定点诱变G6PD基因的表达及突变酶的生物学功能研究

首次将9种人工定点诱变的G6PD基因转化至G6PD缺陷的大肠杆菌HB351(DE3)中表达,并对突变酶的生物学功能进行研究。初步证实G6PD基因m1376 G to T(Arg 459keu),1388 G to A(Arg 463 His)突变可降低酶活性并引起酶动力学改变。这可能与取代氨基酸的化学结构、所带电荷的性质及极性有关。这两个部位的精氨酸在酶与NADP~ 的结合过程中亦起到重要作用。赖氨酸取代精氨酸对酶与NADP~ 的结合影响不大。引入无义突变,证实G6PD第459位以后的氨基酸对酶活性有重要影响。     

Thrombophilic gene polymorphism studies in G6PD deficient individuals from Saudi population

We performed a study to evaluate the role of three single nucleotide polymorphisms (SNPs), factor V Leiden G1691A (FVL), prothrombin gene mutation G20210A (PRT or FII-G20210A) and methylenotetrahydrofolate reductase variant C677T (MTHFRC677T), as risk factors for G6PD in Saudi populations. Our results did not show any association with the three Thrombophilic genes with FVL gene, no statistical analysis have shown any association with either allele or genotype frequencies OR=0.566, p=.0.667, (95% CI=0.014-22.48) and OR=0.569, p=0.251¸ (95% CI=0.014-22.96).In PRT gene G20210A for G Vs A, p=0.774; OR=0.566 (95%CI; 0.011-29.6); AA+GA Vs GG; p=0.502; OR=0.569 (95%CI=0.010-2969). G and A allele frequencies were similar between cases and controls with no statistical significance. In the MTHFR gene none of the genotypes or allele frequency cannot show any association OR=1.281, p=.0.667, (95% CI=0.414-3.958) and OR=1.1.172, p=0.800¸ (95% CI=0.343-4.008). Similarly, the difference of T allele frequencies between patients and controls was not found any association. In conclusion, our finding indicates that the prevalence of G1691A, G20210A and C677T mutations in G6PD deficient individuals is not statistically different compared to normal subjects and G6PD is not associated with these thrombophilic mutations in Saudi population.     

Analysis of G6PD enzyme deficiency in Saudi population

The evolutionary conservation of a housekeeping gene such as G6PD is greater than that of tissue-specific genes, presumably because the latter may require more specific adaptation to the physiology of individual organisms. The abundance of distinct mutation sites and their clinical manifestations make G6PD ideal for structure-function analysis. Therefore, it is of interest to screen of G6PD deficiency in the blood donors in Kingdom of Saudi Arabia. We report the mean and variation of enzyme activity in a huge set of Suadi to non-Saudi population with reference to the entire population. The sequence level conservation of G6PD among distant species is demonstrated using phylogenetic trees. These observations have implications in the sequence-structure-function understanding of G6PD with reference to its association to several human diseases.     

Glucose 6-phosphate dehydrogenase activity in membranes of erythrocytes from normal individuals and subjects with Mediterranean G6PD deficiency

Unsealed, hemoglobin-free erythrocyte ghosts contain low yet significant levels of Glucose 6-phosphate dehydrogenase (G6PD) activity. This activity is comparable in erythrocyte ghosts obtained from normal individuals and from G6PD-deficient subjects (of Mediterranean type), in spite of the marked differences found in the corresponding cytosolic compartments. The membrane preparations can bind purified human G6PD (type B) to their cytoplasmic surface according to patterns of positive cooperativity. 2.4 × 10⁴ and 1.6 × 10⁴ G6PD-binding sites are present on the inner surface of each ghost obtained from normal and from G6PD-deficient erythrocytes, respectively, the relevant association constants being 2.8 × 10⁶ M^?1 and 0.82 × 10⁶ M^?1. The interaction of G6PD with the ghosts is unaffected by different ionic strengths or by metabolites such as glucose 6-phosphate, NADP and NADPH.     

Molecular analysis of three novel G6PD variants: G6PD Pedoplis-Ckaro, G6PD Piotrkow and G6PD Krakow

We present three novel mutations in the G6PD gene and discuss the changes they cause in the 3-dimensional structure of the enzyme: 573C-->G substitution that predicts Phe to Leu at position 191 in the C-terminus of helix alphae, 851T-->C mutation which results in the substitution 284Val--> -->Ala in the beta+alpha domain close to the C-terminal part of helix alphaj, and 1175T-->C substitution that predicts Ile to Thr change at position 392.     

