Purification of the NF2 tumor suppressor protein from human erythrocytes.
Purification of the NF2 tumor suppressor protein from human erythrocytes.
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从人红细胞中纯化 NF2 肿瘤抑制蛋白。
DOI：
10.1017/s0317167100005357
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Chishti,AtharH
中科院分区：
文献类型：
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作者：
Jindal,HiteshK;Yoshinaga,Kazumi;Seo,Pil-Soo;Lutchman,Mohini;Dion,PatrickA;Rouleau,GuyA;Hanada,Toshihiko;Chishti,AtharH
背景 2 型神经纤维瘤病 (NF2) 是一种常染色体显性遗传疾病，使个体易患脑神经和脊神经肿瘤的风险。 NF2 肿瘤抑制蛋白（称为 Merlin/Schwanomin）是蛋白 4.1 超家族的成员，其功能是连接细胞骨架和质膜。方法使用低离子强度溶液从红细胞质膜（鬼影）中选择性提取膜相关蛋白后，大部分 NF2 蛋白仍然与耗尽了血影蛋白-肌动蛋白的内而外囊泡相关。蛋白质印迹分析显示红细胞质膜中有约 70 kDa 的多肽。此外，使用 1.0 M 碘化钾（一种已知可去除紧密结合的外周膜蛋白的处理方法）实现了从内向外囊泡中定量去除 NF2 蛋白。 结果这些结果表明 NF2 蛋白与红细胞膜结合的一种新模式，与已知的蛋白 4.1 的膜相互作用不同。基于这些生化特性，设计了几种纯化策略来从人红细胞血影中分离天然 NF2 蛋白。使用纯化和重组的 NF2 蛋白作为内标，我们对每个红细胞约 41-65,000 个 NF2 蛋白分子进行了定量。结论我们为人红细胞膜中存在 NF2 蛋白提供了证据。人红细胞膜中 NF2 蛋白的鉴定将使利用哺乳动物细胞中强大的红细胞生物化学和遗传学技术发现 NF2 蛋白的新相互作用成为可能。
BackgroundNeurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane.MethodsUpon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins.ResultsThese results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte.ConclusionWe provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells.