Molecular mechanism for the assembly of red cell membrane skeletons based on pathobiology of congenital hemolytic anemia in cattle

基于牛先天性溶血性贫血病理学的红细胞膜骨架组装分子机制

• 批准号: 12460137
• 负责人: INABA Mutsumi
• 金额: $ 9.15万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12460137/
• 关键词: red cell membranes; membrane skeleton; band 3; ankyrin; congenital hemolytic anemia; proeasome; vesicle trafficking; cattle; 遺伝性溶血性貧; プロテアソーム; 膜蛋白質; 膜移行; 遺伝性バンド3欠損症

Stable transfectants of normal bovine band 3 (bebWT) or the mutant band 3 with R664X mutation (bebRX) were established in K562 cells and HEK293 cells using retoriviral vectors. Transfected cells expressing EGFP-bebWT and EGFP-bebRX, and N-terminal domain of ankyrin (AnkN90) in combination with band 3 proteins were also prepared.The bebWT and EGFP-bebWT showed stable expression on the plasma membrane of the transfected cells, whereas the mutant proteins, bebRX and EGFP-bebRX were degraded by Ub-proteasome system soon after synthesis on the ER or after retrograde transported from the Golgi apparatus to the ER. Stability of the bebWT was extremely reduced when bebRX was co-transfected. AnkN90 showed membrane localization within the cells and was destabilized in the cells that had the mutant band 3.These findings indicate that the mutant band 3 (bebRX) plays a dominant-negative role on the expression of normal band 3 and a partner in the membrane skeleton, ankyrin, and the interaction of band 3 with ankyrin occurs on the ER membrane soon after band 3 synthesis is started during erythroid development.

使用反胎病毒载体在K562细胞和HEK293细胞中建立了正常牛带3（BEBWT）或具有R664X突变（BEBRX）的突变体3的稳定转染物。还制备了表达EGFP-BEBWT和EGFP-BEBRX的转染细胞，并且还制备了与BARS 3蛋白结合的Ankyrin（Ankn90）的N末端结构域（ANKN90）。BEBWT和EGFP-BEBWT在质膜上显示了稳定的质膜表达，均显示出稳定的。突变蛋白，BEBRX和EGFP-BEBRX在ER合成后不久或从高尔基体转运到ER后不久就会通过UB-蛋白酶体系统降解。当Bebrx共转染时，BEBWT的稳定性将极大地降低。 ANKN90显示在细胞中的膜定位，并在具有突变带的细胞中不稳定3.这些发现表明突变带3（BEBRX）在正常带3的表达中起着显性的作用，并且在膜上伴侣在红细胞发育期间开始，骨架3和骨与Ankyrin的相互作用发生在带3合成后的ER膜上。

项目成果

Sato, K, ら7名: "Inherited defects of Na-dependent glutamate transport mediated by GLAST in canine red cells due to a decreased level of transporter protein expression"Journal of Biological Chemistry. 275. 6620-6627 (2000)

Sato, K 等人：“由于转运蛋白表达水平降低，导致犬红细胞中 GLAST 介导的 Na 依赖性谷氨酸转运的遗传缺陷”《生物化学杂志》275. 6620-6627 (2000)。

Shimizu, R., Takahashi, S., Ohneda, K., Engel, J.D., and Yamamoto, M.: "In vivo requirements for GATA-1 functional domains during primitive and definitive erythropoiesis"EMBO J.. 20. 5250-5260 (2001)

Shimizu, R.、Takahashi, S.、Ohneda, K.、Engel, J.D. 和 Yamamoto, M.：“原始和最终红细胞生成过程中 GATA-1 功能域的体内要求”EMBO J.. 20. 5250-5260

Sato,K.,Inaba,M.,Suwa,Y 他4名: "Inherited defects of Na-dependent glutamate transport mediated by glutamate/aspartate transporter in canine red cells due to a decreased level of transporter protein expression."Journal of Biological Chemistry. 275. 6620-6627

Sato, K.、Inaba, M.、Suwa, Y 和其他 4 人：“由于转运蛋白表达水平降低，导致犬红细胞中谷氨酸/天冬氨酸转运蛋白介导的 Na 依赖性谷氨酸转运的遗传性缺陷。”《生物学杂志》化学275。6620-6627

Koshino,I,Inaba,M.,Matsumoto,M 他3名: "Membrane trafficking defect of premature termination mutant band 3 leading to spherocytosis with nearly normal membrane skeletons in cattle."Journal of Biological Chemistry. 276(In press). (2001)

Koshino, I、Inaba, M.、Matsumoto, M 和其他 3 人：“过早终止的突变带 3 的膜运输缺陷导致牛的膜骨架接近正常的球形红细胞增多症”，《生物化学杂志》2001 年。 ）

An, X.-L., ら6名: "Structural and functional characterization of protein 4.1R-phosphatidylserine interaction. Potential role in 4.1R dorting within cells"Journal of Biological Chemistry. 276. 35778-35785 (2001)

An，X.-L.，等：“蛋白质 4.1R-磷脂酰丝氨酸相互作用的结构和功能表征。细胞内 4.1R 休眠的潜在作用”生物化学杂志 276. 35778-35785 (2001)。