Zhong D, Smith K J, Birktoft J J, Bajaj S P
Department of Pathology, Saint Louis University School of Medicine, MO 63104.
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3574-8. doi: 10.1073/pnas.91.9.3574.
Factor IX consists of a gamma-carboxyglutamic acid-rich domain followed by two epidermal growth factor (EGF)-like domains and the C-terminal protease domain. To delineate the function of EGF1 domain in factor IX, we constructed three mutants: an EGF1 domain-deleted mutant (IX delta EGF1), a point mutant (IXQ50P) with a Gln-50-->Pro change, and a replacement mutant (IXPCEGF1) in which the EGF1 domain of factor IX was replaced by that of protein C. These mutants and wild-type (WT) factor IX (IXWT) were expressed in 293 kidney cells by using pRc/CMV vector. The purified proteins had the same gamma-carboxyglutamic acid content as the normal plasma factor IX (IXNP) and were activated normally by factor XIa-Ca2+. In contrast, IX delta EGF1 could not be activated by factor VIIa-tissue factor-Ca2+, and the activation of IXPCEGF1 in this system was markedly slow; however, IXQ50P was activated at a normal rate. In additional studies, both IXWT and IX delta EGF1 were rapidly converted to their respective IX alpha forms by factor Xa-phospholipid-Ca2+. Since this reaction has an absolute requirement for phospholipid, it indicates that the mutants under study are not impaired in their interactions with phospholipid. Relative coagulant activities of factor XIa-activated proteins were IXNP, 100%; IXWT, 75-85%; IX delta EGF1, < or = 1%; IXPCEGF1, < or = 2%; and IXQ50P, 6-10%. We conclude that the EGF1 domain of factor IX is required for its activation by factor VIIa-tissue factor and that the Gln-50 residue is not critical for this activation. Further, the EGF1 domain of factor IX is not essential for phospholipid binding and for its activation by factor XIa. In addition, the low coagulant activities of the activated mutants indicate that the EGF1 domain is also important in factor X activation by factor IXa-factor VIIIa-Ca(2+)-phospholipid complex.
因子IX由富含γ-羧基谷氨酸的结构域、两个表皮生长因子（EGF）样结构域和C端蛋白酶结构域组成。为了阐明EGF1结构域在因子IX中的功能，我们构建了三个突变体：一个缺失EGF1结构域的突变体（IXΔEGF1）、一个发生Gln-50→Pro变化的点突变体（IXQ50P）以及一个将因子IX的EGF1结构域替换为蛋白C的EGF1结构域的替代突变体（IXPCEGF1）。通过使用pRc/CMV载体，这些突变体和野生型（WT）因子IX（IXWT）在293肾细胞中表达。纯化后的蛋白质的γ-羧基谷氨酸含量与正常血浆因子IX（IXNP）相同，并且能被因子XIa-Ca2+正常激活。相比之下，IXΔEGF1不能被因子VIIa-组织因子-Ca2+激活，并且IXPCEGF1在该系统中的激活明显缓慢；然而，IXQ50P以正常速率被激活。在进一步的研究中，IXWT和IXΔEGF1都能被因子Xa-磷脂-Ca2+迅速转化为各自的IXα形式。由于该反应对磷脂有绝对需求，这表明所研究的突变体与磷脂的相互作用未受损。因子XIa激活的蛋白质的相对凝血活性分别为：IXNP，100%；IXWT，75 - 85%；IXΔEGF1，≤1%；IXPCEGF1，≤2%；IXQ50P，6 - 10%。我们得出结论，因子IX的EGF1结构域对于其被因子VIIa-组织因子激活是必需的，并且Gln-50残基对于这种激活并不关键。此外，因子IX的EGF1结构域对于磷脂结合及其被因子XIa激活并非必不可少。另外，激活后的突变体的低凝血活性表明EGF1结构域在因子IXa-因子VIIIa-Ca(2+)-磷脂复合物激活因子X的过程中也很重要。
