scholarly journals Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

2020 ◽  
Vol 21 (14) ◽  
pp. 4831
Author(s):  
Laura Morales-Luna ◽  
Beatriz Hernández-Ochoa ◽  
Edson Jiovany Ramírez-Nava ◽  
Víctor Martínez-Rosas ◽  
Paulina Ortiz-Ramírez ◽  
...  
Keyword(s):  
Trichomonas Vaginalis ◽  
Enzyme Stability ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Oligomeric State ◽  
Biochemical Assays ◽  
Heterologous System ◽  
G6pd Gene ◽  
Effects Of Temperature ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Temperature Susceptibility

This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

Molecular Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in the Shenzhen Population

2021 ◽  
pp. 1-7
Author(s):  
Jian Gao ◽  
Sheng Lin ◽  
Shiguo Chen ◽  
Qunyan Wu ◽  
Kaifeng Zheng ◽  
...  
Keyword(s):  
G6pd Deficiency ◽  
Amplification Refractory Mutation System ◽  
Gene Variants ◽  
Coding Region ◽  
G6pd Gene ◽  
Mutation System ◽  
Hotspot Mutations ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Generation Sequencing

<b><i>Background:</i></b> Glucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. This study aimed to characterize the G6PD gene variant distribution in Shenzhen of Guangdong province. <b><i>Methods:</i></b> A total of 33,562 individuals were selected at the hospital for retrospective analysis, of which 1,213 cases with enzymatic activity-confirmed G6PD deficiency were screened for G6PD gene variants. Amplification refractory mutation system PCR was first used to screen the 6 dominant mutants in the Chinese population (c.1376G&#x3e;T, c.1388G&#x3e;A, c.95A&#x3e;G, c.1024C&#x3e;T, c.392G&#x3e;T, and c.871G&#x3e;A). If the 6 hotspot variants were not found, next-generation sequencing was then performed. Finally, Sanger sequencing was used to verify all the mutations. <b><i>Results:</i></b> The incidence of G6PD deficiency in this study was 3.54%. A total of 26 kinds of mutants were found in the coding region, except for c.-8-624T&#x3e;C, which was in the noncoding region. c.1376G&#x3e;T and c.1388G&#x3e;A, both located in exon 12, were the top 2 mutants, accounting for 68.43% of all individuals. The 6 hotspot mutations had a cumulative proportion of 94.02%. <b><i>Conclusions:</i></b> This study provided detailed characteristics of G6PD gene variants in Shenzhen, and the results would be valuable to enrich the knowledge of G6PD deficiency.

scholarly journals Generation and Export of Red Blood Cell ATP in Health and Disease

2021 ◽  
Vol 12 ◽  
Author(s):  
Timothy J. McMahon ◽  
Cole C. Darrow ◽  
Brooke A. Hoehn ◽  
Hongmei Zhu
Keyword(s):  
Blood Cell ◽  
Red Blood Cell ◽  
Dynamic Control ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Antioxidant Systems ◽  
Atp Production ◽  
Allosteric Effector ◽  
The Face ◽  
Differential Control ◽  
Glucose 6 Phosphate Dehydrogenase

Metabolic homeostasis in animals depends critically on evolved mechanisms by which red blood cell (RBC) hemoglobin (Hb) senses oxygen (O2) need and responds accordingly. The entwined regulation of ATP production and antioxidant systems within the RBC also exploits Hb-based O2-sensitivity to respond to various physiologic and pathophysiologic stresses. O2 offloading, for example, promotes glycolysis in order to generate both 2,3-DPG (a negative allosteric effector of Hb O2 binding) and ATP. Alternatively, generation of the nicotinamide adenine dinucleotide phosphate (NADPH) critical for reducing systems is favored under the oxidizing conditions of O2 abundance. Dynamic control of ATP not only ensures the functional activity of ion pumps and cellular flexibility, but also contributes to the availability of vasoregulatory ATP that can be exported when necessary, for example in hypoxia or upon RBC deformation in microvessels. RBC ATP export in response to hypoxia or deformation dilates blood vessels in order to promote efficient O2 delivery. The ability of RBCs to adapt to the metabolic environment via differential control of these metabolites is impaired in the face of enzymopathies [pyruvate kinase deficiency; glucose-6-phosphate dehydrogenase (G6PD) deficiency], blood banking, diabetes mellitus, COVID-19 or sepsis, and sickle cell disease. The emerging availability of therapies capable of augmenting RBC ATP, including newly established uses of allosteric effectors and metabolite-specific additive solutions for RBC transfusates, raises the prospect of clinical interventions to optimize or correct RBC function via these metabolite delivery mechanisms.

scholarly journals Prevalence and Genetic Characterization of Glucose-6-Phosphate Dehydrogenase Deficiency in Anemic Subjects from Uttar Pradesh, India

