Isolation and characterization of a UDP–glucuronosyltransferase (UGT1A01) cloned from female rhesus monkey

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Abstract

An isoform (rhesus UGT1A01) orthologus to the human UGT1A1 was cloned and sequenced from female rhesus monkey liver cDNA using primers designed from the human nucleotide sequences. Open reading frame analysis of the PCR-generated product encodes a 533-amino acid protein with a proposed 27-residue signal peptide. Nucleotide sequence comparison of rhesus UGT1A01 to other rhesus UGT1A isoforms detected a single-transition mutation at nucleotide 1520 (T→C), resulting in a neutral F to S substitution at position 507. Rhesus UGT1A01 was greater than 99 and 95% identical to cynomolgus UGT1A01 and human UGT1A1, respectively. The rhesus UGT1A01 was expressed in HK-293 cells for functional analysis. Catalytic activity of UGT1A01 was determined with 7-hydroxy-4-(trifluoromethyl)-coumarin and more specific human UGT1A1 substrates (1-naphthol, β-estradiol, 17α-ethinylestradiol, and bilirubin). Expression of UGT1A01 protein was also detected by a Western blot utilizing a polyclonal antibody developed against the human UGT1A family.

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Materials and methods

Materials. RNeasy, PolyFect transfection reagent, and other molecular biology products were obtained through Qiagen (Valencia, CA). M-MLV Reverse Transcriptase (RNase H minus) and the pGEM-T Easy vector were purchased from Promega (Madison, WI). The E-gels, tris–acetate NuPAGE gels, Dulbecco's modified eagle's media (DMEM), heat-inactivated fetal bovine serum, Hepes, geneticin (G418), and the pcDNA3.1 vector were obtained through Invitrogen (Carlsbad, CA). AmpliTAQ Gold was purchased through

Results

Detection and isolation of novel UGTs from rhesus monkey liver cDNA were performed using primers for human UGT isoforms based on the high degree of homology between primates. Screening of various primer combinations has led to the detection of a 1645-bp PCR product using the human UGT1A1 primers. This cDNA was sequenced and data analysis confirmed existence of a 1599-bp open reading frame encoding a 533-amino acid translational product (Fig. 1). The deduced amino acid sequence revealed greater

Discussion

This paper describes the isolation and characterization of female rhesus UGT1A01, an ortholog of human UGT1A1 and cynomolgus monkey UGTlA01 [32] and the first UGT to be sequenced and characterized from this species. The amplified UGT1A01 cDNA encodes for a functional full-length enzyme of 533 amino acids possessing UGT characteristics such as an ER-directing signal peptide (residues 1–27), a transmembrane-spanning domain (residues 490–508), a UDPGA-binding domain (residues 354–402), and

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