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

研究了红细胞发育过程中细胞膜骨架的“单元装配假说”。利用逆转录病毒载体在K562和HEK293细胞中构建了红细胞跨膜蛋白主要成分的稳定转染体。带3和糖蛋白通过内质网/高尔基体途径转运到质膜。在带3转染的细胞中，锚蛋白带3结合域（AnkN90）和蛋白4.1在细胞质中呈现膜性定位，与内质网和高尔基体标记共定位，尽管红细胞分化不同。内源性膜骨架蛋白包括锚蛋白、蛋白4.1和谱蛋白也呈膜状分布。这些结果表明，在红细胞发育的早期阶段，膜骨架蛋白的组装发生在内质网表面，而不是在质膜的内表面。band 3的稳定表达使K562细胞的增殖率降低到未转染细胞的70%左右。血红蛋白的减少表明红细胞分化也被观察到。然而，在两相液体培养体系中，牛外周血红前体细胞中未观察到涉及谱蛋白的骨蛋白合成并组装到内质网，随后合成并定位到带3的质膜，表明骨膜蛋白组装到内质网发生在生理性红细胞生成过程中。本研究表明，红细胞骨架的组装是发生在内质网红细胞发育早期的一个事件。在细胞内囊泡上形成的小骨架单元被整合到质膜上的确切机制尚不清楚。

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.