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PubMed1993

Pentose-phosphate pathway in Saccharomyces cerevisiae: analysis of deletion mutants for transketolase, transaldolase, and glucose 6-phosphate dehydrogenase.

Deletion mutants for the yeast transketolase gene TKL1 were constructed by gene replacement. Transketolase activity was below the level of detection in mutant crude extracts. Transketolase protein could be detected as a single protein band of the expected size by Western-blot analysis in wild-type strains but not in the deletion mutant. Deletion of TKL1 led to a reduced but distinct growth in synthetic medium without an aromatic amino-acid supplement. We also isolated double and triple mutants for transketolase (tkl1), transaldolase (tal1), and glucose 6-phosphate dehydrogenase (zwf1) by crossing the different mutants. A tal1 tkl1 double mutant grew nearly like wild-type in rich medium. Only the tkl1 zwf1 double and the tal1 tkl1 zwf1 triple mutant grew more slowly than the wild-type in rich medium. This growth defect could be partly alleviated by the addition of xylulose but not ribose. The triple mutant still grew slowly on a synthetic mineral salts medium without a supplement of aromatic amino acids. This suggests the existence of an alternative but limited source of pentose phosphates and erythrose 4-phosphate in the tkl1 zwf1 double mutants. Hybridization with low stringency showed the existence of a sequence with homology to transketolase, possibly a second gene.

Base Sequence↗
PubMed2007

The Arxula adeninivorans ATAL gene encoding transaldolase-gene characterization and biotechnological exploitation.

The yeast Arxula adeninivorans provides an attractive expression platform and can be exploited as gene source for biotechnologically interesting proteins. In the following study, a striking example for the combination of both aspects is presented. The transaldolase-encoding A. adeninivorans ATAL gene, including its promoter and terminator elements, was isolated and characterized. The gene includes a coding sequence of 963 bp encoding a putative 321 amino acid protein of 35.0 kDa. The enzyme characteristics analyzed from isolates of native strains and recombinant strains overexpressing the ATAL gene revealed a molecular mass of ca. 140 kDa corresponding to a tetrameric structure, a pH optimum of ca. 5.5, and a temperature optimum of 20 degrees C. The preferred substrates for the enzyme include D-erythrose-4-phosphate and D-fructose-6-phosphate, whereas D-glyceraldehyde is not converted. The ATAL expression level under salt-free conditions was observed to increase in media supplemented with 5% NaCl rendering the ATAL promoter attractive for moderate heterologous gene expression under high-salt conditions. Its suitability was assessed for the expression of a human serum albumin (HSA) reporter gene.

Fungal Proteins↗
PubMed2006

Transaldolase deficiency: a new cause of hydrops fetalis and neonatal multi-organ disease.

Transaldolase (TALDO) deficiency is a newly recognized metabolic disease, which has been reported so far in 2 patients presenting with liver failure and cirrhosis. We report a new sibship of 4 infants born to the same consanguineous parents; all presented at birth or in the antenatal period with dysmorphic features, cutis laxa and hypertrichosis, hepatomegaly, splenomegaly, liver failure, hemolytic anemia, thrombocytopenia, and genitourinary malformations. The clinical courses were variable: the first child died of liver failure at 4 months of age; the second pregnancy was medically terminated at 28 weeks gestation because of hydrops fetalis with oligohydramnios. The third child is doing well at age 7 with liver fibrosis and mild kidney failure. The fourth child is now 21 months old and has hepatosplenomegaly, mild anemia, and thrombocytopenia. Urine assessment of polyols showed elevations of erythritol, arabitol, and ribitol consistent with TALDO deficiency. TALDO activity was undetectable in the patients' tissues, and mutation in the TALDO1 gene was found in the 4 patients.

Biomarkers↗
PubMed2005

Transaldolase is part of a supramolecular complex containing glucose-6-phosphate dehydrogenase in human neutrophils that undergoes retrograde trafficking during pregnancy.

