SULT1A3及HEK-SULT1A3细胞催化甘草素(7-OH)磺酸化代谢动力学的研究

7-hydroxy sulfonation of liquiritigenin by recombinant SULT1A3 enzyme and HEK-SULT1A3 cells

该文采用酶孵育法,测定了甘草素在SULT亚型酶中的代谢活性,并通过活性-蛋白表达相关性分析,评价了SULT1A3在甘草素(7-OH) SULT代谢中的作用。结果发现各亚型酶均能催化甘草素生成7-O-SULT代谢物,代谢动力学均符合米氏方程模型。根据内在清除率(CLint),不同亚型酶催化甘草素(7-OH)发生SULT代谢的活性大小依次为SULT1C4>SULT1A3>SULT1E1>SULT1A1>SULT1A2>SULT1B1>SULT1C2>SULT2A1。相关性分析结果显示,甘草素(7-OH)代谢速率与SULT1A3蛋白水平呈显著性相关(P<0. 05)。此外,构建了SULT1A3高表达的HEK293细胞(HEK-SULT1A3细胞),并能代谢甘草素生成代谢物。甘草素在HEK-SULT1A3细胞中的代谢动力学同样符合米氏方程模型[Vmax为(0. 315±0. 009)μmol·min-1·g-1,Km为(7. 04±0. 680)μmol·L-1,CLint为(0. 045±0. 005) L·min-1·g-1],且HEK-SULT1A3细胞催化甘草素(7-OH)的代谢动力学特征与SULT1A3重组酶催化甘草素(7-OH)的代谢动力学特征具有显著相关性(P<0. 001),验证了该细胞具有SULT1A3的功能。结果表明甘草素易于发生SULT代谢,SULT1A3在甘草素(7-OH) SULT代谢中发挥关键作用,SULT代谢可能是导致甘草素生物利用度低的重要原因。此外,成功构建了HEK-SULT1A3细胞,为研究甘草素SULT代谢提供了一个细胞模型,可用于更加准确地预测甘草素体内SULT代谢过程。

In this study,liquiritigenin sulfonation was characterized using recombinant human sulfotransferases(SULTs).The chemical structure of liquiritigenin sulfate was determined by ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS/MS).Then model fitting and parameter estimation were performed using the Graphpad Prism V5 software.Various SULT enzymes(SULT1 A1,1 A2,1 A3,1 B1,1 C2,1 C4,1 E1 and 2 A1)were able to catalyze the formation of liquiritigenin-7-O-sulfate.Sulfonation of liquiritigenin-7-hydroxy(7-OH)by these eight SULT enzymes consistently displayed the classical Michaelis-Menten profile.According to the intrinsic clearance(CLint)value,the sulfonation rates of liquiritigenin-7-OH by expressed SULT enzymes followed the following rank order:SULT1 C4>SULT1 A3>SULT1 E1>SULT1 A1>SULT1 A2>SULT1 B1>SULT1 C2>SULT2 A1.Further,liquiritigenin-7-O-sulfonation was significantly correlated with the SULT1 A3 protein levels(P<0.05).Then,human embryonic kidney(HEK)293 cells over expressing SULT1 A3(named as HEK-SULT1 A3 cells)were conducted.As a result,liquiritigenin-7-O-sulfate(L-7-S)was rapidly generated upon incubation of the cells with liquiritigenin.Consistent with SULT1 A3,sulfonation of liquiritigenin-7-OH in HEK-SULT1 A3 cells also followed the Michaelis-Menten kinetics.The derived Vmaxvalues was(0.315±0.009)μmol·min-1·g-1,Kmwas(7.04±0.680)μmol·L-1,and CLintwas(0.045±0.005)L·min-1·g-1.Moreover,the sulfonation characters of liquiritigenin(7-OH)in SULT1 A3 were strongly correlated with that in HEK-SULT1 A3 cells(P<0.001).The results indicated that HEK-SULT1 A3 cells have shown the catalytic function of SULT1 A3 enzymes.In conclusion,liquiritigenin was subjected to efficient sulfonation,and SULT1 A3 enzyme plays an important role in the sulfonation of liquiritigenin-7-OH.Significant sulfonation should be the main reason for the low bioavailability of liquiritigenin.In addition,HEK-SULT1 A3 cells were conducted and successfully used to evaluate liquiritigenin sulfonation,which will provide an appropriate tool to accurately depict the sulfonation disposition of liquiritigenin in vivo.