TSP2-8和CUB1+2片段对血管性血友病因子裂解酶分泌方向的影响  

Effects of TSP2-8 and CUB1+2 Domains on Secretion Direction of Von Willebrand Factor-cleaving Protease

作　　者：高单萍[1] 刘琼[1] 陈素华[1] 艾继辉[1] 

机构地区：[1]华中科技大学同济医学院同济医院妇产科,湖北武汉430022

出　　处：《中国实验血液学杂志》2011年第4期964-967,共4页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目;编号30671093

摘　　要：本研究旨在探讨羧基端TSP2-8和CUB1+2片段是否决定血管性血友病因子裂解酶(ADAMTS13)合成后细胞内运输的方向,以了解金属蛋白酶ADAMTS13羧基端结构与功能的关系。利用脂质体转染技术将重组质粒pcDNA3.1-ADAMTS13及pcDNA3.1-delTSP2-8CUB1+2ADAMTS13分别转染犬肾上皮极性细胞MDCK,筛选出阳性细胞克隆后传代铺到中间有特殊分子筛膜的双池培养皿内培养,待细胞生长到无空隙后收集上、下池培养液;通过Western blot检测上、下池培养液中ADAMTS13蛋白表达水平,以对比分析ADAMTS13蛋白在极性细胞内的分泌方向。结果表明,稳定表达野生型ADAMTS13的MDCK细胞组,在分子筛膜的上池培养液中检测到ADAMTS13蛋白,而表达缺失TSP2-8CUB1+2区域ADAMTS13的细胞组,在分子膜的上、下池培养液中均检测到ADAMTS13重组蛋白。结论:金属蛋白酶ADAMTS13的分泌是有极性的,且羧基端TSP2-8和CUB1+2结构域与ADAMTS13合成后细胞内的运输方向密切相关。This study was aimed to explore if the intracellular transportation direction of von Willebrand factor-cleaving protease （ADAMTSI3 ,vWF-CP） after synthesis is determined by the carboxyl terminal TSP2-SCUBI ＋ 2 domains of ADAMTS13 and to decipher the relationship between the structure and function of ADAMTS13. The recombinant plasmids pcDNA3.1-ADAMTS13 and pcDNA3.1-delTSP2-8CUB1 ＋ 2 ADAMTS13 were introduced into Madin-Darby canine kidney cells （MDCK） by lipofectamine-mediated DNA transfection. Positive cell clones gained after antibioticscreening were grown on 6-well transwell filter units with a zeolite membrane in the middle layer. The conditioned culture media in both apical and basolateral wells were collected when cells reached confluency and the tight cell monolayer formed. ADAMTS13 proteases in the conditioned media were determined by Western blot, and the direction of ADAMTS13 secretion in polarized cells was comparatively analyzed. The results showed that Madin-Darby canine kid- ney cells stably expressing wild-type ADAMTS13 were grown on 6-well transwell filter units, then ADAMTS13 protease was only determined in the apical area of the transwell filter units by Western blot, but the recombinant ADAMTS13 protease was determined both in the apical and basolateral area of cells in the group of expressing TSP2-SCUB-1 ＋ 2 domain-deleted ADAMTS13. It is concluded that the metailoprotease ADAMTS13 is sorted apically in polarized cells, and the carboxyl-terminal TSP2-8 and CUB1 ＋ 2 domains of ADAMTS13 are important for the direction of ADAMTS13 protease transportation in the cells after being synthesized.

关 键 词：血管性血友病因子裂解酶 TSP2-8 CUB1+2 

分 类 号：R554.1[医药卫生—血液循环系统疾病]