The irreversibility of subunit associations in glucose-6-phosphate dehydrogenase and a suggestion regarding an early step in cellular morphogenesis.

For the purpose of studying the stability of the association of subunits in the dimeric enzyme glucose-6-PO₄ dehydrogenase (G6PD), the formation of ‘hybrid’ G6PD in the presence or absence of translation inhibitors was studied in polyethylene glycol-induced heterokaryons of mouse and human fibroblasts. In heterokaryons cultured in the absence of translation inhibitors, hybrid G6PD made of one monomer each of the mouse and human types was detectable. In the presence of cycloheximide concentrations that inhibited over 90% of protein synthesis, however, no hybrid G6PD could be detected in heterokaryons, despite the presence in such heterokaryons of substantial amounts of the ‘parental’ mouse and human enzymes that had been produced prior to heterokaryon formation. Thus, we conclude that once the subunits form a dimeric G6PD, they remain irreversibly associated in the mature enzyme. This, we propose, may reflect an early step in the morphogenesis of relatively stable intracellular structures.     

Stability of the "two active X" phenotype in triploid somatic cells.

We examined triploid cells of XXY karyotype heterozygous for glucose 6 phosphate dehydrogenase (G6PD) electrophoretic variants with regard to the stability of their X chromosome phenotype. Clonal populations of cells derived from these human fibroblasts maintained a precise 1:2:1 ratio of A:heteropolymer:B isozymes throughout their life span, indicating stability of the two active X chromosomes in these cells. To determine the influence of the autosomal complement on X chromosome expression, we attempted to perturb the relationship. Fusion of these triploid cells with human diploid fibroblasts carrying a novel G6PD variant (B') resulted in heterokaryons exprssing a novel heteropolymer, presumably indicating that all three parental X chromosomes were active. However, no derepression of the inactive X chromosome was observed. Analysis of interspecific hybrids derived from triploid cells and mouse fibroblasts confirmed that activity of parental X chromosomes is maintained. Some human mouse hybrid clones, however, expressed only a single human G6PD isozyme, probably attributable to segregation of the pertinent X chromosome, but elimination of a relevant autosome cannot be excluded. The triploid cells transformed by SV40 showed alterations in LDH pattern and an approximately 10-20% decrease in chromosome number, but maintained the precise G6PD phenotype of the untransformed cell. These studies provide evidence for the stability of the X chromosome phenotype in triploid cells.     

Expression of lysosomal enzymes in human mutant fibroblast-chick erythrocyte heterokaryons

The generation of enzymes located in lysosomes, in cytosol or in endoplasmatic reticulum/Golgi complex is studied in heterokaryons in which chick erythrocyte nuclei are reactivated. The lysosomal enzymes, alpha-glucosidase (alpha-glu) and beta-galactosidase (beta-gal), are synthesized in heterokaryons obtained after fusion of chick erythrocytes with human fibroblasts of patients with Pompe's disease (alpha-glu-deficient) and GM1-gangliosidosis (beta-gal-deficient), respectively. The enzymes appear to be of chick origin and their activities can be detected at first around 4 days after fusion, i.e., at a time when the nucleoli in the erythrocyte nuclei have been reactivated. Maximal activities are reached around 15 days after fusion. No generation of the lysosomal enzyme beta-hexosaminidase is detected in the heterokaryons up to 23 days after fusion of chick erythrocyte with either beta-hexosaminidase A- and B-deficient fibroblasts (Sandhoff's disease) or beta-hexosaminidase A-deficient fibroblasts (Tay-Sachs disease). Similarly no expression of the cytosol enzyme glucose-6-phosphate dehydrogenase (G6PD) is fond up to 30 days after fusion, when chick erythrocytes are fused with fibroblasts from two different G6PD-deficient cell strains (residual activities of 4 and 20% respectively). Indirectly we examined N-acetyl-glucosamine-1-phosphate transferase activity, an enzyme located in the endoplasmic reticulum/Golgi region. This enzyme is needed for the phosphorylation of the lysosomal hydrolases and absence of its activity is the cause of the multiple lysosomal enzyme deficiencies in patients with I-cell disease. The retention of both, chick and human beta-galactosidase in the experiments in which I-cell fibroblasts were fused with chick erythrocytes indicates a reactivation of the gene coding for this phosphorylating enzyme. It also implies that this step in the processing of human lysosomal enzymes is not species-specific.     

