Methenyltetrahydrofolate Synthetase Regulates Folate Turnover and Accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation. Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation. Folate is a generic term that refers to a family of structurally related compounds that contain 2-amino-4-hydroxypteridine linked to p-aminobenzoylglutamate though a methylene group. The reduced tetrahydrofolates (THF) 1The abbreviations used are: THF, tetrahydrofolates; pABG, para-aminobenzoyl(poly)glutamate; αMEM, α-minimal essential medium; PBS, phosphate-buffered saline; MTHFS, methenyltetrahydrofolate synthetase; HPLC, high performance liquid chromatography; HCF, heavy chain ferritin. serve as cofactors that carry one-carbon moieties for the de novo synthesis of purines (supplies the number 2 and number 8 carbon of the purine ring) and thymidylate (for the methylation of dUMP to dTMP), and for the remethylation of homocysteine to methionine (1Shane B. Vitam. Horm. 1989; 45: 263-335Crossref PubMed Scopus (288) Google Scholar, 2Wagner C. Bailey L.B. Folate in Health and Disease. Marcel Dekker, Inc., New York1995: 23-42Google Scholar). Methionine can be adenylated to form S-adenosylmethionine, which is a cofactor and one-carbon donor for numerous cellular methylation reactions (3Selhub J. Annu. Rev. 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Unsubstituted forms of folate, which include THF and dihydrofolate, and the 10-substituted folates, are chemically labile and susceptible to rapid oxidative degradation in vitro (26Lewis G.P. Rowe P.B. Anal. Biochem. 1979; 93: 91-97Crossref PubMed Scopus (34) Google Scholar, 27Suh J.R. Oppenheim E.W. Girgis S. Stover P.J. J. Biol. Chem. 2000; 275: 35646-35655Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar). There are two pathways for folate oxidation: one resulting in the formation of dihydro forms of the cofactor (dihydrofolate, 5-methyldihydrofolate); the other resulting in the oxidative cleavage of the C9 –N10 bond of dihydrofolates and tetrahydrofolates resulting in the formation of the pterin derivative and para-aminobenzoyl(poly)glutamate (pABG) (15Suh J.R. Herbig A.K. Stover P.J. Annu. Rev. Nutr. 2001; 21: 255-282Crossref PubMed Scopus (224) Google Scholar). 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Gynaecol. 2000; 107: 1149-1154Crossref Scopus (59) Google Scholar). Increased rates of folate catabolism during pregnancy are established in rodent models, but results from human studies have been conflicting (34Higgins J.R. Quinlivan E.P. McPartlin J. Scott J.M. Weir D.G. Darling M.R. Br. J. Obstet. Gynaecol. 2000; 107: 1149-1154Crossref Scopus (59) Google Scholar, 35McPartlin J. Halligan A. Scott J.M. Darling M. Weir D.G. Lancet. 1993; 341: 148-149Abstract PubMed Scopus (157) Google Scholar, 36Caudill M.A. Gregory J.F. Hutson A.D. Bailey L.B. J. Nutr. 1998; 128: 204-208Crossref PubMed Scopus (61) Google Scholar). Elucidating the mechanisms that regulate systemic folate concentrations as well as the folate content of individual tissues is critical to understanding the complex relationships among folate status, folate metabolism, and disease. Materials—(6S)-[3′,5′,7,9-3H]5-Formyl-THF (40 Ci/mmol) and [3′,5′,7,9-3H]folic acid (25.5 Ci/mmol) were obtained from Moravek Biochemicals, Inc. (6R, 6S)-5-Formyl-THF was from SAPEC, pABG was obtained from Sigma, and (6S)-5-methyl-THF was from Eprova. Fetal bovine serum, α-minimal essential medium (αMEM), and α-modification lacking sodium bicarbonate, folate, ribosides, ribotides, deoxyribosides, and deoxyribotides (defined αMEM) were obtained from Hyclone Laboratories. Cell Lines and Medium—SH-SY5Y human neuroblastoma cells, and cell lines expressing human methenyltetrahydrofolate synthetase (MTHFS) cDNA (SH-SY5YMTHFS1, SH-SY5YMTHFS2, and SH-SY5YMTHFS5), have been described previously (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The human MCF-7 mammary adenocarcinoma cells (ATCC catalog number HTB22) and cell lines expressing human MTHFS cDNA (MCFMTHFS1) have been reported (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). For folate turnover studies, fetal bovine serum was dialyzed against 10 volumes of phosphate-buffered saline (PBS) at 4 °C for 24 h with buffer changes every 4 h to deplete serum of folate and other small molecules. The serum was then charcoal-treated to remove any remaining folate. Determination of Folate Turnover in