Protective mechanisms against homocysteine toxicity: the role of bleomycin hydrolase

Homocysteine (Hcy) editing by methionyl-tRNA synthetase results in the formation of Hcy-thiolactone and initiates a pathway that has been implicated in human disease. In addition to being cleared from the circulation by urinary excretion, Hcy-thiolactone is detoxified by the serum Hcy-thiolactonase/paraoxonase carried on high density lipoprotein. Whether Hcy-thiolactone is detoxified inside cells was unknown. Here we show that Hcy-thiolactone is hydrolyzed by an intracellular enzyme, which we have purified to homogeneity from human placenta and identified by proteomic analyses as human bleomycin hydrolase (hBLH). We have also purified an Hcy-thiolactonase from the yeast Saccharomyces cerevisiae and identified it as yeast bleomycin hydrolase (yBLH). BLH belongs to a family of evolutionarily conserved cysteine aminopeptidases, and its only known biologically relevant function was deamidation of the anticancer drug bleomycin. Recombinant hBLH or yBLH, expressed in Escherichia coli, exhibits Hcy-thiolactonase activity similar to that of the native enzymes. Active site mutations, C73A for hBLH and H369A for yBLH, inactivate Hcy-thiolactonase activities. Yeast blh1 mutants are deficient in Hcy-thiolactonase activity in vitro and in vivo, produce more Hcy-thiolactone, and exhibit greater sensitivity to Hcy toxicity than wild type yeast cells. Our data suggest that BLH protects cells against Hcy toxicity by hydrolyzing intracellular Hcy-thiolactone.     

Adenosine analogue inhibitors of S-adenosylhomocysteine hydrolase

Elevated plasma homocysteine (Hcy) levels are an independent risk factor for the onset and progression of Alzheimer’s disease. Reduction of Hcy to normal levels therefore presents a new approach for disease modification. Hcy is produced by the cytosolic enzyme S-adenosylhomocysteine hydrolase (AHCY), which converts S-adenosylhomocysteine (SAH) to Hcy and adenosine. Herein we describe the design and characterization of novel, substrate-based S-adenosylhomocysteine hydrolase inhibitors with low nanomolar potency in vitro and robust activity in vivo.     

Cloning, overexpression, purification, and characterization of S-adenosylhomocysteine hydrolase from Corynebacterium efficiens YS-314

The gene encoding S-adenosylhomocysteine hydrolase activity (SAHase: EC 3.3.1.1) from Corynebacterium efficiens (YS-314) was cloned and expressed as a fusion protein in Escherichia coli Rosetta (DE3). The analyzed nucleotide sequence of the cloned gene proved to be identical to those reported on the NCBI database. The recombinant enzyme is a tetramer, showing a molecular weight of approximately 210 kDa, as estimated by gel filtration. The K(M) values of the enzyme for S-adenosylhomocysteine (SAH), adenosine (Ado), and homocysteine (Hcy), were determined to be 1.4, 10, and 45 microM. The overexpression of the recombinant enzyme produced a high level of protein (>40 mg of protein per gram of wet cells) and revealed certain thermostability when characterized at temperatures above 40 degrees C. It also showed a high capacity for the synthesis of SAH, thermal stability, and high kinetic similarity to human SAHase, indicating a high biotechnological and pharmacological potential.     

Intracellular degradation of bleomycin hydrolase in two Chinese hamster cell lines in relation to their peplomycin susceptibility

The Chinese hamster lung (V79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) cell. This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell. The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonal antibody specific for the enzyme. Labelling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 cells, and that 48 kDa subunits comprising bleomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105,000 X g pellet (microsomes) fraction obtained after subcellular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105,000 X g supernatant cytosol fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degraded forms of the enzyme that have lost enzymatic activity are associated.     

