Functional characterization of human UDP-glucuronosyltransferase 1A5 (UGT1A5): expression systems and structure-function
Başlık çevirisi mevcut değil.
- Tez No: 516823
- Danışmanlar: Prof. JOHN O MINERS, Prof. PETER I MACKENZIE
- Tez Türü: Yüksek Lisans
- Konular: Eczacılık ve Farmakoloji, Pharmacy and Pharmacology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2011
- Dil: İngilizce
- Üniversite: Flinders University
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Belirtilmemiş.
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 145
Özet
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Özet (Çeviri)
Conjugation with glucuronic acid is an important metabolic pathway for biologically active xenobiotics (including drugs) and endogenous compounds. The glucuronidation reaction is catalysed by enzymes of the UGT family. Human UGT enzymes exhibit distinct, but frequently overlapping, substrate selectivities, which may be dependant on a limited number of amino acids. UGT1A5, UGT1A4 and UGT1A3 are >85% identical at the amino acid level. However, whereas UGT1A3 glucuronidates phenols but not tertiary amines, UGT1A4 has the capacity to glucuronidate tertiary amines but not phenols. The differences in substrate selectivities between UGT1A4 and UGT1A3 arises from sequence differences at positions 36 (Thr, Ile) and 40 (His, Pro). In contrast, little is known regarding UGT1A5 structure-function relationships. Thus, the present study aimed to characterize the effects of the T36I and H40P mutants on UGT1A5 substrate selectivity and activity. The UGT1A5 T36I and H40P mutants were generated by PCR-site-directed mutagenesis. UGT1A5 and the mutant proteins were expressed initially in HEK293T and COS7 cells. However, expression of UGT1A5 and the two mutant proteins was very low or negligible. Therefore, baculovirus-mediated expression in Sf9 cell was established. Expression of UGT1A5 and its two mutant proteins was confirmed by western blot analysis using an antibody generated in-house that recognizes Histagged proteins. Since UGT1A5 has been reported to glucuronidate 1-hydroxypyrene (1-OHP), an assay for the separation and quantification of 1-OHP and 1-OHP glucuronide (1-OHPG) was developed and validated. Activity of untransfected Sf9 cells was also characterized in preliminary experiments to investigate the presence of endogenous glycosidation. The incubation of untransfected Sf9 cells with UDPGA showed the presence of an other peak which chromatographed with almost the same retention time as 1-OHPG. The mobile phase was modified to separate the second peak, which was identified as 1-OHP glucoside by liquid chromatography-mass spectrometry and by selective enzyme hydrolysis. The modified HPLC assay allowed the simultaneous measurement of 1-OHP glucoside and 1-OHP glucuronide in incubation samples with good sensitivity and reproducibility. UGT1A5 and its two mutant proteins expressed in HEK293T, COS7 and Sf9 cells were screened for glucuronidation activity towards 1-naphthol (1-NP), 4- methylumbellferone (4-MU), trifluoperazine (TFP), lamotrigine (LTG) and 1- hydroxypyrene (1-OHP), but no activity was observed. UGT1A6 and UGT1A4 expressed in HEK293 cells were used as the positive controls for the glucuronidation of 4-MU/1-NP and TFP/LTG, respectively. Untransfected HEK293T, COS7 and Sf9 cells were also screened with the same compounds and did not form a glucuronide conjugate with any of these substrates. Kinetic characterization of 1-OHP and 4-MU glucosidation by untransfected Sf9 cells (lysate and microsomes) was undertaken with UDP-glucose as the co-factor. Incubation of both substrates showed substrate inhibition kinetics, except with Sf9 microsomes and 4-MU where kinetic data were suggestive of substrate inhibition and negative co-operativity. The kinetic results suggest that the active site of the glucosyltransferase enzyme present in Sf9 cells binds two molecules of 1-OHP and 4-MU. Taken together, these data indicate that xenobiotic glucuronidation studies with UGTs expressed in Spodoptera frugiperda should routinely exclude the presence of a glucoside conjugate when chromatographic methods are used for analysis. Furthermore, the results suggest that previous reports of UGT1A5 activity may arise from an experimental artefact.
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