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PDase) and 6-phosphogluconate dehydrogenase form a supramolecular complex in human neutrophils that undergoes retrograde trafficking in cells from pregnant women, but anterograde trafficking in cells from nonpregnant individuals. Using fluorescence resonance energy transfer techniques, we now demonstrate that transaldolase (TALase), a key regulatory enzyme in the nonoxidative branch of the hexose monophosphate shunt, is in close physical proximity with G6PDase, but not with lactate dehydrogenase, thus suggesting the formation of a TALase-G6PDase complex. Moreover, immunofluorescence microscopy demonstrated that TALase undergoes anterograde trafficking in neutrophils from nonpregnant individuals, whereas retrograde trafficking is found during pregnancy. However, pregnancy did not affect lactate dehydrogenase distribution. Colchicine treatment blocked the retrograde distribution of TALase, suggesting that microtubules are involved in TALase trafficking. We suggest that TALase is part of a supramolecular hexose monophosphate shunt complex, which likely increases the efficiency of the shunt via substrate channeling. We further suggest that TALase's retrograde motion contributes to uncoupling the shunt from its source of glucose-6-phosphate at the plasma membrane, thereby blunting nicotinamide adenine dinucleotide phosphate (reduced form) production and downstream oxidant production by neutrophils.

Cell Membrane↗
PubMed1998

Disruption of Escherichia coli transaldolase into catalytically active monomers: evidence against half-of-the-sites mechanism.

Disruption of the hydrogen bonding network at the interface of Escherichia coli transaldolase by substitution of R300 to a glutamic acid residue resulted in a monomeric enzyme at basic pH values, with almost no change in the kinetic parameters. The stability of the R300A and R300E mutants towards urea and thermal inactivation is similar to that of the wild-type enzyme. X-ray analysis showed that no structural changes occurred as a consequence of the side chain replacement. This indicates that the quaternary structure is not required for catalytic activity nor does it contribute significantly to the stability of the enzyme. The results are not consistent with a proposed half-of-the-sites reaction mechanism.

Amino Acid Substitution↗
PubMed2002

Characterization and tissue-specific expression of the Drosophila transaldolase gene.

Transaldolase (TAL) is an enzyme of the non-oxidative part of the pentose phosphate pathway which produces reductive potential in the form of NADPH as well as ribose 5-phosphate for incorporation into nucleotides. Developmental analysis via reverse transcriptase-polymerase chain reaction, immunoblots, enzymatic activity, in situ hybridization and immunocytochemistry demonstrate that TAL is expressed during all developmental stages in Drosophila. The tal locus is unique and contains two small introns. The first two introns in the human gene are situated at the same locations in the coding sequence as in Drosophila. Analysis of TAL protein levels and expression patterns reveals that it is also expressed in all larval tissues examined with particularly high levels in the fat body. Interestingly, we describe specific TAL expression only in non-neuronal cells in the larval brain.

Amino Acid Sequence↗
PubMed2001

Identification of catalytically important residues in the active site of Escherichia coli transaldolase.

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site-directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB-deficient host and analyzed with respect to their 3D structure and kinetic behavior. X-ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6-phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4-phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300-fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4-phosphate, suggesting a role of this residue in carbon-carbon bond cleavage and stabilization of the carbanion/enamine intermediate.

Alanine↗
PubMed2006

Transaldolase is essential for maintenance of the mitochondrial transmembrane potential and fertility of spermatozoa.

Fertility of spermatozoa depends on maintenance of the mitochondrial transmembrane potential (Deltapsi(m)), which is generated by the electron-transport chain and regulated by an oxidation-reduction equilibrium of reactive oxygen intermediates, pyridine nucleotides, and glutathione (GSH). Here, we report that male mice lacking transaldolase (TAL)(-/-) are sterile because of defective forward motility. TAL(-/-) spermatozoa show loss of Deltapsi(m) and mitochondrial membrane integrity because of diminished NADPH, NADH, and GSH. Mitochondria constitute major Ca(2+) stores; thus, diminished mitochondrial mass accounts for reduced Ca(2+) fluxing, defective forward motility, and infertility. Reduced forward progression of TAL-deficient spermatozoa is associated with diminished mitochondrial reactive oxygen intermediate production and Ca(2+) levels, intracellular acidosis, and compensatory down-regulation of carbonic anhydrase IV and overexpression of CD38 and gamma-glutamyl transferase. Microarray analyses of gene expression in the testis, caput, and cauda epididymidis of TAL(+/+), TAL(+/-), and TAL(-/-) littermates confirmed a dominant impact of TAL deficiency on late stages of sperm-cell development, affecting the electron-transport chain and GSH metabolism. Stimulation of de novo GSH synthesis by oral N-acetyl-cysteine normalized the low fertility rate of TAL(+/-) males without affecting the sterility of TAL(-/-) males. Whereas TAL(-/-) sperm failed to fertilize TAL(+/+) oocytes in vitro, sterility of TAL(-/-) sperm was circumvented by intracytoplasmic sperm injection, indicating that TAL deficiency influenced the structure and function of mitochondria without compromising the nucleus and DNA integrity. Collectively, these data reveal an essential role of TAL in sperm-cell mitochondrial function and, thus, male fertility.

