Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans
Identification and functional characterization of a new CYP2C9 variant (CYP2C9*5) expressed among African Americans
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DOI：
10.1124/mol.60.2.382
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发表时间：
2001-08-01
影响因子：
3.6
通讯作者：
Schwarz, UI
中科院分区：
文献类型：
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作者：
Dickmann, LJ;Rettie, AE;Schwarz, UI
CYP 2C 9是一种多态性基因，有四种已知的等位基因变体：CYP 2C 9 *1、CYP 2C 9 *2、CYP 2C 9 *3和CYP 2C 9 *4。在本研究中，从140个欧洲裔美国人和120个非洲裔美国人的DNA进行了检查单链构象多态性和限制性片段长度多态性分析，导致一个新的CYP 2C 9变异体，CYP 2C 9 *5的鉴定。该变体来源于CYP 2C 9外显子7中的C1080 G颠换，导致编码蛋白中的Asp 360 Glu取代。发现CYP 2C 9 *5变体仅在非裔美国人中表达，因此该人群中约3%携带CYP 2C 9 *5等位基因。该变体在用重组杆状病毒感染的昆虫细胞中表达并纯化。使用纯化的野生型蛋白质CYP 2C 9 *1、IIe 359 Leu变体CYP 2C 9 *3和Asp 360 Glu变体CYP 2C 9 *5，以（S）-华法林、双氯芬酸和月桂酸作为底物进行比较动力学研究。Asp 360 Glu突变的主要影响是增加了所有三种底物的Km值（相对于CYP 2C 9 *1）：对于（S）-华法林7-羟基化高12倍，对于艾地氯芬酸的4 ′-羟基化高5倍，并且对于月桂酸的ω-1羟基化高3倍，CYP 2C 9 *1和CYP 2C 9 *5蛋白质之间的V-max值差异小于K-m值。CYP 2C 9 *5的体外固有清除率计算为V-max/K-m比值，范围为CYP 2C 9 *1值的8 - 18%。CYP 2C 9 *3的相应比值为4 - 13%。因此，体外数据表明，相对于表达野生型蛋白的人，CYP 2C 9 *5等位基因携带者将以较慢的速率消除CYP 2C 9底物。
CYP2C9 is a polymorphic gene for which there are four known allelic variants; CYP2C9*1, CYP2C9*2, CYP2C9*3, and CYP2C9*4. In the present study, DNA from 140 European Americans and 120 African Americans was examined by single-strand conformational polymorphism and restriction fragment length polymorphism analyses, resulting in the identification of a new CYP2C9 variant, CYP2C9*5. This variant is derived from a C1080G transversion in exon 7 of CYP2C9 that leads to an Asp360Glu substitution in the encoded protein. The CYP2C9*5 variant was found to be expressed only in African Americans, such that approximately 3% of this population carries the CYP2C9*5 allele. The variant expressed in, and purified from, insect cells infected with a recombinant baculovirus. Comparative kinetic studies using the purified wild-type protein CYP2C9*1; the IIe359Leu variant, CYP2C9*3; and the Asp 360Glu variant CYP2C9*5 were carried out using (S)-warfarin, diclofenac, and lauric acid as substrates. The major effect of the Asp360Glu mutation was to increase the K-m value relative to that of CYP2C9*1 for all three substrates: 12-fold higher for (S)-warfarin 7-hydroxlation, 5-fold higher for the 4'-hydroxylation of ldiclofnac and 3-fold higher for the omega -1 hydroxlation of lauric acid, V-max values differed less that K-m values between the CYp2C9*1 and CYP2C9*5 proteins. In vitro intrinsic clearances for CYP2C9*5 calculated as the ratio of V-max/K-m, ranged fro 8 to 18% of CYP2C9*1 values. The corresponding ratio for CYP2C9*3 was 4 to 13%. Accordingly, the in vitro data suggest that carriers of the CYP2C9*5 allele would eliminate CYP2C9 substrates at slower rates relative to person expressing the wild-type protein.