 than on SKOV3 cells by treatments that disrupt FRα activity, e.g., incubation with excess FA and phosphatidylinositol specific phospholipase C, whereas Probenecid, which preferentially inhibits the carrier-mediated pathway, showed a strong inhibitory effect on both cell lines. These findings suggest that the internalization of 5-methylTHF in these tumor cells depends not only on the level of overexpressed FRα, but another transport route, with features characteristic for RFC, is functional and participates in folate uptake. J. Cell. Biochem. 65:479–491. © 1997 Wiley-Liss Inc.     

Loss of multidrug resistance protein 1 expression and folate efflux activity results in a highly concentrative folate transport in human leukemia cells

We studied the molecular basis of the up to 46-fold increased accumulation of folates and methotrexate (MTX) in human leukemia CEM-7A cells established by gradual deprivation of leucovorin (LCV). CEM-7A cells consequently exhibited 10- and 68-fold decreased LCV and folic acid growth requirements and 23-25-fold hypersensitivity to MTX and edatrexate. Although CEM-7A cells displayed a 74-86-fold increase in the reduced folate carrier (RFC)-mediated influx of LCV and MTX, RFC overexpression per se cannot induce a prominently increased folate/MTX accumulation because RFC functions as a nonconcentrative anion exchanger. We therefore explored the possibility that folate efflux activity mediated by members of the multidrug resistance protein (MRP) family was impaired in CEM-7A cells. Parental CEM cells expressed substantial levels of MRP1, MRP4, poor MRP5 levels, whereas MRP2, MRP3 and breast cancer resistance protein were undetectable. In contrast, CEM-7A cells lost 95% of MRP1 levels while retaining parental expression of MRP4 and MRP5. Consequently, CEM-7A cells displayed a 5-fold decrease in the [(3)H]folic acid efflux rate constant, which was identical to that obtained with parental CEM cells, when their folic acid efflux was blocked (78%) with probenecid. Furthermore, when compared with parental CEM, CEM-7A cells accumulated 2-fold more calcein fluorescence. Treatment of parental cells with the MRP1 efflux inhibitors MK571 and probenecid resulted in a 60-100% increase in calcein fluorescence. In contrast, these inhibitors failed to alter the calcein fluorescence in CEM-7A cells, which markedly lost MRP1 expression. Replenishment of LCV in the growth medium of CEM-7A cells resulted in resumption of normal MRP1 expression. These results establish for the first time that MRP1 is the primary folate efflux route in CEM leukemia cells and that the loss of folate efflux activity is an efficient means of markedly augmenting cellular folate pools. These findings suggest a functional role for MRP1 in the maintenance of cellular folate homeostasis.     

Intracellular trafficking and membrane targeting mechanisms of the human reduced folate carrier in Mammalian epithelial cells

The major pathway for cellular uptake of the water-soluble vitamin folic acid in mammalian cells is via a plasma membrane protein known as the reduced folate carrier (RFC). The molecular determinants that dictate plasma membrane expression of RFC as well as the cellular mechanisms that deliver RFC to the cell surface remain poorly defined. Therefore, we designed a series of fusion proteins of the human RFC (hRFC) with green fluorescent protein to image the targeting and trafficking dynamics of hRFC in living epithelial cells. We show that, in contrast to many other nutrient transporters, the molecular determinants that dictate hRFC plasma membrane expression reside within the hydrophobic backbone of the polypeptide and not within the cytoplasmic NH(2)- or COOH-terminal domains of the protein. Further, the integrity of the hRFC backbone is critical for export of the polypeptide from the endoplasmic reticulum to the cell surface. This trafficking is critically dependent on intact microtubules because microtubule disruption inhibits motility of hRFC-containing vesicles as well as final expression of hRFC in the plasma membrane. For the first time, these data define the mechanisms that control the intracellular trafficking and cell surface localization of hRFC within mammalian epithelia.     

A high-affinity folate binding protein in human amniotic fluid. Radioligand binding characteristics,immunological properties and molecular size

The presence of a folate binding protein of high-affinity type (affinity constant 5 · 10⁹M^–1, maximum folate binding 3 nM) in human amniotic fluid was demonstrated in equilibrium dialysis experiments (37°C, pH 7.4) with the radioligand³H-folate. Dissociation of³H-folate from the binding protein was slow at pH 7.4 but rapid at pH 3.5. By use of rabbit antibodies against low molecular weight folate binding protein from human milk we determined the concentration of folate binding protein in 5 amniotic fluids (range 1.5–2.3 nM) in an Enzyme-Linked Immunosorbent Assay (ELISA). ultrogel AcA 44 chromatography of amniotic fluid showed that immunoreactive and radioligand bound folate binding protein coeluted in two peaks: a major one (M _r 25 000) and a minor one (M _r 100 000).     

