Abstract
Cytoplasmic folate-mediated one carbon (1C) metabolism functions to carry and activate single carbons for the de novo synthesis of purines, thymidylate, and for the remethylation of homocysteine to methionine. C1 tetrahydrofolate (THF) synthase, encoded by Mthfd1, is an entry point of 1Cs into folate metabolism through its formyl-THF synthetase (FTHFS) activity that catalyzes the ATP-dependent conversion of formate and THF to 10-formyl-THF. Disruption of FTHFS activity by the insertion of a gene trap vector into the Mthfd1 gene results in embryonic lethality in mice. Mthfd1gt/+ mice demonstrated lower hepatic adenosylmethionine levels, which is consistent with formate serving as a source of 1Cs for cellular methylation reactions. Surprisingly, Mthfd1gt/+ mice exhibited decreased levels of uracil in nuclear DNA, indicating enhanced de novo thymidylate synthesis, and suggesting that serine hydroxymethyltransferase and FTHFS compete for a limiting pool of unsubstituted THF. This study demonstrates the essentiality of the Mthfd1 gene and indicates that formate-derived 1Cs are utilized for de novo purine synthesis and the remethylation of homocysteine in liver. Further, the depletion of cytoplasmic FTHFS activity enhances thymidylate synthesis, affirming the competition between thymidylate synthesis and homocysteine remethylation for THF cofactors.
Highlights
Folate-mediated one-carbon (1C)3 metabolism is compartmentalized in the cytoplasm, mitochondria, and nucleus of mammalian cells [1]
The 1C carried by methylene-THF is oxidized and hydrolyzed to generate formate by the NAD-dependent methyleneTHF dehydrogenase (MTHFD) and methenyl-THF cyclohydrolase (MTHFC) activities encoded by a single gene, Mthfd2 [7], and 10-formyl-THF synthetase (FTHFS) activity, encoded by Mthfd1L [8]
The Gene Trap Insertion in the Mthfd1 Gene Inactivates 10-Formyl-THF Synthetase Activity—The insertion of the gene trap vector into the Mthfd1 gene was identified by 5Ј rapid amplification of cDNA ends at BayGenomics
Summary
Sheep anti-mouse C1THF synthase antibody was generated from the peptide NYVPDDTKPNGRKVVG (amino acid residues 239 –254) and affinity purified using the same biotin-conjugated peptide This antibody was diluted 1:10,000, and horseradish peroxidase-conjugated rabbit anti-sheep IgG secondary antibody (Pierce) was diluted 1:20,000. Determination of AdoMet and AdoHcy Concentrations— Frozen tissues were sonicated in 500 l of 0.1 M sodium acetate buffer (pH 6), and protein was precipitated by adding 312 l of 10% perchloric acid to each sample. Uracil content in nuclear DNA was analyzed by gas chromatography mass spectrometry, as previously described [5]. Metabolite Profile from Plasma—Total homocysteine, cystathionine, total cysteine, methionine, glycine, serine, ␣-aminobutyric acid, N,N-dimethylglycine, and N-methylglycine were assayed in mouse plasma by stable isotope dilution capillary gas chromatography mass spectrometry as previously described [21, 22].
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