Abstract
The metabolic role of 5-formyltetrahydrofolate is not known; however, it is an inhibitor of several folate-dependent enzymes including serine hydroxymethyltransferase. Methenyltetrahydrofolate synthetase (MTHFS) is the only enzyme known to metabolize 5-formyltetrahydrofolate and catalyzes the conversion of 5-formyltetrahydrofolate to 5,10-methenyltetrahydrofolate. In order to address the function of 5-formyltetrahydrofolate in mammalian cells, intracellular 5-formyltetrahydrofolate levels were depleted in human 5Y neuroblastoma by overexpressing the human cDNA encoding MTHFS (5YMTHFS cells). When cultured with 2 mM exogenous glycine, the intracellular serine and glycine concentrations in 5YMTHFS cells are elevated approximately 3-fold relative to 5Y cells; 5YMTHFS cells do not contain measurable levels of free methionine and display a 30-40% decrease in cell proliferation rates compared with 5Y cells. Medium supplemented with pharmacological levels of exogenous folinate or methionine ameliorated the glycine induced growth inhibition. Analysis of the folate derivatives demonstrated that 5-methyltetrahydrofolate accounts for 30% of total cellular folate in 5Y cells when cultured with 5 mM exogenous glycine. 5YMTHFS cells do not contain detectable levels of 5-methyltetrahydrofolate under the same culture conditions. These results suggest that 5-formyltetrahydrofolate inhibits serine hydroxymethyltransferase activity in vivo and that serine synthesis and homocysteine remethylation compete for one-carbon units in the cytoplasm.
Highlights
5-CHO-H4PteGlu1 normally accounts for 3–10% of total intracellular folate in mammalian cells; its metabolic function in cells has not been elucidated. 5-CHO-H4PteGlu is synthesized from 5,10-CHϩ-H4PteGlu by both the mitochon
In order to determine the role of 5-CHO-H4PteGlu in mammalian metabolism and the metabolic consequences associated with its depletion in the cell, the human Methenyltetrahydrofolate synthetase (MTHFS) cDNA was overexpressed in 5Y neuroblastoma
We cannot rule out the possibility that mitochondrial MTHFS has been increased because we were not able to detect any MTHFS activity in isolated mitochondria from either 5Y or 5YMTHFS cells
Summary
5-CHO-H4PteGlu, 5-formyltetrahydrofolate; H4PteGlu, tetrahydrofolate; 10-CHO-H4PteGlu, 10-formyltetrahydrofolate; 5,10-CHϩ-H4PteGlu, 5,10-methenyltetrahydrofolate; 5,10CH2-H4PteGlu, 5,10-methylenetetrahydrofolate; 5-CH3-H4PteGlu, 5-methyltetrahydrofolate; SHMT, serine hydroxymethyltransferase; cSHMT, cytoplasmic SHMT; mSHMT, mitochondrial SHMT; MTHFS, methenyltetrahydrofolate synthetase; GCS, glycine cleavage system; 5Y, SH-SY5Y neuroblastoma; HPLC, high pressure liquid chromatography; CHO, Chinese hamster ovary cells; MES, morpholineethanesulfonic acid; MEM, minimal essential medium; PBS, phosphate-buffered saline. The major source of one-carbon units in the form of formate are generated from serine in a reaction catalyzed by mSHMT, but formate can be generated in the mitochondria from glycine in cells that contain a GCS. The primary role of cSHMT may not be to generate glycine or one-carbon units but instead may have other metabolic functions including the synthesis of 5-CHO-H4PteGlu. little is known about the regulation of 5-CHOH4PteGlu, it has been reported that valproic acid treatment lowers the intracellular concentration of 5-CHO-H4PteGlu in fetuses [8]. In order to determine the influence of 5-CHO-H4PteGlu depletion on folic acid-mediated one-carbon metabolism and SHMT activity, we have overexpressed the human MTHFS cDNA in 5Y neuroblastoma and determined its effects on cell proliferation, intracellular serine, glycine and methionine concentrations, and the relative distribution of the folic acid onecarbon derivatives
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