Binding of radiolabeled folate and 5-methyltetrahydrofolate to cow's milk folate binding protein at pH 7.4 and 5.0. Relationship to concentration and polymerization equilibrium of the purified protein

Binding of folate (pteroylglutamate) and 5-methyltetrahydrofolate, the major endogenous form of folate, to folate binding protein purified from cow's milk was studied at 7°C to avoid degradation of 5-methyltetrahydrofolate. Both folates dissociate rapidly from the protein at pH 3.5, but extremely slowly at pH 7.4, most likely due to drastic changes in protein conformation occurring after folate binding. Dissociation of 5-methyltetrahydrofolate showed no increase at 37°C suggesting that protein-bound-5-methyltetrahydrofolate is protected against degradation. Binding displayed two characteristics, positive cooperativity and a binding affinity that increased with decreasing concentrations of the protein. The binding affinity of folate was somewhat greater than that of 5-methyl tetrahydrofolate, in particular at pH 5.0. Ligand-bound protein exhibited concentration-dependent polymerization (8-mers formed at 13 M) at pH 7.4. At pH 5.0, only folate-bound forms showed noticeable polymerization. The fact that folate at pH 5.0 surpasses 5-methyltetrahydrofolate both with regard to binding affinity and ability to induce polymerization suggests that ligand binding is associated with conformational changes of the protein which favor polymerization.     

Analysis of membrane topology of the human reduced folate carrier protein by hemagglutinin epitope insertion and scanning glycosylation insertion mutagenesis

The human reduced folate carrier (RFC) is the major membrane transport system for both reduced folates and chemotherapeutic antifolate drugs, such as methotrexate (MTX). Although the RFC protein has been subjected to intensive study in order to identify critical structural and functional determinants of transport, it is impossible to assess the significance of these studies without characterizing the essential domain structure and membrane topology. The primary amino acid sequence from the cloned cDNAs predicts that the human RFC protein has 12 transmembrane domains (TMDs) with a large cytosolic loop between TMDs 6 and 7, and cytosolic-facing N- and C-termini. To establish the RFC membrane topology, a hemagglutinin (HA) epitope was inserted into the individual predicted intracellular and extracellular loops. HA insertions into putative TMD interconnecting loops 3/4, 6/7, 7/8, and 8/9, and the N- and C-termini all preserved MTX transport activity upon expression in transport-impaired K562 cells. Immunofluorescence detection with HA-specific antibody under both permeabilized and non-permeabilized conditions confirmed extracellular orientations for loops 3/4 and 7/8, and cytosolic orientations for loops 6/7 and 8/9, and the N- and C-termini. Insertion of a consensus N-glycosylation site [NX(S/T)] into putative loops 5/6, 8/9, and 9/10 of deglycosylated RFC-Gln⁵⁸ had minimal effects on MTX transport. Analysis of glycosylation status on Western blots suggested an extracellular orientation for loop 5/6, and intracellular orientations for loops 8/9 and 9/10. Our findings strongly support the predicted topology model for TMDs 1-8 and the C-terminus of human RFC. However, our results raise the possibility of an alternative membrane topology for TMDs 9-12.     

Localization of the murine reduced folate carrier as assessed by immunohistochemical analysis.

The reduced folate carrier (RFC1) is a major route for the transport of folates in mammalian cells. The localization of RFC1 in murine tissues was evaluated by immunohistochemical analysis using a polyclonal antibody to the C-terminus of the carrier. There was expression of RFC1 in the brush-border membrane of the jejunum, ileum, duodenum and colon. RFC1 was localized to the basolateral membrane of the renal tubular epithelium. Carrier was detected on the plasma membrane of hepatocytes but not in bile duct epithelial cells. In the choroid plexus RFC1 was highly expressed at the apical surface. It was also expressed in axons and dendrites and on the apical membrane of cells lining the spinal canal. In spleen, RFC1 was detected only in the cells of the red pulp. These data provide insights into the role that RFC1 plays in folate delivery in a variety of tissues. In particular, the localization of carrier may elucidate the role of RFC1 in the vectorial transport of folates across epithelia. The data also indicate that in kidney tubules and choroid plexus the sites of RFC1 expression are different from what has been reported previously for the folate receptor; and while RFC1 is expressed in small intestine, folate receptor is not.     

Characterization of folate uptake by choroid plexus epithelial cells in a rat primary culture model

Reduced derivatives of folic acid (folates) play a critical role in the development, function and repair of the CNS. However, the molecular systems regulating folate uptake and homeostasis in the central nervous system remain incompletely defined. Choroid plexus epithelial cells express high levels of folate receptor α (FRα) suggesting that the choroid plays an important role in CNS folate trafficking and maintenance of CSF folate levels. We have characterized 5-methyltetrahydrofolate (5-MTHF) uptake and metabolism by primary rat choroid plexus epithelial cells in vitro . Two distinct processes are apparent; one that is FRα dependent and one that is independent of the receptor. FRα binds 5-MTHF with high affinity and facilitates efficient uptake of 5-MTHF at low extracellular folate concentrations; a lower affinity FRα independent system accounts for increased folate uptake at higher concentrations. Cellular metabolism of 5-MTHF depends on the route of folate entry into the cell. 5-MTHF taken up via a non-FRα -mediated process is rapidly metabolized to folylpolyglutamates, whereas 5-MTHF that accumulates via FRα remains non-metabolized, supporting the hypothesis that FRα may be part of a pathway for transcellular movement of the vitamin. The proton-coupled folate transporter, proton-coupled folate transporter (PCFT), mRNA was also shown to be expressed in choroid plexus epithelial cells. This is consistent with the role we have proposed for proton-coupled folate transporter in FRα-mediated transport as the mechanism of export of folates from the endocytic compartment containing FRα.     

Oligomeric Structure of the Human Reduced Folate Carrier: IDENTIFICATION OF HOMO-OLIGOMERS AND DOMINANT-NEGATIVE EFFECTS ON CARRIER EXPRESSION AND FUNCTION*

The ubiquitously expressed reduced folate carrier (RFC) is the major transport system for folate cofactors in mammalian cells and tissues. Previous considerations of RFC structure and mechanism were based on the notion that RFC monomers were sufficient to mediate transport of folate and antifolate substrates. The present study examines the possibility that human RFC (hRFC) exists as higher order homo-oligomers. By chemical cross-linking, transiently expressed hRFC in hRFC-null HeLa (R5) cells with the homobifunctional cross-linker 1,3-propanediyl bis-methanethiosulfonate and Western blotting, hRFC species with molecular masses of hRFC homo-oligomers were identified. Hemagglutinin- and Myc epitope-tagged hRFC proteins expressed in R5 cells were co-immunoprecipitated from both membrane particulate and surface-enriched membrane fractions, indicating that oligomeric hRFC is expressed at the cell surface. By co-expression of wild type and inactive mutant S138C hRFCs, combined with surface biotinylation and confocal microscopy, a dominant-negative phenotype was demonstrated involving greatly decreased cell surface expression of both mutant and wild type carrier caused by impaired intracellular trafficking. For another hRFC mutant (R373A), expression of oligomeric wild type-mutant hRFC was accompanied by a significant and disproportionate loss of wild type activity unrelated to the level of surface carrier. Collectively, our results demonstrate the existence of hRFC homo-oligomers. They also establish the likely importance of these higher order hRFC structures to intracellular trafficking and carrier function.Folates are members of the B class of vitamins that are required for the synthesis of nucleotide precursors, serine, and methionine in one-carbon transfer reactions (1). Because mammals cannot synthesize folates de novo, cellular uptake of these derivatives is essential for cell growth and tissue regeneration (2, 3). Folates are hydrophilic anionic molecules that do not cross biological membranes by diffusion alone, so it is not surprising that sophisticated membrane transport systems have evolved to facilitate their accumulation by mammalian cells.The ubiquitously expressed reduced folate carrier (RFC)² is widely considered to be the major transport system for folate co-factors in mammalian cells and tissues (3, 4). RFC plays a generalized role in folate transport and provides specialized tissue functions such as transport across the basolateral membrane of renal proximal tubules (5), transplacental transport of folates (6), and folate transport across the blood-brain barrier (7), although the contribution of RFC to intestinal absorption of folates remains controversial (8, 9). Loss of RFC expression or function portends potentially profound physiologic and developmental consequences associated with folate deficiency (10). RFC is also a major transporter of antifolate drugs used for cancer chemotherapy such as methotrexate (Mtx), pemetrexed, and raltitrexed (4). Loss of RFC expression or synthesis of mutant RFC protein in tumor cells results in antifolate resistance caused by incomplete inhibition of cellular enzyme targets and low levels of antifolate substrate for polyglutamate synthesis (4, 11).Reflecting its particular physiologic and pharmacologic importance, interest in RFC structure and function has been high. Since 1994, when murine RFC was first cloned (12), application of state-of-the-art molecular biology and biochemistry methods for characterizing polytopic membrane proteins has led to a progressively detailed picture of the molecular structure of the carrier, including its membrane topology, N-glycosylation, functionally or structurally important domains and amino acids, and packing of α-helix transmembrane domains (TMDs) (4, 13). Although no crystal structure for RFC has yet been reported, a detailed homology model for human RFC (hRFC) based on the bacterial lactose/proton symporter LacY and glycerol 3-phosphate/inorganic phosphate antiporter GlpT was generated (13, 14) that permits testing of hypotheses related to hRFC structure and mechanism in a manner not previously possible.Considerations of hRFC structure and mechanism to date have all been based on the notion that a single 591-amino acid hRFC molecule is sufficient to mediate concentrative uptake of folate and antifolate substrates. However, a growing literature suggests that quaternary structure involving the formation of higher order oligomers (e.g. dimers, tetramers, etc.) is commonly an important feature of the structure and function of many membrane transporters (15-18). For major facilitator superfamily proteins, both monomeric (e.g. LacY, GlpT, UhpT, and GLUT3) (19-22) and oligomeric (e.g. LacS, AE1, GLUT1, and TetA) (23-28) structures have been reported, establishing the lack of a clear structural consensus for these related proteins.In this report, we explore the question of whether hRFC exists as a homo-oligomeric species composed of multiple hRFC monomers. Based on results with an assortment of biochemical methods with wt and a collection of mutant hRFC proteins, we not only demonstrate the existence of oligomeric hRFC but also establish the probable importance of these higher order structures to intracellular trafficking and carrier function.     