Trypanosoma cruzi: molecular cloning and characterization of the S-adenosylhomocysteine hydrolase

S-Adenosylhomocysteine (AdoHcy) hydrolase has emerged as an attractive target for antiparasitic drug design because of its role in the regulation of all S-adenosylmethionine-dependent transmethylation reactions, including those reactions crucial for parasite replication. From a genomic DNA library of Trypanosoma cruzi, we have isolated a gene that encodes a polypeptide containing a highly conserved AdoHcy hydrolase consensus sequence. The recombinant T. cruzi enzyme was overexpressed in Escherichia coli and purified as a homotetramer. At pH 7.2 and 37 degrees C, the purified enzyme hydrolyzes AdoHcy to adenosine and homocysteine with a first-order rate constant of 1 s(-1) and synthesizes AdoHcy from adenosine and homocysteine with a pseudo-first-order rate constant of 3 s(-1) in the presence of 1 mM homocysteine. The reversible catalysis depends on the binding of NAD(+) to the enzyme. In spite of the significant structural homology between the parasitic and human AdoHcy hydrolase, the K(d) of 1.3 microM for NAD(+) binding to the T. cruzi enzyme is approximately 11-fold higher than the K(d) (0.12 microM) for NAD(+) binding to the human enzyme.     

Paraoxonase 1 and homocysteine metabolism

Paraoxonase 1 (PON1), a component of high-density lipoprotein (HDL), is a calcium-dependent multifunctional enzyme that connects metabolisms of lipoproteins and homocysteine (Hcy). Both PON1 and Hcy have been implicated in human diseases, including atherosclerosis and neurodegeneration. The involvement of Hcy in disease could be mediated through its interactions with PON1. Due to its ability to reduce oxidative stress, PON1 contributes to atheroprotective functions of HDL in mice and humans. Although PON1 has the ability to hydrolyze a variety of substrates, only one of them-Hcy-thiolactone-is known to occur naturally. In humans and mice, Hcy-thiolactonase activity of PON1 protects against N-homocysteinylation, which is detrimental to protein structure and function. PON1 also protects against neurotoxicity associated with hyperhomocysteinemia in mouse models. The links between PON1 and Hcy in relation to pathological states such as coronary artery disease, stroke, diabetic mellitus, kidney failure and Alzheimer's disease that emerge from recent studies are the topics of this review.     

N-Homocysteinylation Induces Different Structural and Functional Consequences on Acidic and Basic Proteins

One of the proposed mechanisms of homocysteine toxicity in human is the modification of proteins by the metabolite of Hcy, homocysteine thilolactone (HTL). Incubation of proteins with HTL has earlier been shown to form covalent adducts with ε-amino group of lysine residues of protein (called N-homocysteinylation). It has been believed that protein N-homocysteinylation is the pathological hallmark of cardiovascular and neurodegenerative disorders as homocysteinylation induces structural and functional alterations in proteins. In the present study, reactivity of HTL towards proteins with different physico-chemical properties and hence their structural and functional alterations were studied using different spectroscopic approaches. We found that N-homocysteinylation has opposite consequences on acidic and basic proteins suggesting that pI of the protein determines the extent of homocysteinylation, and the structural and functional consequences due to homocysteinylation. Mechanistically, pI of protein determines the extent of N-homocysteinylation and the associated structural and functional alterations. The study suggests the role of HTL primarily targeting acidic proteins in eliciting its toxicity that could yield mechanistic insights for the associated neurodegeneration.     

Computational characterization of substrate binding and catalysis in S-adenosylhomocysteine hydrolase.

