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

Abstract

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 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 MetHb binding 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 enabled 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 NADH-dependent reductase activity, and accumulated oxidative membrane damage. These findings have enabled 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 due to its carbonylation, rather than other forms of oxidative damage. This modification leads to dissociation of ankyrin from the band 3, enabling the tetrameric MetHb to cross-link the resulting freely diffusible band 3 dimers, with formation of clusters.

ATP11C is a major flippase in human erythrocytes and its defect causes congenital hemolytic anemia

“…Senescent cells were collected as the densest fraction (0.1% of total cells) by density gradient centrifugation. 23 There was no apparent difference in the proportion of the dense cell populations among the different blood samples studied (data not shown). The percentage of PS-positive cells among total erythrocytes was slightly higher for the patient (8.86%) than in the control (6.32%) and increased further in the patient's senescent erythrocytes: 15.86% versus 9.17% in the control.…”

“…Analysis of phosphatidylserine-exposing cells PS exposed on the erythrocyte cell surface was analyzed by Ca 2+ -dependent binding of fluorescently labeled annexin V. 1 Washed, unfractionated erythrocytes and senescent erythrocytes fractionated by density centrifugation, as we reported previously, 23 were suspended in nine volumes of Tris-buffered saline with glucose (TBS-G). To control the intracellular Ca 2+ concentration, erythrocytes were treated for 20 min at 37 °C with 2 μM A23187, 5 a Ca 2+ ionophore, in TBS-G; accurate final concentrations of free Ca 2+ were determined by adding 1 mM EGTA and calculating the concentration of CaCl 2 using Calcon free software (e.g., 0.958 mM for a final 1 μM).…”

The human Kell blood group binds the erythroid 4.1R protein: new insights into the 4.1R-dependent red cell membrane complex

“…Thus, we suggest that more mobile band 3 may be present at the red cell membrane in the absence of 4.1R protein. Furthermore, it has been shown that the increase of the mobile fraction of band 3 can lead to the formation of band 3 clusters (Arashiki , et al 2013). Using the band 3 antibody that specifically recognizes clustered band 3 (Arashiki , et al 2013), we explored the extent of band 3 clustering in 4.1(−) erythrocytes.…”

Red Blood Cell Storage Duration and Trauma

“…Membrane signals displayed by the damaged RBCs-such as oxidized proteins and lipids, exposure of phosphatidylserine, clustering of RBC structural proteins, and binding sites for free hemoglobin and complement-target the RBCs for clearance by the reticuloendothelial system [52,[62][63][64][65][66]. These damage signals are similar to those displayed by senescent RBCs undergoing eryptosis [67].…”