Deregulation of homocysteine metabolism and consequences for the vascular system

The link between vascular disease and elevated homocysteine levels has been recognized for more than 30 years, and association with moderately elevated levels has been suspected for 20 years. Homocysteine is a sulfhydryl-containing amino acid that is formed by the demethylation of methionine. It is normally catalysed to cystathionine by cystathionine beta-synthase a pyridoxal phosphate-dependent enzyme. Homocysteine is also remethylated to methionine by methionine synthase, a vitamin B12 dependent enzyme and by methylenetetrahydrofolate reductase. Environmental factors such as folate, or vitamin B12, or vitamin B6 deficiencies and genetic defects such as cystathionine beta-synthase or abnormality of methylene-tetrahydrofolate reductase or some vitamin B12 metabolism defects may contribute to increasing plasma homocysteine levels. Normal fasting levels of homocysteine lie within the range 6-16 mumol/l. Apart from differences in assay methods, age, sex and nutritional status may affect the plasma levels. Though it is now well known that homocysteine is an independent risk factor for premature vascular disease, the pathogenesis of homocysteine-induced vascular damage is, for the most part, unknown. It may be multifactorial, including direct homocysteine damage to the endothelium, an enhanced low-density lipoprotein peroxidation, an increase of platelet thromboxane A2, or a decrease of protein C activation.     

Polymorphism of the methionine synthase gene : association with homocysteine metabolism and late-onset vascular diseases in the Japanese population

Methionine synthase and 5,10-methylenetetrahydrofolate reductase (MTHFR) sequentially catalyze the remethylation of homocysteine to methionine. A point mutation in the encoding region of the methionine synthase gene, which results in substitution of an aspartic acid for a glycine residue (D919G), has been identified in patients of the cblG genetic complementation group; these patients exhibit significantly decreased methionine synthase activity. Nevertheless, the D919G mutation has also been reported to be common in the general population. In this study, we analyzed the distribution of methionine synthase D/G polymorphism in the Japanese population and examined the extent to which it is associated with altered homocysteine metabolism and late-onset vascular diseases. We studied 215 patients with coronary artery disease, 251 patients with histories of ischemic stroke, and 257 control subjects. The methionine synthase genotype was analyzed by polymerase chain reaction followed by HaeIII digestion; allele frequencies for the D919G variant of the enzyme proved to be similar in all 3 subject groups (control subjects, 0.17; coronary artery disease patients, 0. 17; and ischemic stroke patients, 0.19). Furthermore, in patients with ischemic stroke, plasma levels of homocyst(e)ine and folate were similar, irrespective of methionine synthase genotype. Thus, the methionine synthase D919G mutation was found to be common in the Japanese general population, and it appears unlikely that this polymorphism has a major effect on homocysteine metabolism and/or the onset of vascular diseases.     

Cobalamin-dependent methionine synthase from Escherichia coli: involvement of zinc in homocysteine activation

Methionine synthase (MetH) is a modular protein with at least four distinct regions; amino acids 2-353 comprise a region responsible for binding and activation of homocysteine, amino acids 345-649 are thought to be involved in the binding and activation of methyltetrahydrofolate, amino acids 650-896 are responsible for binding of the prosthetic group methylcobalamin, and amino acids 897-1227 are involved in binding adensylmethionine and are required for reductive activation of enzyme in the cob(II)alamin form. Previous studies have shown that mutations of Cys310 or Cys311 to either alanine or serine result in loss of all detectable catalytic activity. These mutant proteins retain the ability to catalyze methyl transfer from methyltetrahydrofolate to exogenous cob(I)alamin, but have lost the ability to transfer methyl groups from exogenous methylcobalamin to homocysteine [Goulding, C. W., Postigo, D., and Matthews, R. G. (1997) Biochemistry 36, 8082-8091]. We now demonstrate that both MetH holoenzyme and a truncated MetH(2-649) protein, which lacks a cobalamin prosthetic group, contain 0.9 equiv of zinc, while the Cys310Ser and Cys311Ser mutant proteins contain less than 0.05 equiv of zinc. Addition of l-homocysteine to MetH(2-649) is accompanied by release of 1 equiv of protons/mol of protein, while addition of l-homocysteine to the Cys310Ser and Cys311Ser mutant truncated proteins does not result in proton release. The Cys310Ala and Cys311Ala mutant methylcobalamin holoenzymes have completely lost the ability to transfer the methyl group from methylcobalamin to homocysteine, suggesting that zinc is required for this reaction. Further evidence that zinc is required for catalytic activity comes from experiments in which the zinc is removed from MetH(2-1227). Removal of zinc from methylated wild-type holoenzyme by treatment with methyl methanethiolsulfonate and then with dithiothreitol and EDTA results in loss of the ability of the protein to catalyze methyl transfer from methyltetrahydrofolate to homocysteine. Reconstitution of the zinc-depleted holoenzyme results in incorporation of 0.4 equiv of zinc/mol of protein and partial restoration of the ability of the protein to catalyze homocysteine methylation.     