Enrichment of human heterokaryons by Ficoll gradient for complementation analysis of iduronate sulfatase deficiency.

Ficoll gradients have been used to enrich for heterokaryons in cultures of human skin fibroblasts following polyethylene glycol (PEG) induced fusion. These gradients provide a simple and consistent method for obtaining populations of multinucleated cells, at least twofold greater than those resulting from fusion alone. Formation of glucose-6-phosphate dehydrogenase (G6PD) heteropolymers has been used as a functional assay for the presence of heterokaryons. Analysis of cell populations enriched for multinucleated cells has revealed complementation leading to iduronate sulfatase activity in heterokaryons derived from iduronate sulfatase-deficient fibroblasts expressing the Hunter and multiple sulfatase-deficiency mutations.     

Red-cell glucose-6-phosphate dehydrogenase of “brown-howler” monkeys (Alouatta fusca)

Physico-chemical properties of erythrocyte glucose-6-phosphate dehydrogenase including erythrocyte G6PD activity, Michaelis constants, K_mG6P and NADP, pH optimum, thermostability and molecular weight were investigated in “brown-howler” monkeys and then compared with the values of human G6PD B(+). The values of Michaelis constants (K_mG6P and NADP) pH optimum were the same as the values of human G6PD B(+). The human G6PD has a dimeric form in the assay conditions employed in the present study, monkey enzyme showing great similariy with human one. Otherwise, the thermostability differed from the human G6PD. The simian enzymatic activity was about four times higher than the human G6PD. A comparison of physico-chemical properties of glucose-6-phosphate dehydrogenase among primates is also presented.     

Presence of two active X chromosomes in hybrids between normal human and SV40-transformed fibroblasts from patients with the Lesch-Nyhan syndrome

Skin fibroblasts (LNSV) derived from a hypoxanthine-guanine phosphoribosyltransferase (HGPRT) deficient patient with the Lesch-Nyhan syndrome, who has glucose-6-phosphate dehydrogenase (G6PD) type A, were transformed with SV40 and hybridized with WI38 human diploid fibroblasts derived from a female embryo which have normal HGPRT and G6PD type B activities. The hybrid clones selected in hypoxanthine, aminopterin and thymidine (HAT) medium, were essentially tetraploid and contained three X and one Y chromosomes. These hybrids contained HGPRT, types A and B and the AB heteropolymeric form of G6PD enzymes which were indicative that in these cells X linked genes of both parental cells were fully active. Hybrids back-selected in medium containing 8-azaguanine (8-AG) contained only two X chromosomes. They had no HGPRT activity and they contained only G6PD type A enzyme. It is concluded that the hybrid cells which grew in the presence of 8-AG retained the X chromosome of the LNSV parental cell and apparently the inactive X of the WI 38 cell.     

Glucose-6-phosphate dehydrogenase from Plasmodium berghei: kinetic and electrophoretic characterization

Evidence is given for the existence of a parasite-specific glucose-6-phosphate dehydrogenase (G6PD) in Plasmodium berghei by characterization of its kinetic and electrophoretic properties. From infected rat erythrocytes the parasites were isolated, washed, and lysed. G6PD was purified by affinity chromatography with 2'5'-ADP-Sepharose 4B, although the separation of the malaria-specific enzyme from that of the host cell was not complete. Malarial G6PD significantly differed from the red cell enzyme with respect to its electrophoretic properties. In cellulose acetate electrophoresis, a band with catodic mobility was observed in addition to the anodically mobile host cell enzyme at pH 7.0. The subunits of the parasite-specific G6PD have a molecular weight of 55 kDa in contrast to 59 kDa of red cell G6PD subunits. The enzyme from P. berghei shows no cross-reactivity with polyclonal antibodies against G6PD from rat erythrocytes. Thus, a close evolutionary relationship between both proteins and the presence of proteolytic modifications could be excluded. The Km value for G6P of malarial G6PD is increased by one order of magnitude compared with the host cell enzyme.     

