The mechanism of formation, structure and physiological relevance of covalent hemoglobin attachment to the erythrocyte membrane

Covalent hemoglobin binding to membranes leads to band 3 (AE1) clustering and the removal of erythrocytes from the circulation; it is also implicated in blood storage lesions. Damaged hemoglobin, with the heme being in a redox and oxygen-binding inactive hemichrome form, has been implicated as the binding species. However, previous studies used strong non-physiological oxidants. In vivo hemoglobin is constantly being oxidised to methemoglobin (ferric), with around 1% of hemoglobin being in this form at any one time. In this study we tested the ability of the natural oxidised form of hemoglobin (methemoglobin) in the presence or absence of the physiological oxidant hydrogen peroxide to initiate membrane binding. The higher the oxidation state of hemoglobin (from Fe(III) to Fe(V)) the more binding was observed, with approximately 50% of this binding requiring reactive sulphydryl groups. The hemoglobin bound was in a high molecular weight complex containing spectrin, ankyrin and band 4.2, which are common to one of the cytoskeletal nodes. Unusually, we showed that hemoglobin bound in this way was redox active and capable of ligand binding. It can initiate lipid peroxidation showing the potential to cause cell damage. In vivo oxidative stress studies using extreme endurance exercise challenges showed an increase in hemoglobin membrane binding, especially in older cells with lower levels of antioxidant enzymes. These are then targeted for destruction. We propose a model where mild oxidative stress initiates the binding of redox active hemoglobin to the membrane. The maximum lifetime of the erythrocyte is thus governed by the redox activity of the cell; from the moment of its release into the circulation the timer is set

赤血球膜スペクトリンの脂質過酸化産物4-ヒドロキシ-2-ノネナールによる分子修飾と赤血球膜物性の変化

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. The present study 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 and marked reduction in the membrane mechanical properties. 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.膜脂質の過酸化は、4-hydroxyl-2-nonenal (HNE)をはじめとする種々のアルデヒドやアルケナールを産生する。これらの脂質過酸化産物は、核酸やタンパク質と共有結合して、その構造と機能の障害を生じる。赤血球は、こうした脂質過酸化産物による障害を最も受け得る細胞であり、その老化にともなうHNE の蓄積が知られるが、実際にいかなる分子修飾がどのような影響をもたらすのかは不明である。本研究は、その解明を目的に、主にヒト赤血球膜におけるHNE 付加タンパク質と修飾部位の同定、ならびに膜物性に対する影響の解析を行った。まず、抗HNE 抗体を用いたイムノブロッティングの結果、膜骨格網状構造の主体をなすタンパク質、α-、ならびにβ-スペクトリンにHNE 付加体のシグナルが検出された。赤血球膜ゴーストをHNE と孵置すると、アクチンやバンド３をはじめとする他の主要膜タンパク質にHNE 付加が生じたが、無傷赤血球にHNE を作用させた場合にはスペクトリンにのみ、特にβ-スペクトリンにHNE 付加体の著しい増加が認められ、α-、ならびにβ-スペクトリンがHNE の主たる標的分子であることが明らかになった。飛行時間型質量分析装置を用いた解析から、その修飾部位は、β-スペクトリン分子N 末端ドメイン(βN)中のIle110-…-Arg127 配列など、膜脂質と直接に相互作用する領域を中心に複数存在し、Cys、His、ならびにLys 残基のMichael 付加体として、またLys 残基では加えてSchiff 塩基付加体として存在すると推定された。一方、ヒト以外の赤血球膜でも、HNE との孵置によりHNE-スペクトリンの著増がみられたが、自然付加体の量はヒト赤血球に比べて少なく、また動物種により差異が認められた。これらの知見は、スペクトリン分子と膜脂質との相互作用がHNE 修飾に影響することを示唆するものである。そこで、赤血球におけるスペクトリン-膜脂質間結合を保つMgATP の存在／非存在下に膜ゴーストを調製し、これらのHNE 付加を検討した。その結果、MgATP 存在下では、HNEとの孵置でスペクトリンの不可溶性凝集体形成が生じ、α鎖よりもβ-スペクトリンの修飾が優位であった。対照的に、MgATP 非存在下では、HNE-α-スペクトリンの生成がβ鎖のそれを上回り、明瞭な凝集はみられなかった。また、比重遠心で分画した赤血球では、予想に反して全ての分画の赤血球でスペクトリンのHNE 修飾がみられ、最下層の老化赤血球では軽度のHNE-スペクトリン減少が認められた。これらの知見は、スペクトリンの膜への組み込み後早期に一定量のHNE 付加が生じることを示すとともに、赤血球の末梢循環過程で限定的なHNE-スペクトリン凝集体の形成と膜小胞としての除去が生じることを示唆している。さらに、HNE 付加による赤血球膜の機械的特性の変化を知るために、エクタサイトメーターで赤血球の変形能を解析したところ、変形能指数は、HNE の濃度と孵置時間に応じて対照赤血球の値の40%～80%に低下した。また、HNE-スペクトリンの赤血球反転小胞への結合定数は対照スペクトリンの約1/2 とわずかではあるが低下を示した。したがって、一時的なHNE 付加体の増加は、スペクトリン-アクチン膜骨格の変形能を低下させて局所的な膜の断片化を生じ得ると推測され、その一因として赤血球膜との相互作用の強まりが考えられた。以上のように、本研究の成績は、酸化ストレス下、赤血球内外で生成するHNE が赤血球膜に作用してスペクトリンと共有結合付加体を形成し、スペクトリンの性状と機能を変化させることを通して赤血球膜の変形能を低下させることを明らかにしたものである。これらの知見は赤血球の老化や様々な疾患病態にともなう赤血球膜物性変化に新しい視点を与えるとともに、酸化ストレスが関わる疾患の細胞病態に、膜インターフェースにおける脂質過酸化産物による骨格タンパク質の構造・機能修飾が関わる可能性を示すものである

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

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

Structural implications of the EL(K/Q)(L/C)LD(A/G)DD sequence in the C-terminal cytoplasmic tail for proper targeting of anion exchanger 1 to the plasma membrane

While the C-terminal cytoplasmic tail of anion exchanger 1 (AE1, band 3) has been reported to possess important physiological roles, including one for proper membrane trafficking, its precise characteristics remain unclear. To clarify the overall structural consequences of the conserved sequence EL(K/Q)(L/C)LD(A/G)DD, containing the core binding sequence LDADD for carbonic anhydrase II, in the C-terminal region, we analyzed the membrane expression and turnover of bovine AE1 with a series of truncation and substitution mutations in HEK293 cells. Immunofluorescence microscopy and cell-surface biotinylation demonstrated that truncation mutants missing 18 C-terminal residues targeted the plasma membrane, but the one lacking the conserved region, by truncation of 28 amino acid residues, was retained inside the cells. Substitutions of Ala for Glu901, Leu902, Leu905, and Asp906 in the sequence E901L(K/Q)(L/C)LDADD909 of bovine AE1 or those in the corresponding murine sequence also caused intracellular retention, though these mutants had half-lives comparable to that for wild-type AE1. These data demonstrate that the conserved amino acid residues Glu1, Leu2, Leu5, and Asp6 in the EL(K/Q)(L/C)LD(A/G)DD region have essential structural consequences in stable expression of AE1 at the plasma membrane regardless of the ability in binding to carbonic anhydrase II of this region

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