Homocysteine: relations to ischemic cardiovascular diseases

Homocysteine, a sulfur-containing amino acid, is an intermediate metabolite of methionine. Patients with homocystinuria and severe hyperhomocysteinemia develop premature arteriosclerosis and arterial thrombotic events, and venous thromboembolism. Studies suggest that moderate hyperhomocysteinemia can be considered as an independent risk factor in the development of premature cardiovascular disease. In vitro, homocysteine has toxic effects on endothelial cells. Homocysteine can promote lipid peroxidation and damage vascular endothelial cells. Moreover, homocysteine interferes with the natural anticoagulant system and the fibrinolytic system. Homocysteinemia should be known in patients with premature vascular diseases, especially in subjects with no risk factors. Folic acid, vitamin B6 can lower homocysteine levels.     

High plasma homocysteine: a risk factor for vascular disease in the elderly

Homocysteine is an intermediate compound formed during the metabolism of methionine. Several studies have shown that plasma homocysteine concentrations rise with age. Overt or borderline deficiencies of folate, vitamin B12 or B6 and possibly age-related kidney dysfunction are the major causes of homocysteine elevation in the elderly population. Multiple case-control and prospective studies have shown that a high plasma homocysteine concentration is an independent risk factor for cardiovascular diseases; this association persists in the elderly. Supplementation with folic acid either alone or with vitamins B12 and B6 can lower plasma homocysteine. Intervention studies to assess the effects (if any) of such treatment on prognosis are now in progress in patients with vascular disease.     

Hepatic betaine-homocysteine methyltransferase activity in the chicken is influenced by dietary intake of sulfur amino acids, choline and betaine

There is much interest in the metabolism of homocysteine, because elevated plasma homocysteine [hyperhomocyst(e)inemia] is an independent risk factor for the development of cardiovascular disease. Four chick assays were conducted to determine the effects of varying dietary sulfur amino acids, choline and betaine on the activity of hepatic betaine-homocysteine methyltransferase (BHMT), an enzyme likely to be important in modulating plasma homocysteine. In Experiment 1, chicks were fed a purified crystalline amino acid diet containing adequate sulfur amino acids and choline. Excess dietary methionine, or the combination of excess cystine with choline or betaine, caused a small increase (P < 0.05) in BHMT activity. In Experiment 2, use of a methionine-deficient purified diet resulted in a threefold increase (P < 0.05) in BHMT activity, and addition of choline or betaine further increased (P < 0.05) BHMT activity. In Experiment 3, use of a methionine-deficient corn-peanut meal diet increased BHMT (P < 0.05) relative to that of chicks supplemented with adequate methionine, and addition of surfeit choline to the methionine-deficient basal diet caused a further increase (P < 0.05). In Experiment 4, addition of both surfeit choline and surfeit betaine to the methionine-deficient corn-peanut meal diet caused an increase (P < 0.05) in BHMT activity relative to that observed in chicks fed the methionine-deficient basal diet. These assays show that large increases in BHMT activity can be produced under methionine-deficient conditions, especially in the presence of excess choline or betaine.     

Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone

A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase. The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone. Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected. Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme. The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase. However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids. Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA. This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.     

Renal uptake and excretion of homocysteine in rats with acute hyperhomocysteinemia

BACKGROUND: Elevated plasma total homocysteine, an independent risk factor for cardiovascular disease, is commonly observed in renal patients. We have previously shown that the kidney is a major site for the removal of plasma homocysteine in the rat. The present investigation was performed to further characterize the capacity of the kidney to handle acute elevations in plasma homocysteine concentrations. METHODS: Acute hyperhomocysteinemic conditions (4- to 7-fold > controls) in rats were produced by either a primed-continuous infusion of L-homocysteine or exposure to 80:20% nitrous oxide:oxygen, which results in the inhibition of methionine synthase. RESULTS: At physiological homocysteine concentrations, approximately 15% of the arterial plasma homocysteine was removed on passage through the kidney. Renal homocysteine uptake was approximately 85% of the filtered load. The urinary excretion of homocysteine was negligible (<2%). During acute hyperhomocysteinemia produced by the infusion of L-homocysteine, renal homocysteine uptake was increased fourfold and was equivalent to 50% of the infused dose, while urinary excretion remained negligible. Renal homocysteine uptake during nitrous oxide-induced hyperhomocysteinemia increased threefold, with urinary excretion remaining negligible. CONCLUSIONS: These results provide strong evidence that the kidney has a significant capacity for metabolizing acute elevations in plasma homocysteine, and support a very limited role for the re-methylation pathway in renal homocysteine metabolism.     

