Increased Urinary 3-Mercaptolactate Excretion and Enhanced Passive Systemic Anaphylaxis in Mice Lacking Mercaptopyruvate Sulfurtransferase, a Model of Mercaptolactate-Cysteine Disulfiduria.

Mercaptopyruvate sulfurtransferase (Mpst) and its homolog thiosulfate sulfurtransferase (Tst = rhodanese) detoxify cyanide to thiocyanate. Mpst is attracting attention as one of the four endogenous hydrogen sulfide (H2S)/reactive sulfur species (RSS)-producing enzymes, along with cystathionine ß-synthase (Cbs), cystathionine γ-lyase (Cth), and cysteinyl-tRNA synthetase 2 (Cars2). MPST deficiency was found in 1960s among rare hereditary mercaptolactate-cysteine disulfiduria patients. Mpst-knockout (KO) mice with enhanced liver Tst expression were recently generated as its model; however, the physiological roles/significances of Mpst remain largely unknown. Here we generated three independent germ lines of Mpst-KO mice by CRISPR/Cas9 technology, all of which maintained normal hepatic Tst expression/activity. Mpst/Cth-double knockout (DKO) mice were generated via crossbreeding with our previously generated Cth-KO mice. Mpst-KO mice were born at the expected frequency and developed normally like Cth-KO mice, but displayed increased urinary 3-mercaptolactate excretion and enhanced passive systemic anaphylactic responses when compared to wild-type or Cth-KO mice. Mpst/Cth-DKO mice were also born at the expected frequency and developed normally, but excreted slightly more 3-mercaptolactate in urine compared to Mpst-KO or Cth-KO mice. Our Mpst-KO, Cth-KO, and Mpst/Cth-DKO mice, unlike semi-lethal Cbs-KO mice and lethal Cars2-KO mice, are useful tools for analyzing the unknown physiological roles of endogenous H2S/RSS production.

The Clinical and Molecular Characteristics of Molybdenum Cofactor Deficiency Due to MOCS2 Mutations.

BACKGROUND: We explored the clinical and molecular characteristics of molybdenum cofactor deficiency due to MOCS2 muations. METHODS: We summarize the genetic and clinical findings of previously reported patients with a MOCS2 mutation. We also present a new patient with novel neuroradiological findings associated with molybdenum cofactor deficiency due to a novel homozygous variant in the 5' untranslated region of the MOCS2 gene. RESULTS: The study population comprised 35 patients with a MOCS2 gene mutation. All reported children had delayed motor milestones. The major initial symptom was seizures in neonatal period. Facial dysmorphism was present in 61% of the patients. Only one patient had ectopia lentis. Agenesis of the corpus callosum and an associated interhemispheric cyst in our case are novel neuroradiological findings. CONCLUSIONS: The occurrence of neonatal seizures and feeding difficulties can be the first clinical signs of molybdenum cofactor deficiency. Although there is no effective therapy for this condition, early diagnosis and genetic analysis of these lethal disorders facilitate adequate genetic counseling.

Effect of 3-mercaptopyruvate Sulfurtransferase Deficiency on the Development of Multiorgan Failure, Inflammation, and Wound Healing in Mice Subjected to Burn Injury.

The gaseous transmitter hydrogen sulfide (H2S) has been implicated in various forms of critical illness. Here, we have compared the outcome of scald burn injury in wild-type mice and in mice deficient in 3-mercaptopyruvate sulfurtransferase (3-MST), a mammalian H2S-generating enzyme. Outcome variables included indices of organ injury, clinical chemistry parameters, and plasma levels of inflammatory mediators. Plasma levels of H2S significantly increased in response to burn in wild-type mice, but remained unchanged in 3-MST-/- mice. The capacity of tissue homogenates to produce H2S from 3-mercaptopyruvate was unaffected by burn injury. In 3-MST-/- mice, compared to wild-type controls, there was a significant enhancement in the accumulation of polymorphonuclear cells (as assessed by the quantification of myeloperoxidase) in the liver (but not heart, lung, or skin) at 7 days postburn. Oxidative tissue damage (as assessed by malon dialdehyde content) was comparable between wild-type and 3-MST-deficient mice in all tissues studied. 3-MST-/- and wild-type mice exhibited comparable burn-induced elevations in circulating plasma levels of hepatic injury; however, 3-MST-/- mice exhibited a higher degree of renal injury (as reflected by elevated blood urea nitrogen levels) at 7 days postburn. Inflammatory mediators (eg, TNF-α, IL-1ß, IL-2, IL-6, IL-10, and IL-12) increased in burn injury, but without significant differences between the 3-MST-/- and wild-type groups. The healing of the burn wound was also unaffected by 3-MST deficiency. In conclusion, the absence of the H2S-producing enzyme 3-MST slightly exacerbates the development of multiorgan dysfunction but does not affect inflammatory mediator production or wound healing in a murine model of burn injury.

Effect of endotoxemia in mice genetically deficient in cystathionine-γ-lyase, cystathionine-ß-synthase or 3-mercaptopyruvate sulfurtransferase.

Hydrogen sulfide (H2S) has been proposed to exert pro- as well as anti-inflammatory effects in various models of critical illness. In this study, we compared bacterial lipopolysaccharide (LPS)­induced changes in inflammatory mediator production, indices of multiple organ injury and survival in wild­type (WT) mice and in mice with reduced expression of one of the three H2S­producing enzymes, cystathionine-γ-lyase (CSE), cystathionine-ß-synthase (CBS) or 3-mercaptopyruvate sulfurtransferase (3MST). Mice were injected intraperitoneally (i.p.) with LPS (10 mg/kg). After 6 h, the animals were sacrificed, blood and organs were collected and the following parameters were evaluated: blood urea nitrogen (BUN) levels in blood, myeloperoxidase (MPO) and malondialdehyde (MDA) in the lung, cytokine levels in plasma and the expression of the three H2S­producing enzymes (CBS, CSE and 3MST) in the spleen, lung, liver and kidney. LPS induced a tissue­dependent upregulation of some of the H2S­producing enzymes in WT mice (upregulation of CBS in the spleen, upregulation of 3MST in the liver and upregulation of CBS, CSE and 3MST in the lung). Moreover, LPS impaired glomerular function, as evidenced by increased BUN levels. Renal impairment was comparable in the CSE­/­ and Δ3MST mice after LPS challenge; however, it was attenuated in the CBS+/­ mice. MPO levels (an index of neutrophil infiltration) and MDA levels (an index of oxidative stress) in lung homogenates were significantly increased in response to LPS; these effects were similar in the WT, CBS+/­, CSE­/­ and Δ3MST mice; however, the MDA levels tended to be lower in the CBS+/­ and CSE­/­ mice. LPS induced significant increases in the plasma levels of multiple cytokines [tumor necrosis factor (TNF)α, interleukin (IL)­1ß, IL­6, IL­10, IL­12 and interferon (IFN)γ] in plasma; TNFα, IL­10 and IL­12 levels tended to be lower in all three groups of animals expressing lower levels of H2S­producing enzymes. The survival rates after the LPS challenge did not show any significant differences between the four animal groups tested. Thus, the findings of this study indicate that a deficiency in 3MST does not significantly affect endotoxemia, while a deficiency in CBS or CSE slightly ameliorates the outcome of LPS-induced endotoxemia in vivo.

Reduced alpha-lipoic acid synthase gene expression exacerbates atherosclerosis in diabetic apolipoprotein E-deficient mice.

OBJECTIVES: To study the effects of reduced lipoic acid gene expression on diabetic atherosclerosis in apolipoprotein E null mice (Apoe(-/-)). METHODS AND RESULTS: Heterozygous lipoic acid synthase gene knockout mice (Lias(+/-)) crossed with Apoe(-/-) mice were used to evaluate the diabetic effect induced by streptozotocin on atherosclerosis in the aortic sinus of the heart. While diabetes markedly increased atherosclerotic plaque size in Apoe(-/-) mice, a small but significant effect of reduced expression of lipoic acid gene was observed in diabetic Lias(+/-)Apoe(-/-) mice. In the aortic lesion area, the Lias(+/-)Apoe(-/-) mice exhibited significantly increased macrophage accumulation and cellular apoptosis than diabetic Lias(+/+)Apoe(-/-) littermates. Plasma glucose, cholesterol, and interleukin-6 were also higher. These abnormalities were accompanied with increased oxidative stress including a decreased ratio of reduced glutathione/oxidized glutathione in erythrocytes, increased systemic lipid peroxidation, and increased Gpx1 and MCP1 gene expression in the aorta. CONCLUSIONS: Decreased endogenous lipoic acid gene expression plays a role in development of diabetic atherosclerosis. These findings extend our understanding of the role of antioxidant in diabetic atherosclerosis.

Cell biology of molybdenum in plants and humans.

The transition element molybdenum (Mo) needs to be complexed by a special cofactor in order to gain catalytic activity. With the exception of bacterial Mo-nitrogenase, where Mo is a constituent of the FeMo-cofactor, Mo is bound to a pterin, thus forming the molybdenum cofactor Moco, which in different variants is the active compound at the catalytic site of all other Mo-containing enzymes. In eukaryotes, the most prominent Mo-enzymes are nitrate reductase, sulfite oxidase, xanthine dehydrogenase, aldehyde oxidase, and the mitochondrial amidoxime reductase. The biosynthesis of Moco involves the complex interaction of six proteins and is a process of four steps, which also requires iron, ATP and copper. After its synthesis, Moco is distributed to the apoproteins of Mo-enzymes by Moco-carrier/binding proteins. A deficiency in the biosynthesis of Moco has lethal consequences for the respective organisms. In humans, Moco deficiency is a severe inherited inborn error in metabolism resulting in severe neurodegeneration in newborns and causing early childhood death. This article is part of a Special Issue entitled: Cell Biology of Metals.

Lipoic acid synthetase deficiency causes neonatal-onset epilepsy, defective mitochondrial energy metabolism, and glycine elevation.

Lipoic acid is an essential prosthetic group of four mitochondrial enzymes involved in the oxidative decarboxylation of pyruvate, α-ketoglutarate, and branched chain amino acids and in the glycine cleavage. Lipoic acid is synthesized stepwise within mitochondria through a process that includes lipoic acid synthetase. We identified the homozygous mutation c.746G>A (p.Arg249His) in LIAS in an individual with neonatal-onset epilepsy, muscular hypotonia, lactic acidosis, and elevated glycine concentration in plasma and urine. Investigation of the mitochondrial energy metabolism showed reduced oxidation of pyruvate and decreased pyruvate dehydrogenase complex activity. A pronounced reduction of the prosthetic group lipoamide was found in lipoylated proteins.

Lipoic acid synthase (LASY): a novel role in inflammation, mitochondrial function, and insulin resistance.

OBJECTIVE: Lipoic acid synthase (LASY) is the enzyme that is involved in the endogenous synthesis of lipoic acid, a potent mitochondrial antioxidant. The aim of this study was to study the role of LASY in type 2 diabetes. RESEARCH DESIGN AND METHODS: We studied expression of LASY in animal models of type 2 diabetes. We also looked at regulation of LASY in vitro under conditions that exist in diabetes. Additionally, we looked at effects of LASY knockdown on cellular antioxidant status, inflammation, mitochondrial function, and insulin-stimulated glucose uptake. RESULTS: LASY expression is significantly reduced in tissues from animal models of diabetes and obesity compared with age- and sex-matched controls. In vitro, LASY mRNA levels were decreased by the proinflammatory cytokine tumor necrosis factor (TNF)-alpha and high glucose. Downregulation of the LASY gene by RNA interference (RNAi) reduced endogenous levels of lipoic acid, and the activities of critical components of the antioxidant defense network, increasing oxidative stress. Treatment with exogenous lipoic acid compensated for some of these defects. RNAi-mediated downregulation of LASY induced a significant loss of mitochondrial membrane potential and decreased insulin-stimulated glucose uptake in skeletal muscle cells. In endothelial cells, downregulation of LASY aggravated the inflammatory response that manifested as an increase in both basal and TNF-alpha-induced expression of the proinflammatory cytokine, monocyte chemoattractant protein-1 (MCP-1). Overexpression of the LASY gene ameliorated the inflammatory response. CONCLUSIONS: Deficiency of LASY results in an overall disturbance in the antioxidant defense network, leading to increased inflammation, insulin resistance, and mitochondrial dysfunction.