S-Adenosylhomocysteine (AdoHcy) hydrolase catalyzes the reversible hydrolysis of AdoHcy to adenosine (Ado) and homocysteine (Hcy), playing an essential role in modulating the cellular Hcy levels and regulating activities of a host of methyltransferases in eukaryotic cells. This enzyme exists in an open conformation (active site unoccupied) and a closed conformation (active site occupied with substrate or inhibitor) [Turner, M. A., Yang, X., Yin, D., Kuczera, K., Borchardt, R. T., and Howell, P. L. (2000) Cell Biochem. Biophys. 33, 101-125]. To investigate the binding of natural substrates during catalysis, the computational docking program AutoDock (with confirming calculations using CHARMM) was used to predict the binding modes of various substrates or inhibitors with the closed and open forms of AdoHcy hydrolase. The results have revealed that the interaction between a substrate and the open form of the enzyme is nonspecific, whereas the binding of the substrate in the closed form is highly specific with the adenine moiety of a substrate as the main recognition factor. Residues Thr57, Glu59, Glu156, Gln181, Lys186, Asp190, Met351, and His35 are involved in substrate binding, which is consistent with the crystal structure. His55 in the docked model appears to participate in the elimination of water from Ado through the interaction with the 5'-OH group of Ado. In the same reaction, Asp131 removes a proton from the 4' position of the substrate after the oxidation-reduction reaction in the enzyme. To identify the residues that bind the Hcy moiety, AdoHcy was docked to the closed form of AdoHcy hydrolase. The Hcy tail is predicted to interact with His55, Cys79, Asn80, Asp131, Asp134, and Leu344 in a strained conformation, which may lower the reaction barrier and enhance the catalysis rate.     

Comparative Study on in Vitro Effects of Homocysteine Thiolactone and Homocysteine on HUVEC Cells: Evidence for a Stronger Proapoptotic and Proinflammative Homocysteine Thiolactone

Hyperhomocysteinemia is an independent risk factor for the development of atherosclerosis. However the underlying mechanisms responsible for endothelial cell injury with increased plasma concentration of homocysteine or homocysteine derivatives remains still incompletely elucidated. In this study, we investigated the ability of homocysteine (Hcy) and homocysteine thiolactone (HcyT) to induce cell death and IL-8 secretion in primary human umbilical vein endothelial cells (HUVEC). Hcy and HcyT were both cytotoxic and capable of promoting cell death, as measured by caspase-3 activation and DNA fragmentation. ELISA assays clearly demonstrated that Hcy and HcyT strongly activated IL-8 release. Furthermore, our results showed that HcyT was much more efficient than Hcy in activating caspase-3 or in inducing IL-8 secretion. The use of antioxidants such as vitamin C and vitamin E strongly but not completely reduced programmed cell death and chemokine release suggesting that other pathways different than reactive oxygen species are also involved. This study suggests that Homocysteine derivatives like HcyT might possess stronger cytotoxicity and pro-inflammatory properties and that Hcy derivatives levels should therefore be more taken into account during diagnostics.     

Existence of Molten Globule State in Homocysteine-Induced Protein Covalent Modifications

Homocysteine thiolactone is a toxic metabolite produced from homocysteine by amino-acyl t-RNA synthetase in error editing reaction. The basic cause of toxicity of homocysteine thiolactone is believed to be due to the adduct formation with lysine residues (known as protein N-homocysteinylation) leading to protein aggregation and loss of enzyme function. There was no data available until now that showed the effect of homocysteine thiolactone on the native state structural changes that led to aggregate formation. In the present study we have investigated the time dependent structural changes due to homocysteine thiolactone induced modifications on three different proteins having different physico-chemical properties (cytochrome-c, lysozyme and alpha lactalbumin). We discovered that N-homocysteinylation leads to the formation of molten globule state—an important protein folding intermediate in the protein folding pathway. We also found that the formation of the molten globule state might be responsible for the appearance of aggregate formation. The study indicates the importance of protein folding intermediate state in eliciting the homocysteine thiolactone toxicity.     

Purification of bleomycin hydrolase with a monoclonal antibody and its characterization

We established a hybridoma clone that produced anti-bleomycin hydrolase antibody. The subclass of the monoclonal antibody was immunoglobulin M. The antibody significantly reacted with bleomycin hydrolase from rabbit tissues, mouse livers, sarcoma 180, and adenocarcinoma 755 but not significantly with that from MH 134 and Ehrlich carcinoma. The enzyme from L5178Y cells showed an intermediate reactivity. Bleomycin hydrolase was purified from rabbit liver by immunoaffinity with the monoclonal antibody and DEAE gel chromatography. Approximately 1300-fold-purified bleomycin hydrolase was obtained. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing on a polyacrylamide slab gel of purified bleomycin hydrolase showed a single band with an apparent Mr of 48K and an isoelectric pH of 5.2. The molecular weight of bleomycin hydrolase determined on gel filtration high-performance liquid chromatography was ca. 300K, suggesting a hexameric enzyme. The enzyme showed an optimum pH of 6.8-7.8 and gave a Vmax value of 6.72 mg min-1 mg-1 for peplomycin and 9.24 mg min-1 mg-1 for bleomycin B2 and a Km value of 0.79 mM for both substrates. The enzyme was inhibited by E-64, leupeptin, p-tosyl-L-lysine chloromethyl ketone, N-ethylmaleimide, Fe2+, Cu2+, and Zn2+ but was enhanced by dithiothreitol. The results suggest that bleomycin hydrolase is a thiol enzyme.     

Influence of homocysteine on matrix metalloproteinase-2: activation and activity.

Increased levels of the physiological amino acid homocysteine (Hcy) are considered a risk factor for vascular disease. Hyperhomocysteinemia causes an intense remodelling of the extracellular matrix in arterial walls, particularly an elastolysis involving metalloproteinases. We investigated the activation of the latent elastolytic metalloproteinase proMMP-2 (72 kDa) by Hcy. Hcy was proved to exert a dual effect, activating proMMP-2 at low molar ratio (MR 10:1) and inhibiting active MMP2 at high molar ratio (MR > 1000:1). Methionine and the disulphide homocystine did not activate nor inhibit MMP-2, showing that the activation as well as the inhibition requires the thiol group to be free. The activation of proMMP-2 by Hcy is in accordance with the "cysteine-switch" mechanism, but occurs without further autoproteolysis of the enzyme molecule. In contrast with Hcy, the other physiological thiol compounds cysteine and reduced glutathione did not activate proMMP-2. These results suggest that the direct activation of proMMP2 by Hcy could be one of the mechanisms involved in the extracellular matrix deterioration in hyperhomocysteinemia-associated arteriosclerosis.     

Rational Approaches to the Design of Mechanism-Based Inhibitors of S-Adenosylhomocysteine Hydrolase

Abstract

Crucial to the rational design of inhibitors of S-adenosyl-L-homocysteine (AdoHcy) hydrolase was the elucidation of its mechanism of catalysis by Palmer and Abeles (J. Biol. Chem. 254, 1217–1226, 1979). This mechanism involves an NAD⁺-dependent oxidation (oxidative activity) of the 3′-hydroxyl group of AdoHcy followed by elimination of homocysteine (Hcy) to form 4′,5′-didehydro-3′-keto-Ado. Addition of water at the 5′-position (hydrolytic activity) of this tightly bound intermediate followed by an NADH-dependent reduction results in the formation of adenosine (Ado). Many inhibitors of this enzyme have been shown to serve as substrates [e.g., 9-(trans-2-trans-3-dihydroxycyclopent-4-en-1-yl)adenine, DHCeA)] for the oxidative activity of AdoHcy hydrolase, affording the 3′-keto-derivative (e.g., 3′-keto-DHCeA), which is tightly bound to the enzyme, and converting the enzyme from its active form (NAD⁺) to its inactive form (NADH) (Type I mechanism-based inhibitors; Wolfe and Borchardt, J. Med. Chem. 34, 1521–1530, 1991). More recently, substrates [e.g., (E)-5.,6′-didehydro-6′-deoxy-6′-fluorohomoadenosine, EDDFHA] for the hydrolytic activity of AdoHcy hydrolase have been identified by our laboratories. Identification of hydrolytic substrates affords a new strategy for the design of more potent and more specific inhibitors of AdoHcy hydrolase.     

The disturbance of hemostasis induced by hyperhomocysteinemia; the role of antioxidants

Elevated concentration of homocysteine (Hcy) in human tissues, definied as hyperhomocysteinemia has been correlated with some diseases, such as cardiovascular, neurodegenerative, and kidney disorders. Homocysteine occurs in human blood plasma in several forms, including the most reactive one, the homocysteine thiolactone (HTL) - a cyclic thioester, which represents up to 0.29% of total plasma Hcy. In the article, the effects of hyperhomocysteinemia on the complex process of hemostasis, which regulates the flowing properties of blood, are described. Possible interactions of homocysteine and its different derivatives, including homocysteine thiolactone, with the major components of hemostasis such as endothelial cells, blood platelets, plasmatic fibrinogen and plasminogen, are also discussed. Modifications of hemostatic proteins (N-homocysteinylation or S-homocysteinylation) induced by Hcy or its thiolactone seem to be the main cause of homocysteine biotoxicity in hemostatic abnormalities. It is suggested that Hcy and HTL may also act as oxidants, but various polyphenolic antioxidants are able to inhibit the oxidative damage induced by Hcy or HTL. We also discuss the role of phenolic antioxidants in hyperhomocysteinemia -induced changes in hemostasis.     

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.     

Matrix metalloproteinase-9 in homocysteine-induced intestinal microvascular endothelial paracellular and transcellular permeability

Although elevated levels of homocysteine (Hcy), known as hyperhomocysteinemia (HHcy), is associated with inflammatory bowel disease (IBD), the mechanism of Hcy action is unclear. In the present study, we tested the hypothesis that HHcy activates matrix metalloproteinase-9 (MMP-9), which in turn enhances permeability of human intestinal microvascular endothelial cell (HIMEC) layer by decreasing expression of endothelial junction proteins and increasing caveolae formation. HIMECs were grown in Transwells and treated with 500 μM Hcy in the presence or absence of MMP-9 activity inhibitor. Hcy-induced permeability to FITC-conjugated bovine serum albumin (FITC-BSA) was assessed by measuring fluorescence intensity of solutes in the Transwells' lower chambers. The cell-cell interaction and cell barrier function was estimated by measuring trans-endothelial electrical impedance. Confocal microscopy and flow cytometry were used to study cell junction protein expressions. Hcy-induced changes in transcellular transport of HIMECs were estimated by observing formation of functional caveolae defined as caveolae labeled by cholera toxin and antibody against caveolin-1 and one that have taken up FITC-BSA. Hcy instigated HIMEC monolayer permeability through activation of MMP-9. The increased paracellular permeability was associated with degradation of vascular endothelial cadherin and zona occludin-1 and transcellular permeability through increased caveolae formation in HIMECs. Elevation of Hcy content increases permeability of HIMEC layer affecting both paracellular and transcellular transport pathways, and this increased permeability was alleviated by inhibition of MMP-9 activity. These findings contribute to clarification of mechanisms of IBD development.     

Homocysteine induces VCAM-1 gene expression through NF-kappaB and NAD(P)H oxidase activation: protective role of Mediterranean diet polyphenolic antioxidants

