Mechanism in the secretion of FXI variant with a novel mutation near the C-terminal region

为了阐明FXI W599R分泌的机制，我们进行了表达实验，并利用从rhFXI37O-607-Ecotin复合体(1XXd.PBD)中提取的FXIa单体的坐标可视化了FXIW599R的结构模型。结构模拟表明，W599残基位于FXI分子C-末端的α螺旋上。该残基属于人FXI、兔FXI、牛FXI、大鼠FXI和小鼠FXI序列中的结构保守区。结果表明，FXI基因C末端的α螺旋中的W599可能在细胞分泌过程中具有重要的结构意义。为了研究FXIW599R对FXI生物合成的影响，利用…在COS-1细胞和CHO-K1细胞中进行了表达实验进一步将含有野生型和突变型FXIcDNA的pcDNA3.0 Zeo载体克隆到pc DNA3.0 Zeo载体中。条件培养上清中FXIW599R的FXIc和FXIag水平为1%，胞内FXI的含量为野生型重组蛋白的60%~70%。FXIW599G、FXIW599E和FXIW599F的结果与FXIW599R相同。实时定量转录分析证实野生型pCDNAXI和pCDNAXI599R对培养细胞的基因转录和信使核糖核酸加工没有影响。结果表明，分别转染人XI599R和野生型PcDNAXI的培养细胞中FXI基因的相对表达量是相同的。在非还原条件下，野生型和突变型Fxi均以二硫键连接的二聚体形式存在于细胞中，在非还原条件下，Fxi的大小为160 KDa，少量单体对应于80 KDa。在蛋氨酸脉冲追踪研究中，条件培养液中突变的Fxi在各个时间点均明显低于野生型Fxi。我们分析了内质网糖基化的强效特异性抑制剂衣霉素、蓖麻黄素和诺吉霉素的作用，以及20S蛋白酶体降解的强效特异性抑制剂lactacystin和溶酶体降解的抑制剂亮肽素的作用，以探讨FXI在稳定表达的CHO细胞中积聚和降解的机制。胞内FXI W599R的表达水平不受衣霉素、蓖麻黄素和诺吉霉素的影响。FXI W599R的细胞内水平在亮肽素和lacticystain的存在下没有变化，而突变体Fxi的W599R水平在Brefield din A的存在下降低。

Mechanism in the secretion of congenital FXI variant with a novel mutation near the C-terminal regionTo elucidate the mechanism in the secretion of FXI W599R, we studied the expression experiments.And, the structural model of FXIW599R was visualized using the co-ordinates of FXIa monomer extracted from the rhFXI37O-607-ecotin complex (1XXd.PBD). Structural modeling showed that the W599 residue is positioned on an alpha helix in the C-terminal region of the FXI molecule. This residue belongs to a structurally conserved region among human FXI, rabbit FXI, bovine FXI, rat FXI and mouse FXI sequences. It was suggested that W599 in the alpha helix at the C-terminal region FXI may be structurally important for secretion from cells.FXI cDNA with the unique restriction enzyme sites were amplified by PCR using single strand cDNA from normal human liver. To investigate the influence of the FXIW599R on FXI biosynthesis, the expression experiments were performed in COS-1 cells and CHO-K1 cells usi … More ng the pcDNA3.0 Zeo vector containing either wild type FXIcDNA or mutant FXIcDNA. FXIc and FXIag levels of FXIW599R secreted into the conditioned media were <1%, and the intracellular FXI was present at 60 to 70% of the wild type recombinant protein level in cells. And FXIW599G, FXIW599E and FXIW599F were same results as FXIW599R. The real-time quantitative transcript analysis were performed to confirm that gene transcription and mRNA processing were not impaired in the cultured cells transfected the wild type pcDNA XI and pcDNA XI599R. It was demonstrated that comparative amounts of FXI mRNA were same in cultured cells transfected the pcDNA XI wild type and pcDNA XI599R, respectively. Both wild type FXI and mutant types were present in cells as disulfide bond-linked dimer corresponding to 160KDa with a small amount of monomer corresponding to 80KDa under non-reducing condition on a 7.5% SDS-PAGE using the intracellular FXI labeled with ^<35>S-methionin. The mutant FXI in the conditioned media was much lower than that of wild-type FXI at all time points in pulse-chase studies with [^<35>S] methionine. We analyzed the effects of tunicamycin, castanosperin and nojirimycin, potent specific inhibitor of glycosylation in ER, and the effects of lactacystin, potent specific inhibitor of 20S-proteasome degradation and leupeptin, inhibitor of lysosomal degradation to investigate the mechanism of accumulation and degradation of FXI in stably transfected CHO cells. The intracellular levels of FXI W599R did not change in the presence of tunicamycin, castanosperin and nojirimycin. The intracellular levels of FXI W599R did not change in the presence of leupeptin and lactacystain, whereas those of the mutant FXI decreased in the presence of brefeldin A. Less