Effects of TSP2-8 and CUB1+2 domains on secretion direction of von Willebrand factor-cleaving protease / 中国实验血液学杂志

ABSTRACT

This study was aimed to explore if the intracellular transportation direction of von Willebrand factor-cleaving protease (ADAMTS13, vWF-CP) after synthesis is determined by the carboxyl terminal TSP2-8CUB1+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 antibiotic-screening 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 kidney 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 TSP2-8CUB-1+2 domain-deleted ADAMTS13. It is concluded that the metalloprotease 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.