Evidence for a functional genetic polymorphism of the human mercaptopyruvate sulfurtransferase (MPST), a cyanide detoxification enzyme.

Mercaptopyruvate sulfurtransferase (MPST) plays a central role in both cysteine degradation and cyanide detoxification. Moreover, deficiency in MPST activity has been suggested to be responsible for a rare inheritable disorder known as mercaptolactate-cysteine disulfiduria (MCDU). To date, no mutation of the human MPST gene has been reported. We developed a screening strategy to search for mutations in the MPST gene of 50 unrelated French individuals. Two intronic polymorphisms (IVS1-110C>G and IVS2+39C>T) and a nonsense mutation (Tyr(85)Stop) were identified and their functional consequences were assessed in vivo by measurement of erythrocyte MPST activity and/or in vitro using heterologous expression or transient transfection assay. The nonsense mutation likely leads to the synthesis of a severely truncated protein without enzymatic activity, as supported by our in vitro data. This work constitutes the first report of the existence of a functional genetic polymorphism affecting MPST and should be of great help to investigate certain disorders such as MCDU.

Mutation of human molybdenum cofactor sulfurase gene is responsible for classical xanthinuria type II.

Drosophila ma-l gene was suggested to encode an enzyme for sulfuration of the desulfo molybdenum cofactor for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO). The human molybdenum cofactor sulfurase (HMCS) gene, the human ma-l homologue, is therefore a candidate gene responsible for classical xanthinuria type II, which involves both XDH and AO deficiencies. However, HMCS has not been identified as yet. In this study, we cloned the HMCS gene from a cDNA library prepared from liver. In two independent patients with classical xanthinuria type II, we identified a C to T base substitution at nucleotide 1255 in the HMCS gene that should cause a CGA (Arg) to TGA (Ter) nonsense substitution at codon 419. A classical xanthinuria type I patient and healthy volunteers lacked this mutation. These results indicate that a functional defect of the HMCS gene is responsible for classical xanthinuria type II, and that HMCS protein functions to provide a sulfur atom for the molybdenum cofactor of XDH and AO.

Selective impairment of the synthesis of basic fibroblast growth factor binding domains of heparan sulphate in a COS cell mutant defective in N-sulphotransferase.

N-Sulphation is a key step in the overall sulphation of heparan sulphate. We have isolated a COS cell-derived mutant, CM-15, that is impaired in its ability to bind to basic fibroblast growth factor (bFGF) and has a 2- to 3-fold reduction in N-sulphotransferase activity [Ishihara et al., (1992a) Anal. Biochem., 206, 400-407]. We now provide structural evidence that CM-15 is selectively impaired in the synthesis of highly sulphated regions or 'blocks' that display high-affinity binding to bFGF; these are completely N-sulphated blocks of decasaccharide or greater length that are enriched in O-sulphate groups. The synthesis of sulphated blocks that did not show high affinity to the growth factor was relatively unimpaired in the mutant cells; this included fully N-sulphated octamer (or smaller) blocks and, unexpectedly, decasaccharide or larger blocks that were poorly O-sulphated. In the latter fraction, the failure to form high-affinity binding regions was the result of a failure to stimulate O-sulphation rather than N-sulphation in CM-15 cells. In agreement with other studies, disaccharide analysis of the wild-type-derived sulphated blocks suggested that 2-O-sulphation of iduronate residues in the polymer was a necessary element to produce a high-affinity binding sequence once N-sulphation was completed in the decasaccharide or larger fraction. These results suggest that a selective reduction in both N- and O-sulphation in the larger blocks produced by CM-15 cells is a consequence of the reduction of N-sulphotransferase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Hyperketotic states due to inherited defects of ketolysis.

