Camani C, Gavin O, Bertossa C, Samatani E, Kruithof E K
Division of Angiology and Hemostasis, University Hospital Geneva, Switzerland.
Eur J Biochem. 1998 Feb 1;251(3):804-11. doi: 10.1046/j.1432-1327.1998.2510804.x.
Previously we observed that the finger/growth factor (FG) region of tissue-type plasminogen activator (t-PA) blocked the low-density-lipoprotein-receptor-related protein (LRP)-mediated clearance of t-PA by rat hepatocytes. However, the concentrations needed were much higher than those for intact t-PA. The FG region was expressed in yeast and lacked the fucose on Thr61, which was reported to be important for efficient clearance of t-PA by human hepatoma cells. At position 83 it had a serine, whereas human t-PA has a free cysteine and rodent t-PA an arginine at this position. To understand the reason for the low efficacy of the FG protein we produced in CHO cells chimeric molecules composed of two FG modules linked to the Fc portion of human IgG1 (FG2-Fc). Two variants were studied, one having Ser83, the other Arg83. The two fucosylated FG2-Fc chimeras were compared with each other, with non-fucosylated FG and with intact t-PA with regard to their effect on the clearance of t-PA and t-PA x plasminogen-activator inhibitor type 1 (PAI-1). For this comparison, LRP-specific clearance models were used. In rat hepatoma cells and in mouse embryonic fibroblasts (MEF-1) the clearance of t-PA and of t-PA x PAI-1 was inhibited more than 95% by receptor-associated protein, an inhibitor of LRP-mediated clearance, whereas no t-PA or t-PA x PAI-1 clearance was observed in LRP-deficient PEA-13 mouse embryonic fibroblasts. The Ser83 and Arg83 FG2-Fc chimeras were equally efficient inhibitors in these models. Their efficacies in inhibiting t-PA and t-PA x PAI-1 degradation (IC50 750 nM and 890 nM, respectively) were similar to those of non-fucosylated FG (IC50 1950 nM and 1560 nM) and 75-fold lower than that of intact t-PA (IC50 9.9 nM and 21.1 nM). The results indicate that the presence of a serine or an arginine at position 83 and the presence of a fucose on Thr61 are not of major importance for the LRP-mediated clearance of t-PA.
此前我们观察到，组织型纤溶酶原激活剂（t-PA）的指状/生长因子（FG）区域可阻断大鼠肝细胞通过低密度脂蛋白受体相关蛋白（LRP）介导的t-PA清除。然而，所需浓度远高于完整t-PA的浓度。FG区域在酵母中表达，且苏氨酸61上缺乏岩藻糖，据报道该岩藻糖对人肝癌细胞有效清除t-PA很重要。在第83位它有一个丝氨酸，而人t-PA在此位置有一个游离半胱氨酸，啮齿动物t-PA在此位置有一个精氨酸。为了解我们在CHO细胞中产生的FG蛋白疗效低的原因，我们构建了由两个FG模块与人类IgG1的Fc部分相连的嵌合分子（FG2-Fc）。研究了两个变体，一个在第83位是丝氨酸，另一个在第83位是精氨酸。将两种岩藻糖基化的FG2-Fc嵌合体相互比较，并与非岩藻糖基化的FG以及完整t-PA在对t-PA和t-PA×纤溶酶原激活剂抑制剂1型（PAI-1）清除的影响方面进行比较。为进行此比较，使用了LRP特异性清除模型。在大鼠肝癌细胞和小鼠胚胎成纤维细胞（MEF-1）中，LRP介导清除的抑制剂受体相关蛋白可使t-PA和t-PA×PAI-1的清除率降低95%以上，而在缺乏LRP的PEA-13小鼠胚胎成纤维细胞中未观察到t-PA或t-PA×PAI-1的清除。在这些模型中，第83位为丝氨酸和精氨酸的FG2-Fc嵌合体是同样有效的抑制剂。它们抑制t-PA和t-PA×PAI-1降解的效力（IC50分别为750 nM和890 nM）与非岩藻糖基化的FG（IC50分别为1950 nM和1560 nM）相似，比完整t-PA（IC50分别为9.9 nM和21.1 nM）低75倍。结果表明，第83位存在丝氨酸或精氨酸以及苏氨酸61上存在岩藻糖对于LRP介导的t-PA清除并非至关重要。
