Regulation of Folate-mediated One-carbon Metabolism by 10-Formyltetrahydrofolate Dehydrogenase

Montserrat C Anguera,Martha S Field,Cheryll Perry,Haifa Ghandour,En-Pei Chiang,Jacob Selhub,Barry Shane,Patrick J Stover

doi:10.1074/jbc.m510623200

Abstract

10-Formyltetrahydrofolate dehydrogenase (FDH) catalyzes the NADP(+)-dependent conversion of 10-formyltetrahydrofolate to CO(2) and tetrahydrofolate (THF) and is an abundant high affinity folate-binding protein. Although several activities have been ascribed to FDH, its metabolic role in folate-mediated one-carbon metabolism is not well understood. FDH has been proposed to: 1) inhibit purine biosynthesis by depleting 10-formyl-THF pools, 2) maintain cellular folate concentrations by sequestering THF, 3) deplete the supply of folate-activated one-carbon units, and 4) stimulate the generation of THF-activated one-carbon unit synthesis by channeling folate cofactors to other folate-dependent enzymes. The metabolic functions of FDH were investigated in neuroblastoma, which do not contain detectable levels of FDH. Both low and high FDH expression reduced total cellular folate concentrations by 60%, elevated rates of folate catabolism, and depleted cellular 5-methyl-THF and S-adenosylmethionine levels. Low FDH expression increased the formyl-THF/THF ratio nearly 10-fold, whereas THF accounted for nearly 50% of total folate in neuroblastoma with high FDH expression. FDH expression did not affect the enrichment of exogenous formate into methionine, serine, or purines and did not suppress de novo purine nucleotide biosynthesis. We conclude that low FDH expression facilitates the incorporation of one-carbon units into the one-carbon pool, whereas high levels of FDH expression deplete the folate-activated one-carbon pool by catalyzing the conversion of 10-formyl-THF to THF. Furthermore, FDH does not increase cellular folate concentrations by sequestering THF in neuroblastoma nor does it inhibit or regulate de novo purine biosynthesis. FDH expression does deplete cellular 5-methyl-THF and S-adenosylmethionine levels indicating that FDH impairs the folate-dependent homocysteine remethylation cycle.

Highlights

Tetrahydrofolate (THF)2 polyglutamates are cofactors that function as one-carbon donors and acceptors in a set of reactions known as folate-mediated one-carbon metabolism, which occurs both in the cytoplasm and in mitochondria [1]
These cell lines were used to validate the previously ascribed metabolic roles of Formyltetrahydrofolate dehydrogenase (FDH) in folate-mediated one-carbon metabolism. The effects of both low and high level FDH expression on: 1) intracellular folate concentrations, 2) the 10-formyl-THF/THF ratio, 3) SAM levels, and 4) flux within the folate-mediated one-carbon metabolic network were examined
FDH expression had no effect on the 14C/3H ratio in nuclear DNA or purine deoxynucleosides, demonstrating that, FDH can deplete 10-formyl-THF pools, this activity does not impair the incorporation of formate into purines relative to salvage pathway biosynthesis in neuroblastoma (Table 4)

Summary

EXPERIMENTAL PROCEDURES

Materials—[6S]-[3H]5-Formyl-THF (40 Ci/mmol) was obtained from Moravek Biochemicals, Inc., and [methyl-3H]thymidine was from PerkinElmer Life Sciences. [6R,S]-5-Formyl-THF was from SAPEC, the chemical Division of Cerbios-Pharma SA, Barbengo, Switzerland. Stable SH-SY5Y cell lines expressing the human FDH cDNA from the pcDNA6/TR vector (Invitrogen) were generated. Stable Isotope Tracer Studies—To quantify the enrichment of [2,3,32H3]serine into purines, thymidine, and methionine, cells were plated at 50% confluence in 100-mm plates and cultured in treatment medium, which consisted of defined ␣MEM lacking deoxyribonucleosides, hypoxanthine, and thymidine but supplemented with 0.05 ␮M 5-formyl-THF, 10 ␮M methionine, 0.2 mM glycine, 1 mg/liter pyridoxine, 250 ␮M formate, 26 mg/liter L-[5,5,5-2H3]leucine, and 250 ␮M L-[2,3,3-2H3]serine). To quantify the enrichment of [13C]formate into purines, thymidine, and methionine, cells were plated at 50% confluence in 100-mm plates and cultured in treatment medium, which consisted of defined ␣MEM lacking deoxyribonucleosides, hypoxanthine, and thymidine but was supplemented with 250 ␮M [13C]formate, 0.05 ␮M 5-formyl-THF, 10 ␮M methionine, 0.2 mM glycine, 1 mg/liter pyridoxine, 26 mg/liter L-[5,5,5-2H3]leucine. Peaks corresponding to nucleosides were identified from standards, collected, and isotope levels quantified

RESULTS AND DISCUSSION

Total folate

CONCLUSIONS