Animals↗
PubMed1996

Glutathione levels and sensitivity to apoptosis are regulated by changes in transaldolase expression.

Transaldolase (TAL) is a key enzyme of the reversible nonoxidative branch of the pentose phosphate pathway (PPP) that is responsible for the generation of NADPH to maintain glutathione at a reduced state (GSH) and, thus, to protect cellular integrity from reactive oxygen intermediates (ROIs). Formation of ROIs have been implicated in certain types of apoptotic cell death. To evaluate the role of TAL in this process, Jurkat human T cells were permanently transfected with TAL expression vectors oriented in the sense or antisense direction. Overexpression of TAL resulted in a decrease in glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities and NADPH and GSH levels and rendered these cells highly susceptible to apoptosis induced by serum deprivation, hydrogen peroxide, nitric oxide, tumor necrosis factor-alpha, and anti-Fas monoclonal antibody. In addition, reduced levels of TAL resulted in increased glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase activities and increased GSH levels with inhibition of apoptosis in all five model systems. The effect of TAL expression on susceptibility to apoptosis through regulating the PPP and GSH production is consistent with an involvement of ROIs in each pathway tested. Production of ROIs in Fas-mediated cell death was further substantiated by measurement of intracellular ROI production with oxidation-sensitive fluorescent probes, by the protective effects of GSH precursor, N-acetyl cysteine, free radical spin traps 5,5-dimethyl-1-pyrroline-1-oxide and 3,3,5,5-tetramethyl-1-pyrroline-1-oxide, the antioxidants desferrioxamine, nordihydroguaiaretic acid, and Amytal, and by the enhancing effects of GSH depletion with buthionine sulfoximine. The results provide definitive evidence that TAL has a role in regulating the balance between the two branches of PPP and its overall output as measured by GSH production and thus influences sensitivity to cell death signals.

Apoptosis↗
PubMed1998

Molecular ordering in HIV-induced apoptosis. Oxidative stress, activation of caspases, and cell survival are regulated by transaldolase.

Dysregulated apoptosis may underlie the etiology of T cell depletion by human immunodeficiency virus type 1 (HIV-1). We show that HIV-induced apoptosis is preceded by an exponential increase in reactive oxygen intermediates (ROIs) produced in mitochondria. This leads to caspase-3 activation, phosphatidylserine (PS) externalization, and GSH depletion. Since mitochondrial ROI levels are regulated by the supply of NADPH from the pentose phosphate pathway (PPP), the effect of transaldolase (TAL), a key enzyme of PPP, was investigated. Jurkat and H9 human CD4+ T cells were transfected with TAL expression vectors oriented in the sense or antisense direction. TAL overexpression down-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. Alternatively, decreased TAL expression up-regulated glucose-6-phosphate dehydrogenase activities and GSH levels. HIV-induced 1) mitochondrial ROI production, 2) caspase-3 activation, 3) proteolysis of poly(ADP-ribose) polymerase, and 4) PS externalization were accelerated in cells overexpressing TAL. In contrast, suppression of TAL abrogated these four activities. Thus, susceptibility to HIV-induced apoptosis can be regulated by TAL through controlling the balance between mitochondrial ROI production and the metabolic supply of reducing equivalents by the PPP. The dominant effect of TAL expression on oxidative stress, caspase activation, PS externalization, and cell death suggests that this balance plays a pivotal role in HIV-induced apoptosis.

Apoptosis↗
PubMed1994

Oligodendrocyte-specific expression and autoantigenicity of transaldolase in multiple sclerosis.

