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.

利用逆转录病毒载体在K562细胞和HEK 293细胞中建立了正常牛带3（bebWT）或带3的R664 X突变体（bebRX）的稳定转染子。转染细胞表达EGFP-bebWT和EGFP-bebRX以及锚蛋白N端结构域（AnkN 90）和带3蛋白，bebWT和EGFP-bebWT在细胞质膜上稳定表达，而突变体蛋白bebRX和EGFP-bebRX在内质网合成后或从高尔基体逆行转运至内质网后不久即被Ub-蛋白酶体系统降解。当bebRX共转染时，bebWT的稳定性极大降低。这些结果表明，在红系发育过程中，带3突变体（bebRX）对正常带3和膜骨架中的配偶体锚蛋白（ankyrin）的表达起显性负调控作用，带3与锚蛋白的相互作用发生在带3合成启动后不久的ER膜上。

项目成果

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, 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)。

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。 ）

Matsuki, N. ほか2名: "Catabolism of cytoplasmic and mitochondria adenosine nucleotides in C2C12 skeletal myotube under chemical hypoxia"Journal of Veterinary Medical Science. 64. 341-347 (2002)

Matsuki, N. 和其他 2 人：“化学缺氧下 C2C12 骨骼肌管中细胞质和线粒体腺苷核苷酸的分解代谢”《兽医医学科学杂志》64. 341-347 (2002)。