2019 ◽  
Vol 08 (02) ◽  
pp. 047-053 ◽  
Author(s):  
Poonam Tripathi ◽  
Sarita Agarwal ◽  
Srinivasan Muthuswamy
Keyword(s):  
G6pd Deficiency ◽  
Dehydrogenase Deficiency ◽  
Genetic Characterization ◽  
Gene Mutations ◽  
Uttar Pradesh ◽  
Chromosome X ◽  
G6pd Gene ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Fluorescent Spot

AbstractGlucose-6-phosphate dehydrogenase (G6PD) deficiency is caused by one or more mutations in the G6PD gene on chromosome X. It affects approximately 400 million people worldwide. The purpose of this study was to detect the prevalence of G6PD deficiency and G6PD gene mutations in the hospital-based settings in patients referred for suspected G6PD deficiency. A qualitative fluorescent spot test and dichlorophenol-indolphenol (DCIP) test were performed. G6PD-deficient, positive samples were further processed for mutation analysis by Sanger sequencing. Out of 1,069 cases, 95 (8.8%) were detected as G6PD deficient (by DCIP test) and were sent for molecular analysis. The G6PD Mediterranean mutation (563C > T) is the most common variant among G6PD-deficient individuals followed by the Coimbra (592C→T) and Orissa (131C→G) variants. We concluded that all symptomatic patients (anemic or jaundiced) should be investigated for G6PD deficiency. Our findings will inform our population screening approach and help provide better management for G6PD-deficient patients.

scholarly journals Pathways for the reduction of oxidized glutathione in the Plasmodium falciparum-infected erythrocyte: can parasite enzymes replace host red cell glucose-6-phosphate dehydrogenase?

Blood ◽  
1986 ◽  
Vol 67 (3) ◽  
pp. 827-830 ◽  
Author(s):  
EF Jr Roth ◽  
S Schulman ◽  
J Vanderberg ◽  
J Olson
Keyword(s):  
Plasmodium Falciparum ◽  
Oxidant Stress ◽  
Reduction Rate ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Red Cells ◽  
Host Cells ◽  
Functional Assay ◽  
Red Cell ◽  
Oxidized Glutathione ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract Plasmodium falciparum-infected human red cells possess at least two pathways for the generation of reduced nicotinamide adenine dinucleotide phosphate (NADPH): (1) the glucose-6-phosphate dehydrogenase (G6PD) pathway and (2) the glutamate dehydrogenase (GD) pathway using glutamate as a substrate. Uninfected erythrocytes lack the GD pathway. The NADPH generated can be used to reduce oxidized glutathione (GSSG), which accumulates in the presence of an oxidative stress. In red cell G6PD deficiency, this pathway is reduced or absent, and the host cells as well as the parasites within them are vulnerable to oxidant stress. In view of the presence of the GD pathway in parasitized red cells and the recent description of a parasite-derived G6PD enzyme, we have asked whether the pathways for the reduction of GSSG provided by the parasite can substitute for the host G6PD in red cells deficient in G6PD activity. We have devised a functional assay in which the reduction rate of GSSG is monitored in the presence of buffered infected or control red cell lysates and substrates. Infected G6PD-deficient erythrocytes were obtained from in vitro cultures after a single prior growth cycle of the parasites in G6PD deficient cells to eliminate contaminating normal red cells. The results show that only parasitized red cells can reduce GSSG via the GD pathway. In parasitized G6PD Mediterranean red cells (completely G6PD-deficient), there is a detectable GSSG reduction via the G6PD pathway, not found in uninfected lysates from the same individual. In G6PD A- (African type, featuring partial deficiency), a small increment in the G6PD-dependent reduction of GSSG can also be detected. However, when compared to G6PD normal red cells, the activities from the parasite-derived pathways are small and could not be considered substitutes for normal host enzyme activity. It is concluded that while the plasmodium provides additional pathways for the generation of NADPH that may serve its own metabolic needs, the host red cells and hence the parasite itself remain vulnerable to oxidant stress.

scholarly journals Identification, Characterization, and Stress Responsiveness of Glucose-6-phosphate Dehydrogenase Genes in Highland Barley

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1800
Author(s):  
Ruijun Feng ◽  
Xiaomin Wang ◽  
Li He ◽  
Shengwang Wang ◽  
Junjie Li ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Redox Homeostasis ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reducing Power ◽  
Cellular Redox ◽  
E Coli ◽  
Intermediate Metabolites ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Highland Barley ◽  
Salt And Drought Stresses