Embryonic development in the reduced folate carrier knockout mouse is modulated by maternal folate supplementation

BACKGROUND: The reduced folate carrier (RFC1) is a ubiquitously expressed integral membrane protein that mediates delivery of 5‐methyltetrahydrofolate into mammalian cells. In this study, embryonic/fetal development is characterized in an RFC1 knockout mouse model in which pregnant dams receive different levels of folate supplementation. METHODS: RFC1^+/? males were mated to RFC1^+/? females, and pregnant dams were treated with vehicle (control) or folic acid (25 or 50 mg/kg) by daily subcutaneous injection (0.1 mL/10 g bwt), beginning on E0.5 and continuing throughout gestation until the time of sacrifice. RESULTS: Without maternal folate supplementation, RFC1 nullizygous embryos die shortly postimplantation. Supplementation of pregnant dams with 25 mg/kg/day folic acid prolongs survival of mutant embryos until E9.5–E10.5, but they are developmentally delayed relative to wild‐type littermates, display a marked absence of erythropoiesis, severe neural tube and limb bud defects, and failure of chorioallantoic fusion. Fgfr2 protein levels are significantly reduced or absent in the extraembryonic membranes of RFC1 nullizygous embryos. Maternal folate supplementation with 50 mg/kg/day results in survival of 22% of RFC1 mutants to E18.5, but they develop with multiple malformations of the eyelids, lungs, heart, and skin. CONCLUSIONS: High doses of daily maternal folate supplementation during embryonic/fetal development are necessary for early postimplantation embryonic viability of RFC1 nullizygous embryos, and play a critical role in chorioallantoic fusion, erythropoiesis, and proper development of the neural tube, limbs, lungs, heart, and skin. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells

Although the reduced folate carrierRFC1 and the thiamine transporters THTR-1 and THTR-2 share ~40% oftheir identity in protein sequence, RFC1 does not transport thiamineand THTR-1 and THTR-2 do not transport folates. In the present study,we demonstrate that transport of thiamine monophosphate (TMP), animportant thiamine metabolite present in plasma and cerebrospinalfluid, is mediated by RFC1 in L1210 murine leukemia cells. Transport ofTMP was augmented by a factor of five in cells (R16) that overexpressRFC1 and was markedly inhibited by methotrexate, an RFC1 substrate, butnot by thiamine. At a near-physiological concentration (50 nM), TMP influx mediated by RFC1 in wild-type L1210 cells was ~50% ofthiamine influx mediated by thiamine transporter(s). Within 1 min, the majority of TMP transported into R16 cells was hydrolyzed to thiamine with a component metabolized to thiamine pyrophosphate, the active enzyme cofactor. These data suggest that RFC1 may be one of the alternative transport routes available for TMP in some tissues whenTHTR-1 is mutated in the autosomal recessive disorderthiamine-responsive megaloblastic anemia.

     

The H(+)-dependent reduced folate carrier 1 of humans and the sodium-dependent methotrexate carrier-1 of the rat are orthologs

Previously, two different carrier systems for uptake of reduced folates and the antifolate methotrexate (Mtx) were described: the pH-dependent folate sensitive reduced folate carrier 1 (RFC1) from human, hamster and mouse and a sodium-dependent and folate insensitive Mtx carrier-1 (MTX-1) from rat. It was found that all critical residues of the homologous amino acid sequence were identical. RFC1- as well as MTX-1-mediated uptake of a marker substrate into suitable human and rat cell lines increased with proton concentration, was sodium-dependent at neutral pH, and inhibited by folate at acidic pH. It is concluded that RFC1 and MTX-1 are orthologs.