Fujiwara Ryoichi, Nakajima Miki, Yamanaka Hiroyuki, Nakamura Akiko, Katoh Miki, Ikushiro Shin-ichi, Sakaki Toshiyuki, Yokoi Tsuyoshi
Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan.
Drug Metab Dispos. 2007 May;35(5):747-57. doi: 10.1124/dmd.106.014191. Epub 2007 Feb 9.
We established stable HEK293 cell lines expressing double isoforms, UGT1A1 and UGT1A9, UGT1A4 and UGT1A9, or UGT1A6 and UGT1A9, as well as stable cell lines expressing each single isoform. To analyze the protein-protein interaction between the UGT1As, we investigated the thermal stability and resistance to detergent. UGT1A9 uniquely demonstrated thermal stability, which was enhanced in the presence of UDP-glucuronic acid (>90% of control), and resistance to detergent. Interestingly, UGT1A1, UGT1A4, and UGT1A6 acquired thermal stability and resistance to detergent by the coexpression of UGT1A9. An immunoprecipitation assay revealed that UGT1A6 and UGT1A9 interact in the double expression system. Using the single expression systems, it was confirmed that estradiol 3-O-glucuronide, imipramine N-glucuronide, serotonin O-glucuronide, and propofol O-glucuronide formations are specific for UGT1A1, UGT1A4, UGT1A6, and UGT1A9, respectively. By kinetic analyses, we found that the coexpressed UGT1A9 significantly affected the kinetics of estradiol 3-O-glucuronide formation (decreased Vmax), imipramine N-glucuronide formation (increased Km and Vmax), and serotonin O-glucuronide formation (decreased Vmax) catalyzed by UGT1A1, UGT1A4, and UGT1A6, respectively. On the other hand, the coexpressed UGT1A1 increased Km and decreased the Vmax of the propofol O-glucuronide formation catalyzed by UGT1A9. The coexpressed UGT1A4 and UGT1A6 also increased the Vmax of the propofol O-glucuronide formation by UGT1A9. This is the first study showing that human UGT1A isoforms interact with other isoforms to change the enzymatic characteristics.
我们建立了稳定表达双重同工型（UGT1A1和UGT1A9、UGT1A4和UGT1A9或UGT1A6和UGT1A9）的HEK293细胞系，以及表达每种单一同工型的稳定细胞系。为了分析UGT1A之间的蛋白质-蛋白质相互作用，我们研究了热稳定性和对去污剂的抗性。UGT1A9独特地表现出热稳定性，在存在UDP-葡糖醛酸的情况下热稳定性增强（>对照的90%），并且对去污剂具有抗性。有趣的是，UGT1A1、UGT1A4和UGT1A6通过与UGT1A9共表达而获得热稳定性和对去污剂的抗性。免疫沉淀试验表明UGT1A6和UGT1A9在双重表达系统中相互作用。使用单一表达系统，证实了雌二醇3-O-葡糖醛酸、丙咪嗪N-葡糖醛酸、5-羟色胺O-葡糖醛酸和丙泊酚O-葡糖醛酸的形成分别对UGT1A1、UGT1A4、UGT1A6和UGT1A9具有特异性。通过动力学分析，我们发现共表达的UGT1A9分别显著影响UGT1A1、UGT1A4和UGT1A6催化的雌二醇3-O-葡糖醛酸形成（Vmax降低）、丙咪嗪N-葡糖醛酸形成（Km和Vmax增加）和5-羟色胺O-葡糖醛酸形成（Vmax降低）的动力学。另一方面，共表达的UGT1A1增加了UGT1A9催化的丙泊酚O-葡糖醛酸形成的Km并降低了Vmax。共表达的UGT1A4和UGT1A6也增加了UGT1A9催化的丙泊酚O-葡糖醛酸形成的Vmax。这是第一项表明人类UGT1A同工型与其他同工型相互作用以改变酶学特性的研究。