G6PDH provides intermediate metabolites and reducing power (nicotinamide adenine dinucleotide phosphate, NADPH) for plant metabolism, and plays a pivotal role in the cellular redox homeostasis. In this study, we cloned five G6PDH genes (HvG6PDH1 to HvG6PDH5) from highland barley and characterized their encoded proteins. Functional analysis of HvG6PDHs in E. coli showed that HvG6PDH1 to HvG6PDH5 encode the functional G6PDH proteins. Subcellular localization and phylogenetic analysis indicated that HvG6PDH2 and HvG6PDH5 are localized in the cytoplasm, while HvG6PDH1, HvG6PDH3, and HvG6PDH4 are plastidic isoforms. Analysis of enzymatic activities and gene expression showed that HvG6PDH1 to HvG6PDH4 are involved in responses to salt and drought stresses. The cytosolic HvG6PDH2 is the major isoform against oxidative stress. HvG6PDH5 may be a house-keeping gene. In addition, HvG6PDH1 to HvG6PDH4 and their encoded enzymes responded to jasmonic acid (JA) and abscisic acid (ABA) treatments, implying that JA and ABA are probably critical regulators of HvG6PDHs (except for HvG6PDH5). Reactive oxygen species analysis showed that inhibition of cytosolic and plastidic G6PDH activities leads to increased H2O2 and O2− contents in highland barley under salt and drought stresses. These results suggest that G6PDH can maintain cellular redox homeostasis and that cytosolic HvG6PDH2 is an irreplaceable isoform against oxidative stress in highland barley.

scholarly journals Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) deficiency in patients of Chinese descent and identification of new base substitutions in the human G6PD gene

Blood ◽  
1993 ◽  
Vol 81 (8) ◽  
pp. 2150-2154 ◽  
Author(s):  
DT Chiu ◽  
L Zuo ◽  
L Chao ◽  
E Chen ◽  
E Louie ◽  
...  
Keyword(s):  
G6pd Deficiency ◽  
Point Mutations ◽  
Nucleotide Substitutions ◽  
New Findings ◽  
G6pd Gene ◽  
High Prevalence ◽  
Chinese Descent ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sequencing Procedure

Abstract The underlying DNA changes associated with glucose-6-phosphate dehydrogenase (G6PD)-deficient Asians have not been extensively investigated. To fill this gap, we sequenced the G6PD gene of 43 G6PD- deficient Chinese whose G6PD was well characterized biochemically. DNA samples were obtained from peripheral blood of these individuals for sequencing using a direct polymerase chain reaction (PCR) sequencing procedure. From these 43 samples, we have identified five different types of nucleotide substitutions in the G6PD gene: at cDNA 1388 from G to A (Arg to His); at cDNA 1376 from G to T (Arg to Leu); at cDNA 1024 from C to T (Leu to Phe); at cDNA 392 from G to T (Gly to Val); at cDNA 95 from A to G (His to Arg). These five nucleotide substitutions account for over 83% of our 43 G6PD-deficient samples and these substitutions have not been reported in non-Asians. The substitutions found at cDNA 392 and cDNA 1024 are new findings. The substitutions at cDNA 1376 and 1388 account for over 50% of the 43 samples examined indicating a high prevalence of these two alleles among G6PD-deficient Chinese. Our findings add support to the notion that diverse point mutations may account largely for much of the phenotypic heterogeneity of G6PD deficiency.

scholarly journals Multiple forms with glucose 6-phosphate dehydrogenase activity in Musca domestica L. as revealed by electrophoresis on cellulose acetate gel.

1978 ◽  
Vol 26 (10) ◽  
pp. 846-854 ◽  
Author(s):  
L Cima ◽  
A Malacrida ◽  
G Gasperi ◽  
L Sacchi ◽  
A Grigolo
Keyword(s):  
Musca Domestica ◽  
Nicotinamide Adenine Dinucleotide ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
G6pd Activity ◽  
Nicotinamide Adenine ◽  
Molecular Forms ◽  
Type I ◽  
Coenzyme Specificity ◽  
Low Sensitivity ◽  
Glucose 6 Phosphate Dehydrogenase

Single newly emerged males of Musca domestica, WHO strain, usually show five electrophoretic bands of glucose 6-phosphate dehydrogenase (G6PD) activity. Of these five molecular forms, designated with Roman numerals in order from the origin, we have considered the first three: these have been characterized with respect to their substrate and coenzyme specificity and to their sensitivity to some sulfhydryl inhibitors. The data show band III to be G6P specific, nicotinamide adenine dinucleotide phosphate dependent and to be a type I enzyme according to Kamada and Hori's classification. Bands I and II, on the other hand, show wide substrate specificity and low sensitivity to the sulfhydryl inhibitors assayed. In addition, in the absence of an exogenous substrate and in the presence of nicotinamide adenine dinucleotide as a coenzyme, fairly weak bands, which can be ascribed to the so called "nothing dehydrogenase" effect, are seen in the position I and II. Nevertheless, the data reported do not allow a clear definition of the enzymatic type corresponding to bands I and II of G6PD activity.

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