GNMT expression increases hepatic folate contents and folate-dependent methionine synthase-mediated homocysteine remethylation

Glycine N-methyltransferase (GNMT) is a major hepatic enzyme that converts S-adenosylmethionine to S-adenosylhomocysteine while generating sarcosine from glycine, hence it can regulate mediating methyl group availability in mammalian cells. GNMT is also a major hepatic folate binding protein that binds to, and, subsequently, may be inhibited by 5-methyltetrafolate. GNMT is commonly diminished in human hepatoma; yet its role in cellular folate metabolism, in tumorigenesis and antifolate therapies, is not understood completely. In the present study, we investigated the impacts of GNMT expression on cell growth, folate status, methylfolate-dependent reactions and antifolate cytotoxicity. GNMT-diminished hepatoma cell lines transfected with GNMT were cultured under folate abundance or restriction. Folate-dependent homocysteine remethylation fluxes were investigated using stable isotopic tracers and gas chromatography/mass spectrometry. Folate status was compared between wild-type (WT), GNMT transgenic (GNMT(tg)) and GNMT knockout (GNMT(ko)) mice. In the cell model, GNMT expression increased folate concentration, induced folate-dependent homocysteine remethylation, and reduced antifolate methotrexate cytotoxicity. In the mouse models, GNMT(tg) had increased hepatic folate significantly, whereas GNMT(ko) had reduced folate. Liver folate levels correlated well with GNMT expressions (r = 0.53, P = 0.002); and methionine synthase expression was reduced significantly in GNMT(ko), demonstrating impaired methylfolate-dependent metabolism by GNMT deletion. In conclusion, we demonstrated novel findings that restoring GNMT assists methylfolate-dependent reactions and ameliorates the consequences of folate depletion. GNMT expression in vivo improves folate retention and bioavailability in the liver. Studies on how GNMT expression impacts the distribution of different folate cofactors and the regulation of specific folate dependent reactions are underway.     

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration

Formate can differentiate between hyperhomocysteinemia due to impaired remethylation and impaired transsulfuration. Am J Physiol Endocrinol Metab 301: E000-E000, 2011. First published September 20, 2011; 10.1152/ajpendo.00345.2011.-We carried out a (1)H-NMR metabolomic analysis of sera from vitamin B(12)-deficient rats. In addition to the expected increases in methylmalonate and homocysteine (Hcy), we observed an approximately sevenfold increase in formate levels, from 64 μM in control rats to 402 μM in vitamin B(12)-deficient rats. Urinary formate was also elevated. This elevation of formate could be attributed to impaired one-carbon metabolism since formate is assimilated into the one-carbon pool by incorporation into 10-formyl-THF via the enzyme 10-formyl-THF synthase. Both plasma and urinary formate were also increased in folate-deficient rats. Hcy was elevated in both the vitamin B(12)- and folate-deficient rats. Although plasma Hcy was also elevated, plasma formate was unaffected in vitamin B(6)-deficient rats (impaired transsulfuration pathway). These results were in accord with a mathematical model of folate metabolism, which predicted that reduction in methionine synthase activity would cause increased formate levels, whereas reduced cystathionine β-synthase activity would not. Our data indicate that formate provides a novel window into cellular folate metabolism, that elevated formate can be a useful indicator of deranged one-carbon metabolism and can be used to discriminate between the hyperhomocysteinemia caused by defects in the remethylation and transsulfuration pathways.     

Dietary nickel and folic acid interact to affect folate and methionine metabolism in the rat

A previous experiment using rats indicated that dietary nickel (Ni), folic acid, and their interaction affected variables associated with one-carbon metabolism. That study used diets that produced only mild folate deficiency. Thus, an experiment was performed to determine the effect of a severe folate deficiency on nickel deprivation in rats. A 2×2 factorially arranged experiment used groups of six weanling Sprague-Dawley rats. Dietary variables were nickel, as NiCl₂·6H₂O, 0 or 1 μg/g and folic acid, 0 or 4 mg/kg. All diets contained 10 g succinylsulfathiazole/kg to suppress microbial folate synthesis. The basal diet contained <20 ng Ni/g. After 50 d, an interaction between nickel and folate affected the urinary excretion of formiminoglutamic acid (FIGLU) and the liver concentration of S-adenosylmethionine (SAM). Because of this, it is proposed that the physiological function of nickel is related to the common metabolism shared by SAM and FIGLU. Possibly the physiological function of nickel could be related to the tissue concentration of 5-methyltetrahydrofolate (MTHF) or tetrahydrofolate (THF).     