Although the etiology of multiple sclerosis (MS) is unknown, there is compelling evidence that its pathogenesis is mediated through the immune system. Molecular mimicry, i.e., crossreactivity between self-antigens and viral proteins, has been implicated in the initiation of autoimmunity and MS. Based on homology to human T cell lymphotropic virus type I (HTLV-I) a novel human retrotransposon was cloned and found to constitute an integral part of the coding sequence of the human transaldolase gene (TAL-H). TAL-H is a key enzyme of the nonoxidative pentose phosphate pathway (PPP) providing ribose-5-phosphate for nucleic acid synthesis and NADPH for lipid biosynthesis. Another fundamental function of the PPP is to maintain glutathione at a reduced state and, consequently, to protect sulfhydryl groups and cellular integrity from oxygen radicals. Immunohistochemical analyses of human brain sections and primary murine brain cell cultures demonstrated that TAL is expressed selectively in oligodendrocytes at high levels, possibly linked to production of large amounts of lipids as a major component of myelin, and to the protection of the vast network of myelin sheaths from oxygen radicals. High-affinity autoantibodies to recombinant TAL-H were detected in serum (25/87) and cerebrospinal fluid (15/20) of patients with MS. By contrast, TAL-H antibodies were absent in 145 normal individuals and patients with other autoimmune and neurological diseases. In addition, recombinant TAL-H stimulated proliferation and caused aggregate formation of peripheral blood lymphocytes from patients with MS. Remarkable amino acid sequence homologies were noted between TAL-H and core proteins of human retroviruses. Presence of crossreactive antigenic epitopes between recombinant TAL-H and HTLV-I/human immunodeficiency virus type 1 (HIV-1) gas proteins was demonstrated by Western blot analysis. The results suggest that molecular mimicry between viral core proteins and TAL-H may play a role in breaking immunological tolerance and leading to a selective destruction of oligodendrocytes in MS.

Adult↗
PubMed1997

Comparative analysis of antibody and cell-mediated autoimmunity to transaldolase and myelin basic protein in patients with multiple sclerosis.

Antibody and T cell-mediated immune responses to oligodendroglial autoantigens transaldolase (TAL) and myelin basic protein (MBP) were examined in patients with multiple sclerosis (MS). Immunohistochemical studies of postmortem brain sections revealed decreased staining by MBP- and TAL-specific antibodies in MS plaques, indicating a concurrent loss of these antigens from demyelination sites. By Western blot high titer antibodies to human recombinant TAL were found in 29/94 sera and 16/23 cerebrospinal fluid samples from MS patients. Antibodies to MBP were undetectable in sera or cerebrospinal fluid of these MS patients. Proliferative responses to human recombinant TAL (stimulation index [SI] = 2.47+/-0.3) were significantly increased in comparison to MBP in 25 patients with MS (SI = 1.37+/-0.1; P < 0.01). After a 7-d stimulation of PBL, utilization of any of 24 different T cell receptor Vbeta gene segments in response to MBP was increased less than twofold in the two control donors and six MS patients investigated. In response to TAL-H, while skewing of individual Vbeta genes was also less than twofold in healthy controls, usage of specific Vbeta gene segments was differentially increased ranging from 2.5 to 65.9-fold in patients with MS. The results suggest that TAL may be a more potent immunogen than MBP in MS.

Adult↗
PubMed2003

Profiling of pentose phosphate pathway intermediates in blood spots by tandem mass spectrometry: application to transaldolase deficiency.