Knockdown of glucose-6-phosphate dehydrogenase (G6PD) following cerebral ischemic reperfusion: the pros and cons

NADPH derived from glucose-6-phosphate dehydrogenase (G6PD), the rate-limiting enzyme of the pentose phosphate pathway, has been implicated not only to promote reduced glutathione (GSH) but also enhance oxidative stress in specific cellular conditions. In this study, the effects of G6PD antisense oligodeoxynucleotides (AS-ODNs) was examined on the CA1 pyramidal neurons following transient cerebral ischemia. Specifically knockdown of G6PD protein expression in hippocampus CA1 subregion at early reperfusion period (1-24 h) with a strategy to pre-treated G6PD AS-ODNs significantly reduced G6PD activity and NADPH level, an effect correlated with attenuation of NADPH oxidase activation and superoxide anion production. Concomitantly, pre-treatment of G6PD AS-ODNs markedly reduced oxidative DNA damage and the delayed neuronal cell death in rat hippocampal CA1 region induced by global cerebral ischemia. By contrast, knockdown of G6PD protein at late reperfusion period (48-96 h) increased oxidative DNA damage and exacerbated the ischemia-induced neuronal cell death in hippocampal CA1 region, an effect associated with reduced NADPH level and GSH/GSSG ratio. These findings indicate that G6PD not only plays a role in oxidative neuronal damage but also a neuroprotective role during different ischemic reperfusion period. Therefore, G6PD mediated oxidative response and redox regulation in the hippocampal CA1 act as the two sides of the same coin and may represent two potential applications of G6PD during different stage of cerebral ischemic reperfusion.     

Maintaining Specimen Integrity for G6PD Screening by Cytofluorometric Assays

Cytochemical staining remains an efficient way of identifying females who are heterozygous for the X chromosome-linked glucose-6-phosphate dehydrogenase (G6PD) gene. G6PD is highly polymorphic with certain alleles resulting in low intracellular G6PD activity in red blood cells. Low intracellular G6PD activity is associated with a risk of severe hemolysis when exposed to an oxidative stress such as fava beans, certain drugs and infections. Heterozygous females express the enzyme from both X-chromosome alleles resulting in two red blood cell populations each with G6PD enzyme characteristics representative of each allele; for example, normal and deficient. Cytochemical staining is the only way to determine the relative representation of each allele in red blood cells, a feature that is critical when assessing the risk for severe hemolysis when exposed to an oxidant such as the anti-malarial drug primaquine. This letter discusses red blood cell integrity with respect to the cytofluorometric assays for G6PD activity. An approach to making this test more robust is suggested. The approach makes this test more reliable and extends its use to a broader range of blood specimens.     

Primary cultures of hepatocytes in serum and hormone-free medium: Identification of conditions which stimulate an in vivo-like induction of G6PD

Summary Recent results from several laboratories suggest that complex interactions between hormones and dietary carbohydrate may be responsible for regulating the induction of several hepatic lipogenic enzymes. Elucidation of these interactions requires the ability to culture hepatocytes for several days in serum-free medium where the hormones or carbohydrate or both present is strictly controlled. The functional response of primary adult rat hepatocytes was examined in a medium without exposure to serum, hormones, or carbohydrates and on three substrata commonly used to culture cells in a defined medium. Hepatocytes cultured on a floating collagen gel in which is embedded a nylon mesh possess cell attachment and morphologic characteristics superior to either cells cultured on a collagen-coated or fibronectin(Fn)-coated substratum. Cells cultured on the gel-mesh system retain insulin responsivity, as measured by protein synthesis rates and glucose-6-phosphate dehydrogenase (G6PD) induction, for at least 6 d in culture. Under these conditions, insulin, dexamethasone, and fructose increase G6PD specific activity to levels comparble to that seen in an induced animal. Hepatocytes cultured on the gel-mesh system tolerate restricted medium conditions better than cells cultured on collagen or Fn-coated substratum, and remain viable for sufficient times to allow, for the first time, full expression and maximal induction (i.e. like in vivo), of G6PD in cultured cells. This system represents a satisfactory model for in vivo liver metabolism and a superior system for studying the effects of hormones and metabolites on G6PD levels, as well as other nutritional-hormonally regulated enzymes.     

