Microsoft Word - 50394953-file00

List of Nonstandard Abbreviations: UGT, UDP-glucuronosyltransferase; M3G, morphine-3-ß-glucuronide: M6G, morphine-6-ß-glucuronide; DEPC, diethyl pyrocarbonate; PCR, polymerase chain reaction; HEK293, human embryonic kidney 293; DMEM, Dulbecco's modified Eagle's medium; FBS, fetal bovine serum; HPLC, high performance liquid chromatography; N-glycosylation, asparagine-linked glycosylation. 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-β-glucuronide (M3G) and morphine-6-β-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 to 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 HEK293 cells. The UGT activity toward morphine was assessed by the rate of formation of M3G and M6G by HPLC. Eight baboon UGT2Bs were cloned and identified: UGT2B41 and UGT2B42, that are 90% homologous to human UGT2B4; UGT2B43 that is 93% homologous to human UGT2B15; and UGT2B39, 40, 44, 45, and 46 that 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 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. This article has not been copyedited and formatted. The final version may differ from this version.