Increased methyl esterification of altered aspartyl residues in erythrocyte membrane proteins in response to oxidative stress

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Abstract
Protein‐l‐isoaspartate (d‐aspartate) O‐methyltransferase (PCMT; EC 2.1.1.77) catalyses the methyl esterification of the free α‐carboxyl group of abnormal l‐isoaspartyl residues, which occur spontaneously in protein and peptide substrates as a consequence of molecular ageing. The biological function of this transmethylation reaction is related to the repair or degradation of age‐damaged proteins. Methyl ester formation in erythrocyte membrane proteins has also been used as a marker reaction to tag these abnormal residues and to monitor their increase associated with erythrocyte ageing diseases, such as hereditary spherocytosis, or cell stress (thermal or osmotic) conditions. The study shows that levels of l‐isoaspartyl residues rise in membrane proteins of human erythrocytes exposed to oxidative stress, induced by t‐butyl hydroperoxide or H2O2. The increase in malondialdehyde content confirmed that the cell membrane is a primary target of oxidative alterations. A parallel rise in the methaemoglobin content indicates that proteins are heavily affected by the molecular alterations induced by oxidative treatments in erythrocytes. Antioxidants largely prevented the increase in membrane protein methylation, underscoring the specificity of the effect. Conversely, we found that PCMT activity, consistent with its repair function, remained remarkably stable under oxidative conditions, while damaged membrane protein substrates increased significantly. The latter include ankyrin, band 4.1 and 4.2, and the integral membrane protein band 3 (the anion exchanger). The main target was found to be particularly protein 4.1, a crucial element in the maintenance of membrane‐cytoskeleton network stability. We conclude that the increased formation/exposure of l‐isoaspartyl residues is one of the major structural alterations occurring in erythrocyte membrane proteins as a result of an oxidative stress event. In the light of these and previous findings, the occurrence of isoaspartyl sites in membrane proteins as a key event in erythrocyte spleen conditioning and hemocatheresis is proposed.

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