Methionine and Serine Formation in Control and Mutant Human Cultured Fibroblasts: Evidence for Methyl Trapping and Characterization of Remethylation Defects

Abstract

The conversion of labeled formate to methionine and serine, as a measure of remethylation of homocysteine to methionine and folate coenzyme cycling, has been studied in control and mutant human fibroblasts. Fibroblasts in monolayer culture were incubated with [14C]formate, and labeled methionine sulfone and serine were determined in hydrolysates of oxidized cell proteins. In control cells, methionine and serine were clearly measurable (n = 21, 1.7-5.5 and 2.4-9.7 nmol/mg protein/16 h, respectively). In contrast, methionine formation was reduced in cells from patients with methylenetetrahydrofolate reductase (MR) deficiency (MR mutant, n = 11, 0.05-0.44), combined methylmalonic aciduria/homocystinuria [cobalamin(cbl)C/D mutant, n = 12, 0.014-0.13), and methionine synthase deficiency (MS mutant, n = 3, 0.04-0.23). Furthermore, serine formation was low in cblC/D mutant (0.08-0.98) and MS mutant (0.17-0.94) cells, but normal or high in MR mutant cells (5.2-11.4). Growth of cblC/D mutant cells in medium supplemented with high concentrations of hydroxo-cbl resulted in significant increases of both methionine and serine formation. Taken together these findings provide clear evidence for the existence of the formate to serine pathway described by W. B. Strong and V. Schirch in cultured fibroblasts and indicate that disturbed MS function due to a specific genetic disorder is associated with reduced serine formation in vitro, which reflects availability of reduced folate coenzymes. The correction of this defect by vitamin B12 alone, in cblC/D mutant cell lines, correlates well with the clinical response in the patients and fits in well with the idea that reduced availability of folate coenzymes occurs in functional MS deficiency, in agreement with the methyl trap hypothesis.

Cytoplasmic Serine Hydroxymethyltransferase Mediates Competition between Folate-dependent Deoxyribonucleotide andS-Adenosylmethionine Biosyntheses

“…In fibroblasts where MS activity was deficient due to a variety of genetic mutations, serine formation was low compared with control cells, consistent with the formation of a folate methyl trap. Moreover, mutant fibroblasts with diminished MTHFR activity exhibited normal to high serine formation, indicating that MTHFR deficiency increased the availability of 5,10-methyleneTHF for cSHMT-catalyzed synthesis of serine from glycine (15).…”

Folate restriction and methylenetetrahydrofolate reductase 677T polymorphism decreases adoMet synthesis via folate-dependent remethylation in human-transformed lymphoblasts

“…7 In the present study, we investigated the impacts of MTHFR 677C-T on the partitioning of one-carbon units between methionine regeneration and de novo purine and thymidylate syntheses under different folate conditions. MTHFR deficient fibroblasts were reported to synthesize normal or high serine concentrations from formate, 41 suggesting that MTHFR could alter the availability of reduced folate coenzymes. Excessive L-methionine inhibited folate-dependent thymidylate synthesis in Raji cells 42 because more one-carbon units were used in the synthesis of serine needed for homocysteine detoxification.…”

“…These studies suggested that cellular methyleneTHF concentration regulates the flux of this metabolite into pathways that biosynthesize nucleotides and regenerate methionine, and the thymidine synthase activity is highly dependent on methyleneTHF availability. 41,43 It was suggested that MTHFR and thymidine synthase directly compete for a common cellular pool of methyleneTHF, and folate coenzymes are preferentially directed toward adoMet-dependent methylation reactions at low cellular folate concentrations. 43,44 MTHFR activity was found less sensitive to changes in methyleneTHF availability, whereas thymidine synthase activity was highly dependent on them, 43,44 suggesting that adoMet has a higher metabolic priority when folate was limited.…”

Regulation of Folate-Mediated One-Carbon Metabolism by Glycine <i>N</i>-Methyltransferase (GNMT) and Methylenetetrahydrofolate Reductase (MTHFR)

“…MTHFRdeficient fibroblasts can synthesize normal or high serine from formate (27) suggesting that MTHFR not only regulates the availability of reduced folate coenzymes but also changes the partitioning of 1C moieties. Excessive L-methionine inhibits thymidine synthesis in Raji cells (28), presumably due to a shift of 1C unit utilization.…”

“…Excessive L-methionine inhibits thymidine synthesis in Raji cells (28), presumably due to a shift of 1C unit utilization. Thymidine synthase activity is highly dependent on methyleneTHF availability (27,29); therefore MTHFR may modulate thymidine synthesis. It has been proposed that genetic variations in MTHFR may increase the size of the thymidine pool and improve the quality of DNA synthesis, which may confer the protective effect against the development of leukemia, but direct evidence was needed.…”