Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells
Oxidized and poorly glycosylated band 3 is selectively phosphorylated by Syk kinase to form large membrane clusters in normal and G6PD-deficient red blood cells
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DOI：
10.1042/bj20081557
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发表时间：
2009-03-01
影响因子：
4.1
通讯作者：
Turrini, Franco
中科院分区：
文献类型：
--
作者：
Pantaleo, Antonella;Ferru, Emanuela;Turrini, Franco
涉及带3（阴离子交换器1）的氧化事件与通过结合nab（自然产生的抗体）去除RBC（红细胞）有关；然而，潜在的机制只是部分表征。除了直接诱导膜蛋白氧化修饰外，氧化处理还会特异性触发3带酪氨酸残基的磷酸化。本研究报道，二胺，一种巯基氧化剂，诱导低糖基化带3的二硫交联，低聚带3部分在G6PD（葡萄糖-6-磷酸脱氢酶）缺陷和对照红细胞中选择性酪氨酸磷酸化。这种现象在缺乏g6pd的红细胞中是不可逆的，而在对照红细胞中则是暂时受限的。二胺处理导致p72syk磷酸化并易位到膜上。二胺还诱导p72 Syk共免疫沉淀，聚集带3。此外，对Triton x -100提取的膜蛋白进行尺寸排除分离后发现，Syk仅存在于含有低聚/磷酸化带3的高分子质量部分。Src家族抑制剂有效地消除了3带酪氨酸磷酸化、3带聚集和nab与红细胞表面的结合，表明这些事件之间存在因果关系。用非渗透交联剂BS3（双磺基琥珀酰亚磺酸）进行的实验表明，带3酪氨酸磷酸化增强了其形成大聚集体的能力。本研究的结果表明，Syk选择性酪氨酸磷酸化氧化带3可能在对nab具有高亲和力的大膜聚集体中氧化带3的募集中发挥作用，导致红细胞从循环中清除。
Oxidative events involving band 3 (Anion Exchanger 1) have been associated with RBC (red blood cell) removal through binding of NAbs (naturally occurring antibodies); however, the underlying mechanism has been only partially characterized. In addition to inducing direct membrane protein oxidative modification, oxidative treatment specifically triggers the phosphorylation of band 3 tyrosine residues. The present study reports that diamide, a thiol group oxidant, induces disulfide cross-linking of poorly glycosylated band 3 and that the oligomerized band 3 fraction is selectively tyrosine phosphorylated both in G6PD (glucose-6-phosphate dehydrogenase)-deficient and control RBCs. This phenomenon is irreversible in G6PD-deficient RBCs, whereas it is temporarily limited in control RBCs. Diamide treatment caused p72 Syk phosphorylation and translocation to the membrane. diamide also induced p72 Syk co-immunoprecipitation with aggregated band 3. Moreover, following size-exclusion separation of Triton X-100-extracted membrane proteins, Syk was found only in the high-molecular-mass fraction containing oligomerized/phosphorylated band 3. Src family inhibitors efficiently abrogated band 3 tyrosine phosphorylation, band 3 Clustering and NAbs binding to the RBC surface, suggesting a causal relationship between these events. Experiments performed with the non-permeant cross-linker BS3 (bis-sulfosuccinimidyl-suberate) showed that band 3 tyrosine phosphorylation enhances its capability to form large aggregates. The results Of the present Study Suggest that selective tyrosine phosphorylation of oxidized band 3 by Syk may play a role in the recruitment of Oxidized band 3 in large membrane aggregates that show a high affinity to NAbs, leading to RBC removal from the circulation.