ARASHIKI Nobuto    Department   School of Medicine, School of Medicine    Position   Assistant Professor
Article types	Original article
Language	English
Peer review	Peer reviewed
Title	Membrane Peroxidation and Methemoglobin Formation are Both Necessary for Band 3 Clustering: Mechanistic Insights into Human Erythrocyte Senescence
Journal	Formal name：Biochemistry Abbreviation：Biochemistry ISSN code：00062960
Volume, Issue, Page	52,pp.5760-5769
Author and coauthor	Arashiki Nobuto†, Kimata Naoki, Manno Sumie, Mohandas Narla, Takakuwa Yuichi*
Authorship	Lead author
Publication date	2013/07
Summary	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.