Expression and release of the a and b subunits for human coagulation factor XIII in baby hamster kidney (BHK) cells.
Expression and release of the a and b subunits for human coagulation factor XIII in baby hamster kidney (BHK) cells.
复制标题
人凝血因子 XIII 的 a 和 b 亚基在幼仓鼠肾 (BHK) 细胞中的表达和释放。
DOI：
10.1093/oxfordjournals.jbchem.a021336
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发表时间：
1996
影响因子：
2.7
通讯作者：
Ichinose,A
中科院分区：
文献类型：
--
作者：
Kaetsu,H;Hashiguchi,T;Foster,D;Ichinose,A
凝血因子XIII的a亚基缺乏从细胞分泌的疏水信号序列，而B亚基具有典型的信号序列。为了确定α亚基是否可以合成和释放，将含有任一亚基的cDNA的表达载体转染到幼仓鼠肾（BHK）细胞中。蛋白质印迹分析和凝胶过滤层析证明重组的a和B亚基（rXIIIb和rXIIIb）具有与天然分子相同的分子量和亚基结构（rXIIIb 2、b2和rXIIIb 2b2）。当被凝血酶激活时，rXIII具有酶活性。通过ELISA测量，大多数rXIIIb被分泌，而通过亚细胞分级分离在胞质溶胶中检测到大多数rXIIIb。在相同的细胞中与rXIIIb共表达不会促进rXIIIb的释放。用布雷菲德菌素A（一种有效的蛋白质转运抑制剂）处理细胞，阻断了rXIIIb的分泌，尽管它对rXIIIb的释放没有影响。几种药物和热应激诱导rXIII？的释放，这与细胞质乳酸脱氢酶的释放直接相关。这些结果表明，α亚基从细胞中释放作为细胞损伤的结果，这是不依赖于经典的分泌途径。
The a subunit of coagulation factor XIII lacks a hydrophobic signal sequence for secretion from cells, while the b subunit has a typical signal sequence. To determine whether the a subunit can be synthesized and released, expression vectors containing the cDNA for either subunit were transfected into baby hamster kidney(BHK) cells. Western blotting analysis and gel filtration chromatography demonstrated that the recombinant a and b subunits (rXIIIƒ¿ and rXIIIb) had the same molecular weights and subunit structures(ƒ¿ 2, b2, and ƒ¿ 2b2) as the native molecules. rXIIIƒ¿ was enzymatically active when activated by thrombin. Most rXIIIb was secreted as measured by ELISA, while most rXIIIƒ¿ was detected in the cytosol by subcellular fractionation. Co-expression with rXIIIb in the same cells did not promote the release of rXIIIƒ¿. Treatment of the cells with brefeldin A, a potent inhibitor of protein transportation, blocked the secretion of rXIIIb, although it had no effect on the release of rXIIIƒ¿. Several drugs and heat stress induced the release of rXIIIƒ¿, which correlated directly with that of cytoplasmic lactate dehydrogenase. These results suggest that the a subunit is released from cells as a consequence of cell injury, which is indepen dent of the classical secretory pathway.