Cultured Cells—Cell monolayers at 75% confluence were washed with PBS, then labeled for 12 h in αMEM lacking folate and glycine but supplemented with 25 nm (6S)-[3H]5-formyl-THF or 120 nm [3H]folic acid. For the chase, cell monolayers were washed with 10 ml of PBS, trypsinized, and pelleted by centrifugation. The cells were seeded in triplicate (0.3–1 × 106) into 100-mm culture plates containing 10 ml of αMEM supplemented with 2 μm folic acid. Cells were harvested at defined time points, the medium was removed, and the tritium in the medium was quantified. The cell monolayers were washed with PBS and lysed with 0.2 m ammonium hydroxide. Tritium remaining in the cells was quantified using a Beckman LS 8100 liquid scintillation counter. Western Blot Analysis of Human MTHFS—Cells were cultured to 75% confluence in αMEM, washed with 1× PBS, then harvested by trypsinization. Cell pellets were lysed using 10 mm Tris, pH 7.5, 150 mm sodium chloride, 5 mm EDTA, 1% Triton X-100, 20 mm 2-mercaptoethanol, 1 mm phenylmethylsulfonyl fluoride, centrifuged, and protein concentration of the supernatant was determined. Protein extracts (60 μg/lane) were run on a 12% SDS-PAGE gel, then transferred to a polyvinylidene fluoride microporous membrane (Millipore) using a MiniTransblot apparatus (Bio-Rad). For detection of MTHFS, the membrane was incubated overnight at 4 °C with primary antiserum (1: 10,000 dilution) consisting of polyclonal antibodies generated in sheep against a highly conserved peptide sequence of human MTHFS (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). The membrane was washed with 0.1% Tween 20 in PBS, then incubated for 2 h with horseradish peroxidase-conjugated rabbit anti-sheep secondary antibody (1:6500 dilution, Pierce) and detected using the Super Signal West Pico chemiluminescent detection system (Pierce). The protein bands were quantified using ChemiImager 4400 from Alpha Innotech Corp. (San Leandro, CA). MTHFS Activity Assays—MTHFS activity was measured from cell extracts using a procedure described previously (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Cell pellets were lysed by sonication and clarified by centrifugation. Cell extract was added to a cuvette containing 100 μm (6S)-5-formyl-THF, 10 mm MgATP, and 50 mm potassium phosphate buffer, pH 6.0, and the rate of 5,10-methenyl-THF formation was determined by monitoring the increase in absorbance at 360 nm in a spectrophotometer. Determination of Radiolabeled Folate Incorporation in Cultured Cells—Cell monolayers at 70% confluence in 6-well plates were labeled for 12 h in αMEM lacking folate and glycine supplemented with 25 nm (6S)-[3H]5-formyl-THF. Cells were harvested by washing with 5 ml of PBS, trypsinized, and pelleted by centrifugation and viable cells (determined by their ability to exclude trypan blue) were quantified. The cell pellets were lysed with 0.2 m ammonium hydroxide, and the intracellular tritium was quantified using a Beckman LS 8100 scintillation counter. Determination of Total Folate Content and Polyglutamate Chain Lengths—Total cellular folate and folate polyglutamate chain lengths were determined by affinity/reverse phase HPLC as described elsewhere (38Bagley P.J. Selhub J. Clin. Chem. 2000; 46: 404-411Crossref PubMed Scopus (116) Google Scholar). This two-column HPLC method first purifies folate derivatives from the extract using a folate-binding protein resin, and the second phenyl column separates the individual folate derivatives (38Bagley P.J. Selhub J. Clin. Chem. 2000; 46: 404-411Crossref PubMed Scopus (116) Google Scholar). Folates are identified with a four-channel coulometric electrochemical detector. Measurements were made from cells cultured to 70% confluence in αMEM that contains 2 μm folic acid. Determination of pABG and 5-Formyl-THF Levels in Culture Media—Cells were seeded (1.45 × 107) into 100-mm Primaria culture dishes (Falcon) containing 10 ml of αMEM. Following a 2-h incubation to allow the cells to adhere to the plate, the medium was replaced with defined αMEM containing 25 nm (6S)-[3H]5-formyl-THF. Aliquots of medium were taken at various time points, clarified by centrifugation, then transferred to a new tube and stored at –80 °C until HPLC analyses. Reverse phase HPLC was used to separate folate degradation products from labeled folates present in the culture medium. Medium samples were spiked with a mixture of