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Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.


Activity assays of UGT1A1*N or UGT1A1*N-UGT1A1*N on the glucuronidation of quercetin.The concentration of quercetin in the incubation mixture was 100 μmol/L. ☐ indicates single expression and ▄ indicates double expression. Data are presented as mean ± SD from three independent determinations, and the asterisks indicate differences that are statistically significant (**P < 0.005, *P < 0.05).
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f4: Activity assays of UGT1A1*N or UGT1A1*N-UGT1A1*N on the glucuronidation of quercetin.The concentration of quercetin in the incubation mixture was 100 μmol/L. ☐ indicates single expression and ▄ indicates double expression. Data are presented as mean ± SD from three independent determinations, and the asterisks indicate differences that are statistically significant (**P < 0.005, *P < 0.05).

Mentions: The hydrolyzed glucuronides were quantified using a modified high performance liquid chromatography (HPLC) method. The quercetin glucuronidation activity was measured in terms of the peak area of targeted glucuronide conjugated compounds. Quercetin was incubated with UGT1A1*1/*1b and UGT1A9*1/*2/*3/*5 from single-expression system and kinetic parameters were estimated. The glucuronidation positions of quercetin monoglucuronides were accorded to our previous study15. As a result, quercetin was converted into three main monoglucuronides (M1: 7-glucuronide, M3: 4′-glucuronide, and M4: 3′-glucuronide) by UGT1A1 (Fig. S4A), and the splice mutant UGT1A1*1b showed no enzymatic activity (Fig. 4).


Dimerization of human uridine diphosphate glucuronosyltransferase allozymes 1A1 and 1A9 alters their quercetin glucuronidation activities.

Liu YQ, Yuan LM, Gao ZZ, Xiao YS, Sun HY, Yu LS, Zeng S - Sci Rep (2016)

Activity assays of UGT1A1*N or UGT1A1*N-UGT1A1*N on the glucuronidation of quercetin.The concentration of quercetin in the incubation mixture was 100 μmol/L. ☐ indicates single expression and ▄ indicates double expression. Data are presented as mean ± SD from three independent determinations, and the asterisks indicate differences that are statistically significant (**P < 0.005, *P < 0.05).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837415&req=5

f4: Activity assays of UGT1A1*N or UGT1A1*N-UGT1A1*N on the glucuronidation of quercetin.The concentration of quercetin in the incubation mixture was 100 μmol/L. ☐ indicates single expression and ▄ indicates double expression. Data are presented as mean ± SD from three independent determinations, and the asterisks indicate differences that are statistically significant (**P < 0.005, *P < 0.05).
Mentions: The hydrolyzed glucuronides were quantified using a modified high performance liquid chromatography (HPLC) method. The quercetin glucuronidation activity was measured in terms of the peak area of targeted glucuronide conjugated compounds. Quercetin was incubated with UGT1A1*1/*1b and UGT1A9*1/*2/*3/*5 from single-expression system and kinetic parameters were estimated. The glucuronidation positions of quercetin monoglucuronides were accorded to our previous study15. As a result, quercetin was converted into three main monoglucuronides (M1: 7-glucuronide, M3: 4′-glucuronide, and M4: 3′-glucuronide) by UGT1A1 (Fig. S4A), and the splice mutant UGT1A1*1b showed no enzymatic activity (Fig. 4).

Bottom Line: Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans.Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity.SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances.

View Article: PubMed Central - PubMed

Affiliation: Institute of Drug Metabolism and Pharmaceutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou 310058, China.

ABSTRACT
Uridine diphosphate glucuronosyltransferase 1A (UGT1A) is a major phase II drug-metabolism enzyme superfamily involved in the glucuronidation of endobiotics and xenobiotics in humans. Many polymorphisms in UGT1A genes are reported to inhibit or decrease UGT1A activity. In this study, two UGT1A1 allozymes, UGT1A1 wild-type and a splice mutant, as well as UGT1A9 wild-type and its three UGT1A9 allozymes, UGT1A9*2(C3Y), UGT1A9*3(M33T), and UGT1A9*5(D256N) were single- or double-expressed in a Bac-to-Bac expression system. Dimerization of UGT1A1 or UGT1A9 allozymes was observed via fluorescence resonance energy transfer (FRET) and co-immunoprecipitation analysis. SNPs of UGT1A altered the ability of protein-protein interaction, resulting in differential FRET efficiencies and donor-acceptor r distances. Dimerization changed the chemical regioselectivity, substrate-binding affinity, and enzymatic activity of UGT1A1 and UGT1A9 in glucuronidation of quercetin. These findings provide molecular insights into the consequences of homozygous and heterozygous UGT1A1 and UGT1A9 allozymes expression on quercetin glucuronidation.

No MeSH data available.