Dataset Information

Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1.

ABSTRACT: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, recessive condition that causes intermittent jaundice or hemolytic anemia because of low NADPH levels in red blood cells. We performed steady-state enzyme kinetics with the recombinant C. elegans ortholog of human G6PD, GSPD-1, and two mutants containing amino acid changes found in human patients. The KM values for glucose-6-phosphate were 100 ± 27 µM, 80 ± 22 µM, and 1000 ± 300 µM for the wild-type, D60N, and R252L GSPD-1 enzymes, respectively. The specific activities of the D60N and R252L mutants were 59% and 11%, respectively, of the wild-type value. Protein homology modeling suggested that the R252L mutation was more severe because the mutation caused a shift in the position of some active site residues. The D60N mutation may have affected the conformation of an outer loop of the enzyme. These data demonstrate that GSPD-1 is a promising model for human G6PD deficiencies, with the advantage that potential treatments could be studied in vivo in C. elegans.

SUBMITTER: Loges LN

PROVIDER: S-EPMC8438584 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1.

Loges Luiza N LN Walstrom Katherine M KM

microPublication biology 20210910

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked, recessive condition that causes intermittent jaundice or hemolytic anemia because of low NADPH levels in red blood cells. We performed steady-state enzyme kinetics with the recombinant C. elegans ortholog of human G6PD, GSPD-1, and two mutants containing amino acid changes found in human patients. The K M values for glucose-6-phosphate were 100 ± 27 µM, 80 ± 22 µM, and 1000 ± 300 µM for the wild-type, D6 ...[more]

PMID: 34532700

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Project description:Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively.

Dataset Information

Modeling human glucose-6-phosphate dehydrogenase mutations using C. elegans GSPD-1.

Publications

Modeling human glucose-6-phosphate dehydrogenase mutations using <i>C. elegans</i> GSPD-1.

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