The study of methionine uptake in Saccharomyces cerevisiae reveals a new family of amino acid permeases

The screening of mutants resistant to the oxidized analogues of methionine (methionine sulphoxide and ethionine sulphoxide) allowed the characterisation of a yeast mutant strain lacking the high affinity methionine permease and defining a new locus that was called MUP1. The study of MUP1 mutants showed that methionine is transported into yeast cells by three different permeases, a high affinity and two low affinity permeases. The MUP1 gene was cloned and was shown to encode an integral membrane protein with 13 putative membrane-spanning regions. Database comparisons revealed that the yeast genome contains an ORF whose product is highly similar to the MUP1 protein. This protein is shown here to encode very low affinity methionine permease and the corresponding gene was thus called MUP3. It has previously been suggested that the amino acid permeases from yeast all belong to a single family of highly similar proteins. The two methionine permeases encoded by genes MUP1 and MUP3 are only distantly related to this family and thus define a new family of amino acid transporters.     

Cloning, sequencing, and heterologous expression of rat methionine synthase cDNA

Methionine synthase catalyzes cobalamin-dependent methyl transfer reaction from 5-methyltetrahydrofolate to homocysteine, forming methionine. Rat methonine synthase cDNA was cloned and analyzed by RT-PCR, 3'- and 5'-RACE techniques. The cDNA consists of a 0.3-kb upstream untranslated region, a 3.8-kb coding region, and a 0.4-kb downstream untranslated region. The open reading frame encoded a polypeptide of 1,253 amino acid residues with a calculated molecular weight of 139,162. This molecular weight was in good agreement with the observed one (143,000) of the purified rat liver enzyme. The deduced amino acid sequence was 53, 92, and 64% identical with those of the Escherichia coli, human, and presumptive Caenorhabditis elegans enzymes, respectively. All the fingerprint sequences, forming parts of the cobalamin- and S-adenosylmethionine-binding sites, were completely conserved in the rat methionine synthase. A high-level expression of catalytically active enzyme in insect cells was done by infection with a baculovirus containing the rat methionine synthase cDNA.     

Modulation of potassium channel function by methionine oxidation and reduction

Oxidation of amino acid residues in proteins can be caused by a variety of oxidizing agents normally produced by cells. The oxidation of methionine in proteins to methionine sulfoxide is implicated in aging as well as in pathological conditions, and it is a reversible reaction mediated by a ubiquitous enzyme, peptide methionine sulfoxide reductase. The reversibility of methionine oxidation suggests that it could act as a cellular regulatory mechanism although no such in vivo activity has been demonstrated. We show here that oxidation of a methionine residue in a voltage-dependent potassium channel modulates its inactivation. When this methionine residue is oxidized to methionine sulfoxide, the inactivation is disrupted, and it is reversed by coexpression with peptide methionine sulfoxide reductase. The results suggest that oxidation and reduction of methionine could play a dynamic role in the cellular signal transduction process in a variety of systems.     

Homocysteinaemia after methionine overload as a coronary artery disease risk factor: importance of age and homocysteine levels

BACKGROUND: Homocysteinaemia is now accepted as an independent risk factor for coronary artery disease (CAD). Our goal was to study the influence of age plasma homocysteine level on the CAD risk attributable to homocysteinaemia. METHODS: We studied a group of 98 patients under 55 years of age who had suffered a myocardial infarction 3-12 months before the study. The patients were matched by sex and age with a group of 98 controls without vascular disease. We measured the plasma homocysteine levels 6h after a methionine overload of 0.1 g/kg body weight in patients and controls. Afterwards, the odds ratio for homocysteinaemia was determined by homocysteine level, and that for hyperhomocysteinaemia (homocysteine level > 34 mumol/l) by age group. RESULTS: After methionine loading, the homocysteine odds ratio varied from 0.47 (homocysteine level < 23 mumol/l) to 2.88 (homocysteine level > 34 mumol/l). In patients under the age of 46 the odds ratio for hyperhomocysteinaemia was 18.6. In patients between 46 and 55 years of age the odds ratio for hyperhomocysteinaemia was 1.2. CONCLUSIONS: Low homocysteine levels are protective against CAD, and the higher the homocysteine level the higher the coronary risk appears to be. This clearly means that heterozygosity for cystathionine beta synthase deficiency alone is not enough to explain the vascular risk associated with homocysteinaemia. Hyperhomocysteinemia was shown to be a significant risk factor only in patients under the age of 46 years old.     

