Molecular cloning of the baboon UDP-glucuronosyltransferase 2B gene family and their activity in conjugating morphine

Abstract

Glucuronidation by UDP-glucuronyltransferase 2B enzymes (UGT2Bs) is a major pathway for the elimination of endobiotics and xenobiotics, including therapeutic drugs. Morphine, a probe drug for UGT2B7, is metabolized to morphine-3-beta-glucuronide (M3G) and morphine-6-beta-glucuronide (M6G) in humans. Morphine has been used in a series of experiments in the baboon to characterize developmental changes in fetal glucuronidation. This study identifies the baboon UGT2B family of enzymes, compares them with that of the human and the monkey (Macaca fascicularis), and measures the activity of the individual baboon UGT2Bs toward morphine. UGT2B cDNAs were cloned from the liver of adult and newborn baboons and expressed in human embryonic kidney 293 cells. The UGT activity toward morphine was assessed by the rate of formation of M3G and M6G by high-performance liquid chromatography. Eight baboon UGT2Bs were cloned and identified: UGT2B41 and UGT2B42, which are 90% homologous to human UGT2B4; UGT2B43, which is 93% homologous to human UGT2B15; and UGT2B39, UGT2B40, UGT2B44, UGT2B45, and UGT2B46, which are 89 to 91% homologous to human UGT2B7. Homology between baboon and monkey UGT2B ranged from 92.6 to 99.1%, with the primary protein structure of UGT2B43 being 99.1% identical to monkey UGT2B20, including a unique R96I substitution. Gene conversion interfered with the phylogenetic signal in the baboon UGT2B7-like and the monkey UGT2B4-like groups and led to concerted evolution of these enzymes. All of the baboon UGT2Bs metabolized morphine to both M3G and M6G. This study lays the foundation for investigating the regulation of UGT2B enzymes during fetal and neonatal development in the baboon.

Fig. 1.

Neighbor-joining (NJ) reconciled phylogenetic tree with baboon, human, and monkey (M. fascicularis) UGT2B enzymes. The sequences for human and monkey UGT2B genes were obtained from the GenBank. The protein coding regions were aligned using MEGA4.0, and an NJ distance-based phylogenetic tree using p distances was generated. Robustness of bifurcations is estimated by bootstrap analysis with 1000 replicates, and the consensus tree is shown. Numbers at bifurcations indicate bootstrap values in percent. Annotation: bab (baboon, P. anubis), h (human), and mon (cynomolgus monkey, M. fascicularis).

Fig. 2.

Multiple alignments of deduced baboon UGT2B protein amino acid sequences were aligned using the Multalin web page. Identical amino acids are shown in black background, and >80% conservation is illustrated by boxes. Residue 96 is indicated by a star.

Fig. 3.

Morphine glucuronidation in heavy membranes from HEK293 cells expressing individual baboon UGT2B isoforms or HEK293 vector control. Morphine-3-UGT and morphine-6-UGT activity was measured at a morphine concentration of 5 mM. Each bar represents the mean of duplicate measurements.