Evaluation of DNA damage in leukocytes of G6PD-deficient Iranian newborns (Mediterranean variant) using comet assay

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is the most common inherited disease, which causes neonatal hemolytic anemia and jaundice. Recent studies of our group showed that the Mediterranean variant of this enzyme (Gd-Md) is the predominant G6PD in Iranian male infants suffering from jaundice; this variant is classified as severe G6PD deficiency. Considering the importance of G6PD reaction and its products NADPH and glutathione (GSH) against oxidative stress, we hypothesized the failure of detoxification of H(2)O(2) in G6PD-deficient white blood cells that could probably induce primary DNA damage. For the evaluation of DNA damage, we analyzed mononuclear leukocytes of 36 males suffering from the Gd-Md deficiency using alkaline single cell gel electrophoresis (SCGE) or comet assay. The level of DNA damage was compared with the level of basal DNA damage in control group represented by healthy male infant donors (of the same age group). Visual scoring was used for the evaluation of DNA damages. The results showed that the mean level of the DNA strand breakage in mononuclear leukocytes of 36 male G6PD-deficient (Gd-Md) infants was significantly higher (P < 0.001) than those observed in the normal lymphocytes. In conclusion, this investigation indicates that the mononuclear leukocytes of the Gd-Md samples may be exposed to DNA damage due to oxidative stress. This is the first report using comet assay for evaluation of DNA damage in severe G6PD deficiency samples.     

Distribution of abnormal hemoglobins and glucose-6-phosphate dehydrogenase variants in 1200 school children of Bahia,Brazil

School children from Bahia, Brazil were studied for hemoglobin and glucose-6-phosphate dehydrogenase electrophoretic variants. Eighty-nine heterozygotes Hb AS and 41 Hb AC were identified out of 1200 children. In a subsample of 369 male children there were 38 Gd A^?, 38 Gd A, and six Gd Med. An example of Gd MG was identified and evidence is added to the suggestion that this allele is not rare in Brazil.     

Selective precipitation of phosphofructokinase,glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase from rat erythrocyte hemolysates by polyethylene glycol

Precipitation profiles of phosphofructokinase, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase have been established in the range of 0–16% PEG at different pH (5–7) values. Precipitation generally occurred between narrow limits of polyethylene glycol. The polymer concentration needed to reach any level of enzyme precipitation is dependent on pH. Particular conditions (% PEG and pH) for the selective enzyme enrichment have been determined.     

Molecular analysis of G6PD variants in northern Italy: a study on the population from the Ferrara district

In the Ferrara district, an area south of the Po delta, four different variants of glucose-6-phosphate dehydrogenase (G6PD;E.C.1.1.49) have been described as a result of biochemical characterization of the enzyme protein: one was G6PD Mediterranean (G6PD Med) and three were local variants named Ferrara I, II, and III. The Ferrara I variant was recently analysed at the DNA level and shown to correspond to G6PD A^376G/202A, while the mutations causing the variants II and III, still remain unknown. We analysed the G6PD coding region of 18 apparently unrelated G6PD deficient subjects, whose families have lived in the Ferrara district for at least three generations: 12 subjects had G6PD Med^563T/1311T, 3, G6PD Santamaria^376G/542T and 2, G6PD A-^376G/202A. In one subject we found a new mutation, a GA transition at nucleotide 242 causing an ArgHis amino acid replacement at position 81. We named this new variant G6PD Lagosanto^{242 A}. Phenotypically the enzyme has nearly normal kinetic properties and appears different from the variants Ferrara II and III.     

Elastic Properties of Zinc-blende G a N,A l N and I n N from Molecular Dynamics

Molecular dynamics calculations of the adiabatic elastic constants of group III-Nitrides for temperatures ranging from 300 to 900 K have been performed. The results show good agreement with first-principles calculations. The moduli decreased with increasing temperature. The structural properties of zinc-blende GaN, AlN and InN are reported. Good agreement between the calculated and experimental values of the lattice constant, the cohesion energy, and the bulk modulus and its derivative are obtained.     

Molecular dynamics simulation and bioinformatics study on yeast aquaporin Aqy1 from Pichia pastoris

In the present study, an equilibrated system for the Aqy1 tetramer was developed, and molecular biophysics modeling showed that the Aqy1 channel was blocked by Tyr-31 in the N-terminus, which was also supported by the free energy profiles. However, bioinformatics analysis of the amino acid sequence of Aqy1 indicated this Tyr-31 is not conserved across all fungi. Analysis of the equilibrated structure showed that the central pore along the four-fold axis of the tetramers is formed with hydrophobic amino acid residues. In particular, Phe-90, Trp-198, and Phe-202 form the narrowest part of the pore. Therefore, water molecules are not expected to translocate through the central pore, a hypothesis that we confirmed by molecular dynamics simulations. Each monomer of the Aqy1 tetramers forms a channel whose walls consist mostly of hydrophilic residues, transporting through the selectivity filter containing Arg-227, His-212, Phe-92, and Ala-221, and the two conserved Asn-Pro-Ala (NPA) motifs containing asparagines 224 and 112. In summary, not all fungal aquaporins share the same gating mechanism by a tyrosine residue in the N-terminus, and the structural analysis in the present study should aid our understanding of aquaporin structure and its functional implications.     