BACKGROUND: Recently, several patients with abnormal polyol profiles in body fluids have been reported, but the origins of these polyols are unknown. We hypothesized that they are derived from sugar phosphate intermediates of the pentose phosphate pathway (PPP), and we developed a semiquantitative method for profiling of pentose phosphate pathway intermediates. METHODS: Sugar phosphates in blood spots were simultaneously analyzed by liquid chromatography-tandem mass spectrometry using an ion-pair-loaded C(18) HPLC column. The tandem mass spectrometer was operated in the multiple-reaction monitoring mode. Enzymatically prepared D-[(13)C(6)]glucose 6-phosphate was used as internal standard. The method was used to study sugar phosphates abnormalities in a patient affected with a deficiency of transaldolase (TALDO1; EC 2.2.1.2). RESULTS: In control blood spots, dihydroxyacetone phosphate, pentulose 5-phosphates, pentose 5-phosphates, hexose 6-phosphates, and sedoheptulose 7-phosphate were detected. Detection limits ranged from approximately 100 to approximately 500 nmol/L. Glyceraldehyde 3-phosphate and erythrose 4-phosphate were undetectable. Intra- and interassay imprecision (CVs) were 10-17% and 12-21%, respectively. In blood from the TALDO1-deficient patient, sedoheptulose 7-phosphate was increased. CONCLUSIONS: The new method allows investigation of patients in whom a defect in the PPP is suspected. Measurements of sugar phosphate intermediates of the PPP may provide new insights into metabolic defects underlying the accumulating polyols.

Adolescent↗
PubMed2005

CD8+ T cell-mediated HLA-A*0201-restricted cytotoxicity to transaldolase peptide 168-176 in patients with multiple sclerosis.

Transaldolase (TAL) is expressed at selectively high levels in oligodendrocytes and targeted by autoreactive T cells of patients with multiple sclerosis (MS). Among 14 TAL peptides with predicted HLA-A2 binding, TAL 168-176 (LLFSFAQAV, TALpep) exhibited high affinity for HLA-A2. Prevalence of HLA-A2-restricted CD8+ T cells specific for TALpep was increased in PBMC of HLA-A2+ MS patients, as compared with HLA-A2- MS patients, HLA-A2+ other neurological disease patients, and HLA-A2+ healthy donors. HLA-A*0201/TALpep tetramers detected increased frequency of TAL-specific CD8+ T cells, and precursor frequency of TAL-specific IFN-gamma-producing T cells was increased in each of seven HLA-A2+ MS patients tested. Stimulation by TALpep or rTAL of PBMC from HLA-A2+ MS patients elicited killing of TALpep-pulsed HLA-A2-transfected HmyA2.1 lymphoma cells, but not HLA-A3-transfected control HmyA3.1 targets. Without peptide pulsing of targets, HLA-A2-transfected, but not control MO3.13 oligodendroglial cells, expressing high levels of endogenous TAL, were also killed by CD8+ CTL of MS patients, indicating recognition of endogenously processed TAL. TCR Vbeta repertoire analysis revealed use of the TCR Vbeta14 gene by T cell lines (TCL) of MS patients generated via stimulation by TAL- or TALpep-pulsed APCs. All TAL-specific TCL-binding HLA-A*0201/TALpep tetramers expressed TCR Vbeta14 on the cell surface. Moreover, Ab to TCR Vbeta14 abrogated cytotoxicity by HLA-A2-restricted TAL-specific TCL. Therefore, TAL-specific CTL may serve as a novel target for therapeutic intervention in patients with MS.

Adult↗
PubMed1982

[Changes in the transaldolase activity in the liver, spleen and bone marrow of rats after total X-ray irradiation].

The paper deals with changes in the activity of transaldolase in tissues of the liver, spleen and bone marrow of rats 4.24 h and on the 3 d, 7th, 18th, 30th, 45th, and 60th day after total X-ray irradiation in a dose of 600 R. The ionizing irradiation causes a stable inhibition of the enzyme activity in All tissues. The observed changes are homogeneous in their direction but have their own peculiarities for each tissue. Differences in the degree and extent of the enzyme activity changes at various stages of the X-ray lesion development in the tissues under study may be explained by their different sensitivity to the penetrating radiation.

Animals↗
PubMed1996

Crystallization and preliminary X-ray crystallographic analysis of recombinant transaldolase B from Eschericha coli.

Recombinant transaldolase from Escherichia coli, an enzyme of the pentose phosphate pathway has been crystallized by the vapor-diffusion method using polyethylene glycol 6000 as precipitant. The crystals are orthorhombic, space group P2(1)2(1)2(1) with cell dimensions a = 68.9, b = 91.3 and c = 130.5 A and diffract to 2 A resolution on a conventional X-ray source. The asymmetric unit very likely contains two subunits, corresponding to a packing density of 2.9 A(3) Da(-1).

Journal Article↗