The covalent modification of spectrin in red cell membranes by the lipid peroxidation product 4-hydroxy-2-nonenal

Abstract

Spectrin strengthens the red cell membrane through its direct association with membrane lipids and through protein-protein interactions. Spectrin loss reduces the membrane stability and results in various types of hereditary spherocytosis. However, less is known about acquired spectrin damage. Here, we showed that α-and β-spectrin in human red cells are the primary targets of the lipid peroxidation product 4-hydroxy-2-nonenal (HNE) by immunoblotting and mass spectrometry analyses. The level of HNE adducts in spectrin (particularly α-spectrin) and several other membrane proteins was increased following the HNE treatment of red cell membrane ghosts prepared in the absence of MgATP. In contrast, ghost preparation in the presence of MgATP reduced HNE adduct formation, with preferential β-spectrin modification and increased cross-linking of the HNE-modified spectrins. Exposure of intact red cells to HNE resulted in selective HNE-spectrin adduct formation with a similar preponderance of HNE-β-spectrin modifications. These findings indicate that HNE adduction occurs preferentially in spectrin at the interface between the skeletal proteins and lipid bilayer in red cells and suggest that HNE-spectrin adduct aggregation results in the extrusion of damaged spectrin and membrane lipids under physiological and disease conditions.

Membrane Peroxidation and Methemoglobin Formation Are Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence

“…We suggest that the observed carbonylation of spectrin is a consequence of endogenous modification with 4-hydroxy-2-nonenal (HNE), a carbonylation reaction in a broad sense, because DNPH also reacts with HNE-modified proteins generated by Michael reaction and Schiff-base formation (8, 9). We showed previously that only α- and β-spectrin were endogenously modified with HNE in erythrocyte membrane proteins and HNE-modified spectrin readily formed aggregates (40). The signals of carbonylated spectrin corresponding to the original position of spectrin were shifted to upper edge of the gel (high molecular weight region) by t -BHP-treatment.…”

Differential carbonylation of cytoskeletal proteins in blood group O erythrocytes: Potential role in protection against severe malaria

“…As DNPH exposes global carbonylation while anti-HNE antibodies only recognize carbonylation caused by 4-HNE, the signal difference suggests that only a small fraction of general carbonylation was mediated by 4-HNE under normal physiological conditions. Although differences can be observed between individual samples, a double-band pattern characteristic of heterodimers of a and b-spectrins around 250 kDa (Arashiki et al, 2010) can be recognized.…”

4-Hydroxynonenal (HNE) and hepatic injury related to chronic oxidative stress

“…HNE has high reactivity and interacts easily with thiol (-SH) or amino (-NH 2 ) groups of glutathione and proteins, and at high concentration, with DNA. HNE forms covalent adducts with proteins in the cell, including ones in the plasma membrane, with the modification of specific cellular functions [16,17]. Plasma membrane proteins represent the first targets for adduct formation.…”

“…In addition to the plasma membranes, both mitochondria and the endoplasmic reticulum are markedly susceptible to HNE-induced damage [7,[13][14][15]. HNE is not only a marker of oxidative stress but can act as a mediator of oxidative damage by modifying specific cellular functions [16]. Therefore, HNE is considered to be a toxic messenger that can deepen and expand oxidative damage in cells [8].…”