Structural defects underlying protein dysfunction in human glucose-6-phosphate dehydrogenase A(-) deficiency

The enzyme variant glucose-6-phosphate dehydrogenase (G6PD) A(-), which gives rise to human glucose-6-phosphate dehydrogenase deficiency, is a protein of markedly reduced structural stability. This variant differs from the normal enzyme, G6PD B, in two amino acid substitutions. A further nondeficient variant, G6PD A, bears only one of these two mutations and is structurally stable. In this study, the synergistic structural defect in recombinant G6PD A(-) was reflected by reduced unfolding enthalpy due to loss of beta-sheet and alpha-helix interactions where both mutations are found. This was accompanied by changes in inner spatial distances between residues in the coenzyme domain and the partial disruption of tertiary structure with no significant loss of secondary structure. However, the secondary structure of G6PD A(-) was qualitatively affected by an increase in beta-sheets substituting beta-turns related to the lower unfolding enthalpy. The structural changes observed did not affect the active site of the mutant proteins, since its spatial position was unmodified. The final result is a loss of folding determinants leading to a protein with decreased intracellular stability. This is suggested as the cause of the enzyme deficiency in the red blood cell, which is unable to perform de novo protein synthesis.     

Transfer of the human X chromosome to human--Chinese hamster cell hybrids via isolated HeLa metaphase chromosomes.

Evidence is presented for the uptake of the human X chromosome by human-Chinese hamster cell hybrids which lack H P R T activity, following incubation with isolated human HeLa S3 chromosomes. Sixteen independent clonal cell lines were isolated in H A T medium, all of which contained a human X chromosome as determined by trypsin-Giemsa staining. The frequency of H A T-resistant clones was 32 x 10(-6) when 10(7) cells were incubated with 10(8) HeLa chromosomes. Potential reversion of the hybrid cells in H A T medium was less than 5 x 10(-7). The 16 isolated cell lines all contained activity of the human X-linked marker enzymes H P R T, P G K,alpha-Gal A, and G6PD, as determined by electrophoresis. The phenotype of G6PD was G6PD A, corresponding to G6PD A in HeLa cells. The human parental cells used in the fusion to form the hybrids had the G6PD B phenotype. The recipient cells gave no evidence of containing human X chromosomes. These results indicate that incorporation and expression of HeLa X chromosomes is accomplished in human-Chinese hamster hybrids which lack a human X chromosome.     

Frequency of reactivation and variability in expression of X-linked enzyme loci.

A mouse-human cell hybrid clone retaining an inactive human X chromosome was treated with 5-azacytidine. Following treatment, expression of the X-linked enzyme markers, hypoxanthine-guanine phosphoribosyltransferase (HPRT), glucose-6-phosphate dehydrogenase (G6PD), phosphoglycerate kinase (PGK), and alpha-galactosidase A (GLA) was examined. Results presented here show that 45 of the 62 clones positive for human HPRT expressed human GLA, while only four of 68 clones negative for human HPRT expressed human GLA. These results strongly suggest that there is coordinate reactivation of GLA and HPRT. Reactivated expression of G6PD was studied in detail. The studies show that 5-azacytidine can induce heritable changes in the inactive human X chromosome resulting in the expression of G6PD activity at a level lower than that from an active human X chromosome.     

X-chromosome inactivation in the human cytotrophoblast

Preferential paternal X-chromosome inactivation occurs in the cell lineages that differentiate first within the female rodent blastula (trophectoderm and extraembryonic endoderm). The present studies were designed to test the nature of X-chromosome inactivation (XCI) in the earliest differentiating cell lineage of the human placenta, the cytotrophoblast. Using glucose-6-phosphate-dehydrogenase (G6PD) polymorphisms as a marker system, term placentae were obtained from 13 female heterozygotes where parental allelic contributions could be determined. Chorionic villi were enzymatically digested and centrifuged in a Percoll density gradient to isolate a pure population of cytotrophoblasts, which was ascertained by cell culture, differentiation to syncytiotrophoblasts, and histochemical staining for alpha-human chorionic gonadotrophin (alpha-HCG). On electrophoresis, all 13 samples exhibited exclusive or near exclusive expression of the maternally derived X-linked enzyme variant, regardless of whether it was G6PD A or G6PD B. No intermediate bands were seen, indicating a single active G6PD locus per cell. The stromal cells of the villi, which derive from the mesoderm and differentiate later than the cytotrophoblast, exhibit random XCI. These findings establish preferential paternal XCI in the cytotrophoblast, the cell type first to differentiate within the human blastula.     