氧化应激条件下G6PD对Raji细胞内GSH水平的影响

目的：观察体外培养的Burkit淋巴瘤（Raji）细胞在氧化应激条件下细胞内葡萄糖-6-磷酸脱氢酶（G6PD）对还原型谷胱甘肽（GSH）水平的影响。方法：体外培养Raji细胞,分别在G6PD活性被抑制及不抑制的情况下,检测细胞在酚嗪甲酸硫酯（PMS）作用后60min及360min时G6PD、谷胱甘肽还原酶（GR）、谷胱甘肽过氧化物酶（GPx）活性及GSH水平。结果：在PMS作用下,Raji细胞内GSH水平在60min时显著下降（P〈0．01）而360min时可上升至对照组水平,G6PD及GPx活性持续显著升高（P〈0．01）而GR活性在360min时有显著升高（P〈0．01）;使用脱氢表雄酮（DHEA）抑制G6PD活性后,Raji细胞再在PMS作用下,细胞内各指标与PMS处理组比较,GSH水平显著降低（P〈0．01）,GPx活性在60min时显著增高（P〈0．05）而GR活性在360min时显著降低（P〈0．01）。结论：细胞在氧化应激条件下G6PD可能是Raji细胞内影响GSH水平的一个关键因子,对维持胞内GSH水平起重要的调节作用。     

Low oxygen tension alleviates oxidative damage and delays cellular senescence in G6PD-deficient cells

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are under increased oxidative stress and undergo premature cellular senescence. The present study demonstrates that G6PD-deficient cells cultured under 3% oxygen concentration had an extended replicative lifespan, as compared with those cultured under atmospheric oxygen level. This was accompanied by a reduction in the number of senescence-associated β-galactosidase (SA-β-Gal) positive and morphologically senile cells at comparable population doubling levels (PDL). Concomitant with the extension of lifespan was decreased production of reactive oxygen species. Additionally, lifespan extension was paralleled by the greatly abated formation of such oxidative damage markers as 8-hydroxy-deoxyguanosine (8-OHdG) as well as the oxidized and cross-linked proteins. Moreover, the mitochondrial mass increased, but the mitochondrial membrane potential ΔΨm decreased in cells upon serial propagation. These changes were inhibited by lowering the oxygen tension. Our findings provide additional support to the notion that oxidative damage contributes to replicative senescence of G6PD-deficient cells and reduction of oxidative damage by lowering oxygen tension can delay the onset of cellular senescence.     

Low oxygen tension alleviates oxidative damage and delays cellular senescence in G6PD-deficient cells

Previous studies have shown that glucose-6-phosphate dehydrogenase (G6PD)-deficient cells are under increased oxidative stress and undergo premature cellular senescence. The present study demonstrates that G6PD-deficient cells cultured under 3% oxygen concentration had an extended replicative lifespan, as compared with those cultured under atmospheric oxygen level. This was accompanied by a reduction in the number of senescence-associated β-galactosidase (SA-β-Gal) positive and morphologically senile cells at comparable population doubling levels (PDL). Concomitant with the extension of lifespan was decreased production of reactive oxygen species. Additionally, lifespan extension was paralleled by the greatly abated formation of such oxidative damage markers as 8-hydroxy-deoxyguanosine (8-OHdG) as well as the oxidized and cross-linked proteins. Moreover, the mitochondrial mass increased, but the mitochondrial membrane potential ΔΨm decreased in cells upon serial propagation. These changes were inhibited by lowering the oxygen tension. Our findings provide additional support to the notion that oxidative damage contributes to replicative senescence of G6PD-deficient cells and reduction of oxidative damage by lowering oxygen tension can delay the onset of cellular senescence.     

Polymorphism at the G6pd and 6Pgd loci in Drosophila melanogaster. IV. Genetic factors modifying enzyme activity

Different homozygous lines of similar genotype with respect to G6pd and 6Pgd were shown to have different enzyme activities for G6PD and 6PGD. Crosses between high and low lines suggested that there were modifying genes present on the autosomes, while others were probably located on the X chromosome. Allelic variation within each electrophoretic class of G6pd and 6Pgd might, however, also have contributed to this variation. An experiment on adaptation to sodium octanoate demonstrated that in adapted flies selection for lower enzyme activity had occurred, which provided further evidence for the existence of genetic differences in activity. Furthermore, a strong positive correlation between the activities of G6PD and 6PGD was found for each genotype. Since no correlation was found between MDH and the two enzymes G6PD and 6PGD, it could be concluded that this correlation was probably rather specific for G6PD and 6PGD. Interaction between genotypes with respect to activity was also found. It was shown that the variation at 6Pgd influenced the activity of G6PD within a genotype. The data are discussed in relation to fitness differences presented in foregoing articles.     

Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol

Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx(m2) ). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx(m2) with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx(m2) -mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxC113S exchange in Trx(m2) . Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes.