cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome p450
cDNA cloning and initial characterization of CYP3A43, a novel human cytochrome p450
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DOI：
10.1124/mol.59.2.386
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发表时间：
2001-02-01
影响因子：
3.6
通讯作者：
Zaphiropoulos, PG
中科院分区：
文献类型：
--
作者：
Domanski, TL;Finta, C;Zaphiropoulos, PG
RACE 扩增技术用于在人类 CYP3A 基因表达的逆转录酶/聚合酶链反应分析过程中检测到的新型 CYP3A 样外显子 1 序列。这导致了包含 CYP3A 基因亚家族新成员 CYP3A43 完整编码序列的 cDNA 的鉴定。有趣的是，大多数鉴定的 cDNA 都具有选择性剪接事件的特征，例如外显子跳跃和完全或部分内含子包含。在肝脏、肾脏、胰腺和前列腺中检测到 CYP3A43 表达。氨基酸序列与CYP3A4和CYP3A5的氨基酸序列有75%相同，与CYP3A7有71%相同。 CYP3A43 与 CYP3A4 的不同之处在于六个氨基酸残基，这些残基位于 CYP3A4 的推定底物识别位点内，已知这些残基是底物选择性的决定因素。 CYP3A43 的 N 末端经过修饰，以便在大肠杆菌中有效表达该蛋白，并在 C 末端添加 6X 组氨酸标签以方便纯化。 CYP3A43 给出还原的一氧化碳差异光谱，在 450 nm 处具有最大吸光度。异源表达水平显着低于 CYP3A4 和 CYP3A5 观察到的水平。免疫印迹分析显示，CYP3A43 在聚丙烯酰胺凝胶电泳中与 CYP3A4 共迁移，但与 CYP3A5 分离。单加氧酶测定在多种条件下进行，其中几种条件产生了可重复的睾酮羟化酶活性，尽管其活性较低。这项研究的结果表明，人体组织中表达了一种新的 CYP3A 成员，尽管其对药物代谢的相对贡献尚未确定。
The RACE amplification technology was used on a novel CYP3A-like exon 1 sequence detected during the reverse transcriptase/polymerase chain reaction analysis of human CYP3A gene expression. This resulted in the identification of cDNAs encompassing the complete coding sequence of a new member of the CYP3A gene subfamily, CYP3A43. Interestingly, the majority of the cDNAs identified were characterized by alternative splicing events such as exon skipping and complete or partial intron inclusion. CYP3A43 expression was detected in liver, kidney, pancreas, and prostate. The amino acid sequence is 75% identical to that of CYP3A4 and CYP3A5 and 71% identical to CYP3A7. CYP3A43 differs from CYP3A4 at six amino acid residues, found within the putative substrate recognition sites of CYP3A4, that are known to be determinants of substrate selectivity. The N terminus of CYP3A43 was modified for efficient expression of the protein in Escherichia coli, and a 6X histidine tag was added at the C terminus to facilitate purification. CYP3A43 gave a reduced carbon monoxide difference spectra with an absorbance maximum at 450 nm. The level of heterologous expression was significantly lower than that observed for CYP3A4 and CYP3A5. Immunoblot analyses revealed that CYP3A43 comigrates with CYP3A4 in polyacrylamide gel electrophoresis but does separate from CYP3A5. Monooxygenase assays were performed under a variety of conditions, several of which yielded reproducible, albeit low, testosterone hydroxylase activity. The findings from this study demonstrate that there is a novel CYP3A member expressed in human tissues, although its relative contribution to drug metabolism has yet to be ascertained.