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.     

Evidence for balancing selection from nucleotide sequence analyses of human G6PD

Glucose-6-phosphate dehydrogenase (G6PD) mutations that result in reduced enzyme activity have been implicated in malarial resistance and constitute one of the best examples of selection in the human genome. In the present study, we characterize the nucleotide diversity across a 5.2-kb region of G6PD in a sample of 160 Africans and 56 non-Africans, to determine how selection has shaped patterns of DNA variation at this gene. Our global sample of enzymatically normal B alleles and A, A-, and Med alleles with reduced enzyme activities reveals many previously uncharacterized silent-site polymorphisms. In comparison with the absence of amino acid divergence between human and chimpanzee G6PD sequences, we find that the number of G6PD amino acid polymorphisms in human populations is significantly high. Unlike many other G6PD-activity alleles with reduced activity, we find that the age of the A variant, which is common in Africa, may not be consistent with the recent emergence of severe malaria and therefore may have originally had a historically different adaptive function. Overall, our observations strongly support previous genotype-phenotype association studies that proposed that balancing selection maintains G6PD deficiencies within human populations. The present study demonstrates that nucleotide sequence analyses can reveal signatures of both historical and recent selection in the genome and may elucidate the impact that infectious disease has had during human evolution.     

The extent of linkage disequilibrium caused by selection on G6PD in humans

The gene coding for glucose-6-phosphate dehydrogenase (G6PD) is subject to positive selection by malaria in some human populations. The G6PD A- allele, which is common in sub-Saharan Africa, is associated with deficient enzyme activity and protection from severe malaria. To delimit the impact of selection on patterns of linkage disequilibrium (LD) and nucleotide diversity, we resequenced 5.1 kb at G6PD and approximately 2-3 kb at each of eight loci in a 2.5-Mb region roughly centered on G6PD in a diverse sub-Saharan African panel of 51 unrelated men (including 20 G6PD A-, 11 G6PD A+, and 20 G6PD B chromosomes). The signature of selection is evident in the absence of genetic variation at G6PD and at three neighboring loci within 0.9 Mb from G6PD among all individuals bearing G6PD A- alleles. A genomic region of approximately 1.6 Mb around G6PD was characterized by long-range LD associated with the A- alleles. These patterns of nucleotide variability and LD suggest that G6PD A- is younger than previous age estimates and has increased in frequency in sub-Saharan Africa due to strong selection (0.1 < s < 0.2). These results also show that selection can lead to nonrandom associations among SNPs over great physical and genetic distances, even in African populations.     

An embryoprotective role for glucose-6-phosphate dehydrogenase in developmental oxidative stress and chemical teratogenesis.

The primary recognized health risk from common deficiencies in glucose-6-phosphate dehydrogenase (G6PD), a cytoprotective enzyme for oxidative stress, is red blood cell hemolysis. Here we show that litters from untreated pregnant mutant mice with a hereditary G6PD deficiency had increased prenatal (fetal resorptions) and postnatal death. When treated with the anticonvulsant drug phenytoin, a human teratogen that is commonly used in pregnant women and causes embryonic oxidative stress, G6PD-deficient dams had higher embryonic DNA oxidation and more fetal death and birth defects. The reported G6PD gene mutation was confirmed and used to genotype fetal resorptions, which were primarily G6PD deficient. This is the first evidence that G6PD is a developmentally critical cytoprotective enzyme for both endogenous and xenobiotic-initiated embryopathic oxidative stress and DNA damage. G6PD deficiencies accordingly may have a broader biological relevance as important determinants of infertility, in utero and postnatal death, and teratogenesis.     

Response surface methodology for the evaluation of glucose-6-phosphate dehydrogenase enrichment process by soybean lecithin reversed micelles

Glucose-6-phosphate dehydrogenase (G6PD) present in Saccahromyces cerevisiae is an enzyme of the pentose pathway. An effective enrichment of this intracellular enzyme can be achieved with the reversed micellar methodology. In this work, this methodology was employed with soybean lecithin, a biocompatible surfactant. A factorial design was used to evaluate the influence of pH (A) and extraction runs (B) on the G6PD purification factor. After statistical analysis and process optimization, a mathematical model representing G6PD enrichment was obtained: Y=4.89-0.83A+0.092B+0.27AB-1.37B2 with an enzyme purification factor of about 5.2.     