Early increase in phosphatidyl choline synthesis by choline and transmethylation pathways in spreading fibroblasts

Phosphatidyl choline (PC) synthesis in trypsinized and reattaching fibroblasts during the spreading state was studied by incorporation of [14C]choline and [methyl-14C]methionine. The choline and phosphatidyl-ethanolamine (PE) transmethylation pathways were both transiently increased about 2-fold during the first 2 h after replating. Maximum increase appeared to be simultaneous with maximum spreading. Incorporation of [32P]orthophosphate showed that the increase in PC synthesis was specific and most probably related to establishment of cell-substrate adhesion sites.     

Double restriction in NK cell recognition is linked to transmethylation and can be triggered by asparagine-linked oligosaccharides on tumor cells

The determinants of tumor cell susceptibility to NK cell-mediated cytolysis were analyzed in a two stage model. The binding of tumor cells to NK effectors was measured by target-effector conjugation and cold target competition in 51Cr-release assays, whereas triggering was measured by assaying phospholipid methylation in NK cells stimulated by intact targets. Representative targets could be grouped into three phenotypes based on the data. Those such as YAC 1.2 could bind and trigger NK cells whereas the mutagenized variant, YAC 6.28.8, could bind but was unable to trigger NK cells and therefore resisted lysis. The third phenotype was represented by HL-60 which could neither bind nor trigger NK cells and was therefore completely NK resistant. The oligosaccharide nature of the triggering molecules was demonstrated by showing that purified, high mannose containing, asparagine-linked oligosaccharides from tumor cell targets were potent stimulators of NK transmethylation at submicromolar levels. Tunicamycin pretreatment of target cells inhibited their triggering capacity but not their NK binding function. These results suggest a double restriction in NK specificity involving two independent but sequential stages in recognition represented in binding and triggering by asn-linked oligosaccharides on the tumor cell surface.     

S‐adenosyl‐l‐methionine usage during climacteric ripening of tomato in relation to ethylene and polyamine biosynthesis and transmethylation capacity

S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.     

Identification of a highly conserved domain in the EcoRII methyltransferase which can be photolabeled with S-adenosyl-L-[methyl-3H]methionine. Evidence for UV-induced transmethylation of cysteine 186

DNA methyltransferases can be photolabeled with S-adenosyl-L-methionine (AdoMet). Specific incorporation of radioactivity has been demonstrated after photolabeling with either [methyl-3H]AdoMet or [35S]AdoMet (Som, S., and Friedman, S. (1990) J. Biol. Chem. 265, 4278-4283). The labeling is believed to occur at the AdoMet binding site. With the purpose of localizing the site responsible for [methyl-3H]AdoMet photolabeling, we cleaved the labeled EcoRII methyltransferase by chemical and enzymatic reactions and isolated the radiolabeled peptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high pressure liquid chromatography. The labeled peptides were identified by amino-terminal sequencing. A common region was localized which accounted for 65-70% of the total label. This region includes a highly conserved core sequence present in all DNA (cytosine 5)-methyltransferases. One such fragment was digested further with chymotrypsin, and amino acid analysis of the resulting 3H-labeled peptide was consistent with the sequence Ala-Gly-Phe-Pro-(Cys)-Gln-Pro-Phe-Ser-Leu. However, the cysteine residue was not recovered as carboxymethylcysteine. The Pro-Cys bond was found to be protected from cleavage at cysteine residues after cyanylation. These results suggest that the cysteine residue is modified by the labeling reaction. The chymotryptic fragment was hydrolyzed enzymatically to single amino acids, and the labeled amino acid was identified as S-methylcysteine by thin layer chromatography. These results indicate that the cysteine residue is located at or close to the AdoMet binding site of EcoRII methyltransferase.     

Nitric oxide inhibits methionine synthase activity in vivo and disrupts carbon flow through the folate pathway

Many of nitric oxide's biological effects are mediated via NO binding to the iron in heme-containing proteins. Cobalamin (vitamin B(12)) is structurally similar to heme and is a cofactor for methionine synthase, a key enzyme in folate metabolism. NO inhibits methionine synthase activity in vitro, but data concerning NO binding to cobalamin are controversial. We now show spectroscopically that NO reacts with all three valency states of cobalamin and that NO's inhibition of methionine synthase activity most likely involves its reaction with monovalent cobalamin. By following incorporation of the methyl moiety of [(14)C]methyltetrahydrofolic acid into protein, we show that NO inhibits methionine synthase activity in vivo, in cultured mammalian cells. The inhibition of methionine synthase activity disrupted carbon flow through the folate pathway as measured by decreased incorporation of [(14)C]formate into methionine, serine, and purine nucleotides. Homocysteine, but not cysteine, attenuated NO's inhibition of purine synthesis, providing further evidence that NO was acting through methionine synthase inhibition. NO's effect was observed both when NO donors were added to cells and when NO was produced physiologically in co-culture experiments. Treating cells with an NO synthase inhibitor increased formate incorporation into methionine, serine, and purines and methyl-tetrahydrofolate incorporation into protein. Thus, physiological concentrations of NO appear to regulate carbon flow through the folate pathway.     