From the description of 2 unrelated patients with succinyl-CoA transferase (3-OAT) deficiency and 1 patient with acetoacetyl-CoA thiolase (AAT) deficiency, we have attempted to draw the clinical and metabolic consequences of such defects. The association of recurrent attacks of severe ketoacidosis with blood glucose levels generally high or normal, low lactacidemia and low ammonemia is the most common presentation of these disorders. In 3-OAT deficiency, a potentially fatal disorder, there is a permanent ketosis with the only excretion of 3-hydroxybutyrate, acetoacetate and 3-hydroxyisovalerate. AAT patients usually excrete, in addition to the usual ketone bodies, 2-methyl-3-hydroxybutyrate and tiglylglycine; 2-methyl-acetoacetate may also be present. Both conditions can be identified by enzymatic analysis in cultured fibroblast. These disorders can mimic diabetic ketoacidosis or salicylism and can easily be missed. The knowledge of these ketolytic defects must severely question the complacent diagnosis of 'fasting ketoacidosis' or 'idiopathic ketotic hypoglycemia', mainly when severe metabolic acidosis is present.

Deficiency of thiosulphate sulphurtransferase (rhodanese) in Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a rare cause of progressive visual failure. Its cause is unknown, but one hypothesis is that patients have a defect in the detoxication of cyanide. One of the enzymes used in this detoxication is thiosulphate sulphurtransferase (rhodanese). The activity of this enzyme was measured in the rectal mucosa of a group of subjects with Leber's hereditary optic neuropathy, and it was found to be considerably reduced compared with that in a group of controls (p less than 0.001). This finding supports the hypothesis of an inborn error of cyanide detoxication in this condition.

Decreased thiosulfate sulfur transferase (rhodanese) in Leber's hereditary optic atrophy.

In mammals the major portion of cyanide is converted to thiocyanate by the liver enzyme thiosulfate sulfur transferase (TST) (rhodanese). We have found a much reduced activity of this enzyme in liver biopsies from two affected males of a family with Leber's hereditary optic atrophy and in two isolated cases of the same disease, (compared to liver biopsies from controls or liver samples obtained at autopsy). In one of the patients we studied the effect of a 3-day thiosulfate infusion. The urinary excretion of thiocyanate which was low prior to the infusion was raised during the thiosulfate treatment; in a healthy control person the same thiosulfate infusion did not alter the thiocyanate excretion rate. This suggests that cyanide detoxification which is suboptimal in patients with Leber's disease may be increased by thiosulfate infusion.

[Sulfated macromolecules in connective tissue and their metabolism]. / Sulfatierte Makromoleküle des Bindegewebes und ihr Stoffwechsel.

Sulfated macromolecules are one of the major constituents of the extracellular matrix of connective tissue. The degree of sulfation is responsible for the physico-chemical properties of the extracellular matrix. Especially the fixed charged groups contribute to the reversible compressibility of the cartilage. The sulfation may play also a role in the regulation of metabolic pathways. The derivatives of sulfate are formed by the help of specific sulfotransferases, transferring the "active" sulfate group from PAPS to the macromolecules. Too low an activity of certain sulfotransferases is responsible for some types of spondyloepiphyseal dysplasia, and too low a concentration of PAPS leads to dwarfism. The sulfate groups of the macromolecules are degraded by specific sulfatases. A genetically caused deficiency in one of these enzymes (mucopolysaccharidoses) is frequently associated with skeletal deformities. The amount and composition of proteoglycans is altered in the ontogenesis. Similar alterations are associated with ageing and arthrotic processes, being difficult to differentiate. The characterization of the alterations in degenerative joint diseases, especially, is important for ethiopathogenetic studies. The most widely used methods for the determination of sulfated macromolecules and particularly analytical parameters of the sulfate metabolism are described.