A new variant of human erythrocyte G6PD occurring at a high frequency amongst the population of two villages in The Gambia,West Africa

Summary During the course of a large survey of red cell G6PD genotypes in The Gambia, a slow electrophoretic variant with reduced enzyme activity was found to occur at a high frequency. This variant, G6PD Gambia, was found in the following genotypic combinations: males; G6PD^Gam, females; G6PD^A+/Gam, G6PD^B+/Gam, and G6PD^A-/Gam. From the electrophoretic mobility and kinetic characteristics it was concluded that G6PD Gambia was a hitherto unreported variant of G6PD. The frequency of the G6PD^Gam gene amongst the 1109 individuals examined was 0.024.     

Reprogramming cell differentiation in the absence of DNA synthesis

We examined whether the activation of muscle gene expression in nonmuscle cells required DNA synthesis. Human fibroblasts from amniotic fluid and fetal lung were fused with differentiated mouse muscle cells in the presence or absence of the DNA synthesis inhibitor, cytosine arabinoside. In the stable heterokaryons formed, the human contractile enzyme, MM-creatine kinase (CK), and the cell surface antigen, 5.1H11, were detected in comparable amounts regardless of whether DNA synthesis had occurred. A single cell analysis revealed that the efficiency of gene activation was high and that DNA synthetic activity was not affected by the ratio of muscle to nonmuscle nuclei in the heterokaryons. In addition, muscle gene expression was not restricted to the G1 phase of the cell cycle. We conclude that cell differentiation can be reprogrammed in heterokaryons regardless of cell cycle phase and in the absence of detectable DNA synthesis.     

Enzyme kinetics and molecular modeling studies of G6PD(Mahidol) associated with acute hemolytic anemia

G6PD(Mahidol) enzyme is the most common variant in the Achang Chinese ethnic group and clinically manifests as class II. In this study, G6PD(Mahidol) enzyme was characterized by molecular modeling to understand its kinetics. G6PD(Mahidol), G6PD(G487A) and G6PD(WT) proteins were heterologously expressed in the G6PD-deficient DF213 E. coli strain, purified and their steady-state kinetic parameters were determined. Compared with G6PD(WT), the Km, and Vmax of NADP+ with G6PD(G487A) were about 28-fold and 12-fold lower, respectively. The Ki values of dehydroepiandrosterone (DHEA), NADPH and ATP with G6PD(G487A) showed 29.5-fold, 2.36-fold reduction and 1.83-fold increase, respectively. A molecular modeling of G6PD(G487A) was performed based on the X-ray structure of human G6PD (PDB: 2BH9). It is suggested that Ser-163 might affect the stability of G6PD(G487A) alpha-helix d and beta-strand E, besides the conformation of beta-strand D. In conclusion, the biochemical and structural properties of G6PD(G487A) and G6PD(WT) enzymes are significantly different, which may be responsible for clinical diversity of G6PD deficiencies.     

Functional expression of human glucose-6-phosphate dehydrogenase in Escherichia coli

The complete coding sequence for human glucose-6-phosphate-dehydrogenase (G6PD) was inserted downstream from the tac promoter of a plasmid, pJF118EH, which also carries the lacIq repressor gene. When Escherichia coli strains (that are unable to grow on glucose due to the absence of functional zwf (G6PD-) and pgi genes) were transformed with this plasmid (pAC1), they were able to grow on glucose as sole carbon source. The rate of growth on glucose was faster in the presence of the inducer of the tac promoter, isopropyl-beta-D-thiogalactopyranoside (IPTG). Extracts of the transformed cells contained a G6PD activity that was not detectable in the parental strains and that was inducible by IPTG. The G6PD activities from normal E. coli and from pAC1-transformed cells comigrated with human G6PD when subjected to electrophoresis on agarose gels. However, when denatured, the G6PD produced by pAC1 was, like the human enzyme, distinguishable from the E. coli-encoded enzyme on the basis of its immunoreactivity with antibody specific for human G6PD. Therefore, human G6PD can be expressed in E. coli and can function to complement the bacterial enzyme deficiency.