Kurachi S, Pantazatos D P, Kurachi K
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109-0618, USA.
Biochemistry. 1997 Apr 8;36(14):4337-44. doi: 10.1021/bi962002v.
The carboxyl-terminal region of factor IX (residues 403-415) contains several natural mutations which result in mild to severe forms of hemophilia B. A battery of factor IX minigene expression vectors carrying various mutations in the C-terminal region were constructed and studied by transient expression assay using HepG2 cells. Mutations included in this study are Y404P, I408N, T412N, T412S, T415G, T415S, T415L, and T415R as well as five selected naturally occurring mutations in the region, R403Q, R403W, Y404H, W407R, and T412K. In comparison to the normal factor IX, these mutations neither significantly affected the factor IX mRNA level nor affected the stability of the secreted factor IX in the culture medium but did decrease to various extents the intracellular and secreted factor IX protein levels as quantified by enzyme-linked immunosorbent assay. T415L, T415S, and T415R showed only minor reductions in the intracellular and minor to moderate reductions in the secreted factor IX levels. T415G showed only minor reduction in the intracellular factor IX level but substantial reduction in the secreted levels. R403Q, R403W, and T412S showed moderate reductions in both intracellular and secreted factor IX levels. Y404H, Y404P, W407R, I408N, T412K, and T412N also showed minor to moderate reductions in the intracellular factor IX levels but very severe reductions in the secreted factor IX level. In one stage clotting assays, secreted factor IX mutants showed specific activities very similar to that of the normal factor IX, suggesting that the carboxyl-terminal region is neither directly involved in the tenase complex formation required for factor X activation nor involved in the activation of factor IX itself. In comparison to the normal factor IX, secreted levels of factor IX mutants with mutations R403Q, Y404H, W407R, and T412K were also very similar to the plasma levels reported for the hemophilia B patients carrying the same mutations. Treatment of cells with proteasome inhibitors (ALLM and ALLN) added to the culture medium at 50 microM resulted in drastic increases of the intracellular mutant factor IX to the levels equivalent to that of the normal factor IX, while the secreted factor IX levels were little or only marginally affected. With a higher concentration of the inhibitors (100 microM), however, both the intracellular and secreted mutant factor IX were fully elevated to the normal factor IX levels. Intracellular and secreted levels of the normal factor IX were little affected by the low inhibitor concentration and only marginally, if at all, at the higher concentration (< or = 10%). Serine protease inhibitors did not significantly affect intracellular and secreted factor IX levels. These results indicate that the carboxyl-terminal region plays a critical role in the cellular secretion of factor IX and that the mutant factor IX proteins carrying specific mutations in this region are subjected to efficient elimination by the proteasome protein degradation mechanism. Furthermore, these results strongly support that HepG2 cells can be utilized as a robust in vitro assay system for studying factor IX biosynthesis, well mimicking the in vivo phenomenon.
凝血因子IX的羧基末端区域（第403 - 415位氨基酸残基）存在多种自然突变，这些突变会导致不同程度的B型血友病。构建了一系列在C末端区域携带各种突变的凝血因子IX小基因表达载体，并利用HepG2细胞通过瞬时表达分析进行研究。本研究中包含的突变有Y404P、I408N、T412N、T412S、T415G、T415S、T415L和T415R，以及该区域五个选定的自然发生的突变R403Q、R403W、Y404H、W407R和T412K。与正常凝血因子IX相比，这些突变既未显著影响凝血因子IX的mRNA水平，也未影响其在培养基中分泌的稳定性，但通过酶联免疫吸附测定法测定，它们在不同程度上降低了细胞内和分泌的凝血因子IX蛋白水平。T415L、T415S和T415R仅使细胞内凝血因子IX有轻微降低，而分泌的凝血因子IX水平有轻微至中度降低。T415G仅使细胞内凝血因子IX水平有轻微降低，但分泌水平大幅降低。R403Q、R403W和T412S使细胞内和分泌的凝血因子IX水平均有中度降低。Y404H、Y404P、W407R、I408N、T412K和T412N也使细胞内凝血因子IX水平有轻微至中度降低，但分泌的凝血因子IX水平有非常严重的降低。在一期凝血试验中，分泌的凝血因子IX突变体的比活性与正常凝血因子IX非常相似，这表明羧基末端区域既不直接参与激活因子X所需的凝血酶原酶复合物的形成，也不参与凝血因子IX自身的激活。与正常凝血因子IX相比，携带R403Q、Y404H、W407R和T412K突变的凝血因子IX突变体的分泌水平也与报道的携带相同突变的B型血友病患者的血浆水平非常相似。向培养基中添加50微摩尔的蛋白酶体抑制剂（ALLM和ALLN）处理细胞，导致细胞内突变型凝血因子IX急剧增加至与正常凝血因子IX相当的水平，而分泌的凝血因子IX水平几乎没有或仅受到轻微影响。然而，使用更高浓度的抑制剂（100微摩尔）时，细胞内和分泌的突变型凝血因子IX均完全升高至正常凝血因子IX水平。低浓度抑制剂对正常凝血因子IX的细胞内和分泌水平影响很小，而在高浓度（≤10%）时影响也微乎其微。丝氨酸蛋白酶抑制剂对细胞内和分泌的凝血因子IX水平没有显著影响。这些结果表明，羧基末端区域在凝血因子IX的细胞分泌中起关键作用，并且在该区域携带特定突变的突变型凝血因子IX蛋白会通过蛋白酶体蛋白降解机制被有效清除。此外，这些结果有力地支持了HepG2细胞可作为研究凝血因子IX生物合成的强大体外检测系统，很好地模拟体内现象。