unlabeled pABG, (6R,6S)-5-formyl-THF, and (6S)-5-methyl-THF, then analyzed on a Shimadzu HPLC equipped with a diode array UV spectrophotometric detector and a Luna 5μ 250 × 4.6-mm C18 column (Phenomenex). A binary buffer gradient was used to separate degradation products from intact folate, with Buffer A consisting of 0.1 m sodium acetate, pH 6.0, 5 mm Pic A reagent (Waters), and 16% methanol, and Buffer B consisting of 100% methanol. The flow rate was 1.0 ml/min. The HPLC method consisted of 100% Buffer A for the first 30 min, 75% Buffer A from 30 to 31 min, then 45% Buffer A from 31 to 42 min. With this method, pABG elutes at 6 min, 5-formyl-THF elutes at 20 min, and 5-methyl-THF elutes at 37 min. For each analysis, 1.0-ml fractions were collected into scintillation tubes, and the tritium was quantified using a Beckman LS 6500 liquid scintillation counter. Determination of Folate Uptake in Cultured Cells—Cell monolayers at 70% confluence in 6-well plates were washed with PBS, then labeled for a total of 10 h in αMEM lacking folate and glycine but supplemented with 25 nm (6S)-[3H]5-formyl-THF. Cells were harvested in triplicate at the defined time points by washing with 5 ml of PBS, then trypsinizing, and pelleting by centrifugation. Viable cells (determined by their ability to exclude trypan blue) were quantified. The cell pellets were lysed with 0.2 m ammonium hydroxide, and the intracellular tritium was quantified using a Beckman LS 8100 scintillation counter. Northern Blot Analyses—The tissue distribution of mouse MTHFS mRNA was determined by Northern analysis using a "Multiple Tissue Northern blot" (Ambion). Each tissue sample contains 2 μg of mRNA. [32P]ATP-labeled probes were generated by asymmetric PCR amplification, using mouse MTHFS cDNA as the template and primers that amplified the first 416 bp of the sequence. The PCR amplification conditions for probe construction were 94 °C for 45 s, 59 °C for 45 s, and 72 °C for 1 min for a total of 30 cycles. The β-actin control probes were generated by linear PCR amplification, using human β-actin cDNA as the template and an antisense primer. PCR cycling conditions were supplied by the manufacturer (Clontech). Hybridization of the blot was performed as directed by the manufacturer (Ambion). The tissue distribution of human MTHFS mRNA was determined by Northern analysis using a Multiple Tissue Northern blot (Clontech). Random [32P]ATP-labeled probes specific for MTHFS and β-actin message were generated from the corresponding human cDNAs following the manufacturer's protocols. The metabolic role of 5-formyl-THF is not known. This folate derivative is unique because it does not directly participate as a cofactor in folate-dependant reactions but rather acts as an inhibitor of various folate-dependent enzymes (39Stover P. Schirch V. Trends Biochem. Sci. 1993; 18: 102-106Abstract Full Text PDF PubMed Scopus (112) Google Scholar). It is synthesized in the cell through a second catalytic activity of the enzyme serine hydroxymethyltransferase (40Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). It is present in high concentrations in seeds, and accumulates to nearly 90% of total cellular folate during the sporulation of Neurospora crassa. Results from these studies have lead to the hypothesis that 5-formyl-THF is a chemically stable storage form of folate (41Kruschwitz H.L. McDonald D. Cossins E.A. Schirch V. J. Biol. Chem. 1994; 269: 28757-28763Abstract Full Text PDF PubMed Google Scholar). Although not thoroughly studied, 5-formyl-THF is not known to accumulate in mammalian cells because of the enzymatic activity of MTHFS (40Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). MTHFS catalyzes reaction 1 in the cytoplasm, a reaction that depletes cellular 5-formyl-THF concentrations (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar) and this is the only reaction known to metabolize 5-formyl-THF. MTHFS is expressed in many organisms ranging from bacteria to humans, which suggests that it may play an important role in one-carbon metabolism (40Holmes W.B. Appling D.R. J. Biol. Chem. 2002; 277: 20205-20213Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 42Roje S. Janave M.T. Ziemak M.J. Hanson A.D. J. Biol. Chem. 2002; 277: 42748-42754Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). 