Biochemical model reactions on the prooxidative activity of homocysteine

The sulfur amino acid homocysteine has recently been addressed as marker for vessel damaging and atherosclerotic dispositions. The atherogenic index has been correlated with the one of cholesterol and is significantly higher in cholesterinemic as compared to normal lipidemic persons. In the present communication biochemical model reactions are presented indicating the prooxidative activity of homocysteine where a cooperative effect with the transition-metals copper and iron is indicated.     

The cytotoxic effect of the vitamin B12 inhibitor cyanocobalamin [c-lactam], and a review of other vitamin B12 antagonists

The vitamin B12 antagonist cyanocobalamin [c-lactam] was cytotoxic to cultured human leukemia cells, grown in methylfolate, homocysteine, and vitamin B12, but not in the presence of methionine. Small concentrations of methionine were effective in restoring the growth rate in a dose-dependent fashion, confirming methionine deficiency as the cytotoxic principle. Cyanocobalamin [c-lactam] prevented utilization of the methyl group of methylfolate, but no evidence of folate deficiency developed in long-term culture. High concentrations of non-methylated folate were unable to reverse the cytotoxicity, excluding a methylfolate 'trap' as the cause. Low concentrations of serine in the medium induced transient biochemical megaloblastosis. Cyanocobalamin [c-lactam] caused this to occur earlier, and persist. In high concentrations of serine, the inhibitor caused only transient changes in deoxyuridine suppression. Homocysteine cannot be remethylated without vitamin B12, and condensation with serine is the only other excretory pathway for this toxic amino acid. We hypothesize that impaired DNA synthesis in vitamin B12 deficiency is the result of diverting serine away from thymidylate synthesis, into homocysteine metabolism.     

Oxidative stress in diabetic macrovascular disease: does homocysteine play a role?

BACKGROUND: Non-insulin-dependent diabetes mellitus (NIDDM) and hyperhomocysteinemia are both associated with increased lipid peroxidation (oxidative stress). This may contribute to the accelerated vascular disease associated with these conditions. It is not known whether the coexistence of elevated homocysteine levels will stimulate oxidative stress further than that caused by diabetes alone. METHODS: Plasma concentrations of thiobarbituric acid reactive substances (TBARS), an index of lipid peroxidation, were measured in patients with NIDDM who had previously had a methionine load test; some of the patients had hyperhomocysteinemia. RESULTS: Plasma TBARS concentrations were elevated in diabetics with vascular disease. The additional presence of hyperhomocysteinemia was not associated with a further increase in plasma TBARS concentrations. CONCLUSIONS: Lipid peroxidation is increased in patients with diabetes mellitus and macrovascular disease and is not further elevated by the coexistence of elevated homocysteine levels. It is possible that diabetes maximally stimulates oxidative stress and any further acceleration of vascular disease in patients who have coexistent hyperhomocysteinemia is mediated through mechanisms other than lipid peroxidation.     

S-methylmethionine metabolism in Escherichia coli

Selenium-accumulating Astragalus spp. contain an enzyme which specifically transfers a methyl group from S-methylmethionine to the selenol of selenocysteine, thus converting it to a nontoxic, since nonproteinogenic, amino acid. Analysis of the amino acid sequence of this enzyme revealed that Escherichia coli possesses a protein (YagD) which shares high sequence similarity with the enzyme. The properties and physiological role of YagD were investigated. YagD is an S-methylmethionine: homocysteine methyltransferase which also accepts selenohomocysteine as a substrate. Mutants in yagD which also possess defects in metE and metH are unable to utilize S-methylmethionine for growth, whereas a metE metH double mutant still grows on S-methylmethionine. Upstream of yagD and overlapping with its reading frame is a gene (ykfD) which, when inactivated, also blocks growth on methylmethionine in a metE metH genetic background. Since it displays sequence similarities with amino acid permeases it appears to be the transporter for S-methylmethionine. Methionine but not S-methylmethionine in the medium reduces the amount of yagD protein. This and the existence of four MET box motifs upstream of yfkD indicate that the two genes are members of the methionine regulon. The physiological roles of the ykfD and yagD products appear to reside in the acquisition of S-methylmethionine, which is an abundant plant product, and its utilization for methionine biosynthesis.     

