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目的:Abba1在体外实验中能介导细胞产生ruffling,其N端的IMD区域中13个赖氨酸为重要的功能氨基酸位点,但Abba1单独的IMD却不足以介导细胞产生ruffling,本研究旨在初步探讨Abba1的C端序列影响细胞产生ruffling的分子机制。方法:先构建Abba1基因重组表达质粒,分别瞬时转染COS7细胞,通过免疫荧光观察细胞形态改变,并用Western blot检测相应GTP-Rac1的蛋白水平变化。结果:过表达全长Abba1的COS7细胞形成ruffling,与对照组相比,细胞中GTP-Rac1蛋白水平升高(P<0.05);过表达Abba1-IMD和Abba1-del-357-722aa-WH2的COS7细胞不形成ruffling,与对照组相比,相应细胞中GTP-Rac1蛋白水平降低(P<0.05)。结论:Abba1的357-722aa这段区域参与介导细胞产生ruffling,而且这种影响与GTP-Rac1蛋白水平的降低有关。
OBJECTIVE: Abba1 mediates ruffling of cells in vitro and 13 lysine residues in the N-terminal IMD region are important functional amino acid sites. However, Abba1 alone is not enough to mediate ruffling in cells. The molecular mechanism of cellular ruffling in Cb sequences affecting Abba1 was preliminary investigated. Methods: The recombinant plasmid of Abba1 gene was constructed and transiently transfected into COS7 cells. The morphological changes of cells were observed by immunofluorescence and the protein level of GTP-Rac1 was detected by Western blot. Results: The overexpression of full-length Abba1 COS7 cells formed ruffling, compared with the control group, the level of GTP-Rac1 protein increased (P <0.05); overexpression of Abba1-IMD and Abba1-del-357-722aa-WH2 COS7 cells did not form ruffling, and compared with the control group, GTP-Rac1 protein level in the corresponding cells was decreased (P <0.05). CONCLUSIONS: The region 357-722aa of Abba1 is involved in mediating ruffling of cells, and this effect is associated with a decrease in GTP-Rac1 protein levels.