Molecular mechanisms for the assembly of the red cell membrane skeleton during erythroid cell development

为探讨红细胞膜骨架在红系发育过程中的“单元组装假说”，利用逆转录病毒载体在K562和HEK 293细胞中构建了红细胞膜骨架主要成分的稳定转染子。带3和血型糖蛋白C通过ER/Golgi途径转运到质膜。带3结合结构域的锚蛋白（AnkN 90）和蛋白4.1表现出膜定位在细胞质中的共定位与标记的ER和高尔基体在带3转染细胞，尽管在红系分化的差异。内源性膜骨架蛋白，包括锚蛋白，蛋白4.1，血影蛋白也表现出膜分布。这些结果表明，膜骨架蛋白的组装发生在红系发育的早期阶段的ER的表面上，而不是在质膜的内表面上。带3的稳定表达引起K562细胞的增殖率降低到约70%的非转染细胞。还观察到血红蛋白产生减少，提示红系分化。牛外周血红系前体细胞在两相液体培养系统中，观察到了包括血影蛋白在内的骨架蛋白的合成和向内质网的组装，以及随后的带3的合成和向质膜的定位，本研究表明，红细胞膜骨架的组装发生在红系发育的早期阶段，急诊室细胞内小泡上形成的一小部分聚结被整合到质膜上的确切机制仍有待阐明。

The "unit-assembly hypothesis" for red cell membrane skeleton during erythroid development was investigated.Stable transfectants of major constituents of red cell transmembrane proteins were developed in K562 and HEK293 cells using retroviral vectors. Band 3 and glycophorinCweretransportedtotheplasmamembranevia the ER/Golgi pathway. Band 3-binding domain of ankyrin (AnkN90) and protein 4.1 showed membranous localization in the cytoplasm with co-localization with markers for the ER and the Golgi apparatus in band 3-transfeted cells despite of the difference in erythroid differentiation. Endogenous membrane skeletal proteins including ankyrin, protein 4.1, and spectrin also exhibited membranous distribution. These results indicate that the assembly of membrane skeletal proteins occurs at an early stage of erythroidal development on the surface of the ER but not on the inner surface of the plasma membrane.Stable expression of band 3 caused a reduction in a proliferation rate of K562 cells to approximately 70% that of non-transfected cells. Reduced production of hemoglobin suggestive of erythroid differentiation was also observed. However, no significant changes in intracellular localization of skeletal proteins and cell morphology was detected Synthesis and assembly to the ER of skeletal proteins involving spectrin and subsequent synthesis and localization to the plasma membrane of band 3 were observed in erythroid precursor cells from peripheral blood of cattle in two phase liquid culture system, indicating that the assembly of membrane skeletal proteins to the ER occurs in physiolosical erythropoiesis.The present study demonstrated that the assembly of the red cell membrane skeleton is an event which takes place at early stages of erythroid development on the ER. The exact mechanism by which a small unit of the ekeleton formed on intracellular vesicles are integrated in the plasma membrane remained to be clarified.