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

Oxidative damage and clustering of band 3 in the membrane have been implicated in the removal of senescent human erythrocytes from the circulation at the end of their 120 day life span. However, the biochemical and mechanistic events leading to band 3 cluster formation have yet to be fully defined. Here we show that while neither membrane peroxidation nor methemoglobin (MetHb) formation on their own can induce band 3 clustering in the human erythrocytes, they can do so when acting in combination. We further show that binding of MetHb to the cytoplasmic domain of band 3 in peroxidized, but not in untreated, erythrocyte membranes induces cluster formation. Age-fractionated populations of erythrocytes from normal human blood, obtained by a density gradient procedure, have allowed us to examine a subpopulation, highly enriched in senescent cells. We have found that band 3 clustering is a feature of only this small fraction, amounting to ∼0.1% of total circulating erythrocytes. These senescent cells are characterized by an increased proportion of MetHb as a result of reduced nicotinamide adenine dinucleotide-dependent reductase activity and accumulated oxidative membrane damage. These findings have allowed us to establish that the combined effects of membrane peroxidation and MetHb formation are necessary for band 3 clustering, and this is a very late event in erythrocyte life. A plausible mechanism for the combined effects of membrane peroxidation and MetHb is proposed, involving high-affinity cooperative binding of MetHb to the cytoplasmic domain of oxidized band 3, probably because of its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from band 3, allowing the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters

An Unrecognized Function of Cholesterol: Regulating the Mechanism Controlling Membrane Phospholipid Asymmetry

An asymmetric distribution of phospholipids in the membrane bilayer is inseparable from physiological functions, including shape preservation and survival of erythrocytes, and by implication other cells. Aminophospholipids, notably phosphatidylserine (PS), are confined to the inner leaflet of the erythrocyte membrane lipid bilayer by the ATP-dependent flippase enzyme, ATP11C, counteracting the activity of an ATP-independent scramblase. Phospholipid scramblase 1 (PLSCR1), a single-transmembrane protein, was previously reported to possess scrambling activity in erythrocytes. However, its function was cast in doubt by the retention of scramblase activity in erythrocytes of knockout mice lacking this protein. We show that in the human erythrocyte PLSCR1 is the predominant scramblase and by reconstitution into liposomes that its activity resides in the transmembrane domain. At or below physiological intracellular calcium concentrations, total suppression of flippase activity nevertheless leaves the membrane asymmetry undisturbed. When liposomes or erythrocytes are depleted of cholesterol (a reversible process in the case of erythrocytes), PS quickly appears at the outer surface, implying that cholesterol acts in the cell as a powerful scramblase inhibitor. Thus, our results bring to light a previously unsuspected function of cholesterol in regulating phospholipid scrambling