The mode of action of homocysteine on mouse brain glutamic decarboxylase and gamma-aminobutyrate aminotransferase

In the belief that homocysteine-induced convulsions might be related to alterations in brain gamma-aminobutyric acid metabolism, we have studied the action of this amino acid on the activity of glutamic decarboxylase (GAD, EC 4.1.1.15) and gamma-aminobutyrate aminotransferase (EC 2.6.1.19) of mouse brain in vitro DL-homocysteine competitively inhibited GAD with respect to both L-glutamate and pyridoxal 5'-phosphate. The respective Ki's were 3.8 mM and 0.3 mM. The activity of GABA-T also was altered in the presence of DL-homocysteine. A competitive inhibition (Ki = 6 mM) was observed with gamma-aminobutyric acid, and an uncompetitive inhibition with respect to pyridoxal 5'-phosphate and alpha-ketoglutarate. These results are explained in terms of a dual action of homocysteine on each of the enzymes: one involving a competition for substrate binding site and the other involving the formation of an inactive inhibitor-cofactor complex. The significance of the inhibition of these enzymes of gamma-aminobutyric acid metabolism is discussed in relation to the convulsant action of homocysteine.     

Determination of plasma and serum homocysteine by high-performance liquid chromatography with fluorescence detection

Determination of homocysteine in plasma or serum is becoming an important diagnostic procedure. Accurate, rapid and low cost methods for measuring homocysteine are therefore required. We have improved an HPLC method and made it suitable for clinical application. The total homocysteine in plasma consists of free homocysteine (i.e., reduced plus oxidized homocysteine in the non-protein fraction of plasma) and protein-bound homocysteine. The method consists of the following steps: reduction of the sample with tri-n-butylphosphine, precipitation of proteins with trichloroacetic acid (10%) and derivatization with ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulfonate. The derivatives are separated by reversed-phase high-performance liquid chromatography followed by fluorescence detection. The concentrations (mean +/- S.D.) of total homocysteine in plasma from 77 normal subjects, 44 male and 33 female adults, were 8.4 +/- 2.15 and 7.1 +/- 1.18 mumol/l, respectively. Serum concentrations were 8.8 +/- 2.6 mumol/l in males and 7.6 +/- 1.5 mumol/l in females.     

PxF, a prenylated protein of peroxisomes

CAAX farnesyltransferase attaches a farnesyl group to proteins that terminate in the sequence CAAX, where C is cysteine, A is an aliphatic amino acid, and X is typically methionine or serine. A limited number of substrates for the CAAX farnesyltransferase have been identified in cultured cells. These include p21ras proteins and the nuclear lamins A and B. We describe here the use of a CAAX farnesyltransferase inhibitor, together with a hamster cell line that exhibits efficient uptake of [3H]mevalonate, as a means of identifying novel farnesylated proteins. One candidate protein was purified and its attached prenyl group identified as farnesyl. The predicted amino acid sequence of this protein, deduced from a cloned cDNA, terminates with the tetrapeptide Cys-Leu-Ile-Met, which conforms to the consensus sequence for recognition by farnesyltransferase. This farnesylated protein, designated PxF, is localized to the outer surface of peroxisomes as determined by indirect immunofluorescence and electron microscopy.     

Extensive modifications for methionine enhancement in the beta-barrels do not alter the structural stability of the bean seed storage protein phaseolin

Common beans are widely utilized as a food source, yet are low in the essential amino acid methionine. As an initial step to overcome this defect the methionine content of the primary bean seed storage protein phaseolin was increased by replacing 20 evolutionarily variant hydrophobic residues with methionine and inserting short, methionine-rich sequences into turn and loop regions of the protein structure. Methionine enhancement ranged from 5 to 30 residues. An Escherichia coli expression system was developed to characterize the structural stability of the mutant proteins. Proteins of expected sizes were obtained for all constructs except for negative controls, which were rapidly degraded in E. coli. Thermal denaturation of the purified proteins demonstrated that both wild-type and mutant phaseolin proteins denatured reversibly at approximately 61 degrees C. In addition, urea denaturation experiments of the wild-type and a mutant protein (with 30 additional methionines) confirmed that the structural stability of the proteins was very similar. Remarkably, these results indicate that the phaseolin protein tolerates extensive modifications, including 20 substitutions and two loop inserts for methionine enhancement in the beta-barrel and loop structures, with extremely small effects on protein stability.