5-Formyl-THF+ATP→5,10-methenyl-THF+ADP+PO4Reaction1(Eq. 1) Previously, we reported the development of stable MCF-7 and SH-SY5Y cell lines that express the human MTHFS cDNA and display increased MTHFS activity (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). To determine whether depletion of 5-formyl-THF impairs folate accumulation, the effects of MTHFS activity on intracellular folate concentrations and folate turnover was determined in these cell lines. Increased expression of human MTHFS protein was verified in these cell lines by Western blot analyses (Fig. 1). Densitometry analysis indicated that MCF-7 cells that express the MTHFS cDNA contained 1.8-fold more MTHFS protein compared with nontransfected cells (Fig. 1). The specific activity of MTHFS in MCF-7 cells is 6.0 ± 0.4 pmol of 5,10-methenyl-THF formed/min/mg cell extract, whereas the specific activity of MTHFS in the MCFMTHFS1 cell line is 11.9 ± 0.8 mol of 5,10-methenyl-THF formed/min/mg cell extract, consistent with densitometry results from Fig. 1. The SH-SY5Y6 cell line that expresses the MTHFS cDNA exhibited a 100-fold elevation in MTHFS enzyme activity levels compared with nontransfected SH-SY5Y cells as described previously (Fig. 1) (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Effect of MTHFS Expression on Folate Accumulation and Turnover—The effect of increased MTHFS expression on folate accumulation was determined in MCF-7 and SH-SY5Y cells. Cells were cultured with [3H]5-formyl-THF for 12 h and the accumulation of radiolabeled folate was determined. [3H]5-Formyl-THF does not accumulate in cells because it equilibrates rapidly into the folate cofactor pool (37Girgis S. Suh J.R. Jolivet J. Stover P.J. J. Biol. Chem. 1997; 272: 4729-4734Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Both MCF-7 and SH-SY5Y cell lines that express the human MTHFS cDNA contained 40% less [3H]folate compared with nontransfected cells (Fig. 2) when cultured in medium containing 25 nm [3H]5-formyl-THF. These results indicate that increased MTHFS activity decreases the capacity of two different cell lines to accumulate folate. Because folate accumulates in cells at levels that approximate the folate binding capacity of the cell (15Suh J.R. Herbig A.K. Stover P.J. Annu. Rev. Nutr. 2001; 21: 255-282Crossref PubMed Scopus (224) Google Scholar), the inhibition of labeled folate accumulation observed above may result from: 1) inhibition of folate uptake, and/or 2) accelerated turnover of newly imported folate, and/or 3) slower rates of folate polyglutamate turnover and thereby decreased concentrations of unliganded or available binding sites for newly imported folate monoglutamates. Mechanism 3 would not be expected to alter total cellular folate concentrations, whereas mechanisms 1 and 2 would be expected to deplete cellular folate concentrations. To distinguish between these mechanisms, the concentration of total cellular folate was determined under the same culture conditions that were used to determine folate accumulation (Fig. 2). The total cellular folate content was decreased by 40 and 28%, respectively, in MCF-7 and SH-SY5Y cell lines expressing the MTHFS cDNA relative to nontransfected cells cultured in medium with high folic acid concentrations (2 μm) (Table I). Therefore, increased MTHFS expression depresses intracellular folate concentrations independent of extracellular folate availability.Table IEffect of MTHFS expression on cellular folate concentrations and folate polyglutamate chain lengthsCell lineTotal folateMean polyglutamate lengthpmol/mg proteinMCF-727.2 ± 2.9 (100%)3.2 ± 0.06MCFMTHFS116.6 ± 5.0 (60%)aValue significantly different from nontransfected cells, p < 0.05 one-tailed Student's t-test.4.4 ± 0.35aValue significantly different from nontransfected cells, p < 0.05 one-tailed Student's t-test.SH-SY5Y39.1 ± 5.1 (100%)5.7 ± 0.12SH-SY5YMTHFS27.9 ± 1.8 (72%)bValue significantly different from nontransfected cells, p < 0.01 one-tailed Student's t-test.6.1 ± 0.10cValue significantly different from nontransfected cells, p < 0.003 one-tailed Student's t-test.a Value significantly different from nontransfected cells, p < 0.05 one-tailed Student's t-test.b Value significantly different from nontransfected cells, p < 0.01 one-tailed Student's t-test.c Value significantly different from nontransfected cells, p < 0.003 one-tailed Student's t-test. Open table in a new tab To determine whether depletion of cellular 5-formyl-THF concentrations influences the stability o