Protein methylation as a marker of aspartate damage in glucose-6-phosphate dehydrogenase-deficient erythrocytes: role of oxidative stress.
The 'Mediterranean' variant of glucose-6-phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme L-isoaspartate (d-aspartate) protein O-methyltransferase (PCMT; EC 2.1.1.77). The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl-labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification. We present here evidence that, in G6PD-deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t-butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid-reactive substances. L-Isoaspartate residues increased dramatically in G6PD-deficient erythrocytes in response to such treatment, compared with baseline conditions. The increased susceptibility of G6PD-deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.
Ingrosso
Diego
D
Department of Biochemistry and Biophysics, School of Medicine, Second University of Naples, Italy. diego.ingrosso@unina2.it
Cimmino
Amelia
A
D'Angelo
Stefania
S
Alfinito
Fiorella
F
Zappia
Vincenzo
V
Galletti
Patrizia
P
D001224:Aspartic Acid / Q000378:metabolism*; D016022:Case-Control Studies; D016922:Cellular Senescence; D004912:Erythrocytes / Q000502:physiology*; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D005955:Glucosephosphate Dehydrogenase Deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008297:Male; D008745:Methylation; D018384:Oxidative Stress / Q000502:physiology; D017354:Point Mutation; D011496:Protein Methyltransferases / Q000502:physiology*
D001224:Aspartic Acid; D005954:Glucosephosphate Dehydrogenase; D011496:Protein Methyltransferases