Hyperhomocysteinemia is a recognized risk factor for vascular disease, but pathogenetic mechanisms involved in its vascular actions are largely unknown. Because VCAM-1 expression is crucial in monocyte adhesion and early atherogenesis, we evaluated the NF-kappaB-related induction of VCAM-1 by homocysteine (Hcy) and the possible inhibitory effect of dietary polyphenolic antioxidants, such as trans-resveratrol (RSV) and hydroxytyrosol (HT), which are known inhibitors of NF-kappaB-mediated VCAM-1 induction. In human umbilical vein endothelial cells (HUVEC), Hcy, at 100 micromol/l, but not cysteine, induced VCAM-1 expression at the protein and mRNA levels, as shown by enzyme immunoassay and Northern analysis, respectively. Transfection studies with deletional VCAM-1 promoter constructs demonstrated that the two tandem NF-kappaB motifs in the VCAM-1 promoter are necessary for Hcy-induced VCAM-1 gene expression. Hcy-induced NF-kappaB activation was confirmed by EMSA, as shown by the nuclear translocation of its p65 (RelA) subunit and the degradation of the inhibitors IkappaB-alpha and IkappaB-beta by Western analysis. Hcy also increased intracellular reactive oxygen species by NAD(P)H oxidase activation, as shown by the membrane translocation of its p47(phox) subunit. NF-kappaB inhibitors decreased Hcy-induced intracellular reactive oxygen species and VCAM-1 expression. Finally, we found that nutritionally relevant concentrations of RSV and HT, but not folate and vitamin B6, reduce (by >60% at 10(-6) mol/l) Hcy-induced VCAM-1 expression and monocytoid cell adhesion to the endothelium. These data indicate that pathophysiologically relevant Hcy concentrations induce VCAM-1 expression through a prooxidant mechanism involving NF-kappaB. Natural Mediterranean diet antioxidants can inhibit such activation, suggesting their possible therapeutic role in Hcy-induced vascular damage.     

Betaine-homocysteine methyltransferase: zinc in a distorted barrel

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.     

Metabolism and neurotoxicity of homocysteine thiolactone in mice: protective role of bleomycin hydrolase

Genetic or nutritional disorders in homocysteine (Hcy) metabolism elevate Hcy-thiolactone and cause heart and brain diseases. Hcy-thiolactone has been implicated in these diseases because it has the ability to modify protein lysine residues and generate toxic N-Hcy-proteins with auto-immunogenic, pro-thrombotic, and amyloidogenic properties. Bleomycin hydrolase (Blmh) has the ability to hydrolyze L-Hcy-thiolactone (but not D-Hcy-thiolactone) to Hcy in vitro, but whether this reflects a physiological function has been unknown. Here, we show that Blmh (-/-) mice excreted in urine 1.8-fold more Hcy-thiolactone than wild-type Blmh (+/+) animals (P = 0.02). Hcy-thiolactone was elevated 2.3-fold in brains (P = 0.004) and 2.0-fold in kidneys (P = 0.047) of Blmh (-/-) mice relative to Blmh (+/+) animals. Plasma N-Hcy-protein was elevated in Blmh (-/-) mice fed a normal (2.3-fold, P < 0.001) or hyperhomocysteinemic diet (1.5-fold, P < 0.001), compared with Blmh (+/+) animals. More intraperitoneally injected L-Hcy-thiolactone was recovered in plasma in Blmh (-/-) mice than in wild-type Blmh (+/+) animals (83.1 vs. 39.3 μM, P < 0.0001). In Blmh (+/+) mice injected intraperitoneally with D-Hcy-thiolactone, D,L-Hcy-thiolactone, or L-Hcy-thiolactone, 88, 47, or 6.3%, respectively, of the injected dose was recovered in plasma. The incidence of seizures induced by L-Hcy-thiolactone injections (3,700 nmol/g body weight) was higher in Blmh (-/-) than in Blmh (+/+) mice (93.8 vs. 29.5%, P < 0.001). Using the Blmh null mice, we provide the first direct evidence that a specific Hcy metabolite, Hcy-thiolactone, rather than Hcy itself, is neurotoxic in vivo. Taken together, our findings indicate that Blmh protects mice against L-Hcy-thiolactone toxicity by metabolizing it to Hcy and suggest a mechanism by which Blmh might protect against neurodegeneration associated with hyperhomocysteinemia and Alzheimer's disease.