Methylenetetrahydrofolate reductase deficiency and low dietary folate increase embryonic delay and placental abnormalities in mice

BACKGROUND: Despite extensive research on mild methylenetetrahydrofolate reductase (MTHFR) deficiency and low dietary folate in different disorders, the association of these metabolic disturbances with a variety of congenital defects and pregnancy complications remains controversial. In this study we investigated the effects of MTHFR and dietary folate deficiency at 10.5 days post coitum (dpc) in our mouse model of mild MTHFR deficiency. METHODS: Mthfr +/+ and +/? female mice were fed a control or folic acid–deficient diet for 6 weeks, then mated with Mthfr +/? males. At 10.5 dpc, embryos were examined and placentae were collected for histologic evaluation. RESULTS: Maternal MTHFR and folate deficiencies resulted in increased developmental delays and smaller embryos. We also observed a low frequency of a variety of embryonic defects in the experimental groups, such as neural tube, heart looping, and turning defects; these results mimic the low incidence and multifactorial nature of these anomalies in humans. Folate‐deficient mice also had increased embryonic losses and severe placental defects, including placental abruption and disturbed patterning of placental layers. Folate‐deficient placentae had decreased ApoA‐I expression, and there was a trend toward a negative correlation between ApoA‐I expression with maternal homocysteine concentrations. CONCLUSIONS: Our study provides biological evidence linking maternal MTHFR and dietary folate deficiencies to adverse pregnancy outcomes in mice. It underscores the importance of folate not only in reducing the incidence of early embryonic defects, but also in the prevention of developmental delays and placental abnormalities that may increase susceptibility to other defects and to reproductive complications. Birth Defects Research (Part A), 2009. © 2009 Wiley‐Liss, Inc.     

Polyglutamylation of folate coenzymes is necessary for methionine biosynthesis and maintenance of intact mitochondrial genome in Saccharomyces cerevisiae

One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes of Saccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by the YMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extension of the glutamate chains of the folate coenzymes, is encoded by the MET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase.     

Increasing community size and connectance can increase stability in competitive communities

The relationship between community complexity and stability has been the subject of an enduring debate in ecology over the last 50 years. Results from early model communities showed that increased complexity is associated with decreased local stability. I demonstrate that increasing both the number of species in a community and the connectance between these species results in an increased probability of local stability in discrete-time competitive communities, when some species would show unstable dynamics in the absence of competition. This is shown analytically for a simple case and across a wider range of community sizes using simulations, where individual species have dynamics that can range from stable point equilibria to periodic or more complex. Increasing the number of competitive links in the community reduces per-capita growth rates through an increase in competitive feedback, stabilising oscillating dynamics. This result was robust to the introduction of a trade-off between competitive ability and intrinsic growth rate and changes in species interaction strengths. This throws new light on the discrepancy between the theoretical view that increased complexity reduces stability and the empirical view that more complex systems are more likely to be stable, giving one explanation for the relative lack of complex dynamics found in natural systems. I examine how these results relate to diversity-biomass stability relationships and show that an analytical solution derived in the region of stable equilibrium dynamics captures many features of the change in biomass fluctuations with community size in communities including species with oscillating dynamics.     

Cell density and airspace patterning in the leaf can be manipulated to increase leaf photosynthetic capacity

The pattern of cell division, growth and separation during leaf development determines the pattern and volume of airspace in a leaf. The resulting balance of cellular material and airspace is expected to significantly influence the primary function of the leaf, photosynthesis, and yet the manner and degree to which cell division patterns affect airspace networks and photosynthesis remains largely unexplored. In this paper we investigate the relationship of cell size and patterning, airspace and photosynthesis by promoting and repressing the expression of cell cycle genes in the leaf mesophyll. Using microCT imaging to quantify leaf cellular architecture and fluorescence/gas exchange analysis to measure leaf function, we show that increased cell density in the mesophyll of Arabidopsis can be used to increase leaf photosynthetic capacity. Our analysis suggests that this occurs both by increasing tissue density (decreasing the relative volume of airspace) and by altering the pattern of airspace distribution within the leaf. Our results indicate that cell division patterns influence the photosynthetic performance of a leaf, and that it is possible to engineer improved photosynthesis via this approach.