Functional Analysis of UGT1A4^P24T and UGT1A4^L48V Variant Enzymes

Abstract

Aim: To investigate the effects of two nonsynonymous SNPs, UGT1A4*2 (rs#: 6755571, 70C>A, P24T) and UGT1A4*3 (rs#: 2011425, 142T>G, L48V), on the function of UGT1A4 against dihydrotestosterone (DHT), transandrosterone (t-AND), lamotrigine (LTG) and tamoxifen (TAM). Materials & methods: Detailed kinetic experiments were conducted with recombinant UGT1A4^wild-type, UGT1A4^P24T and UGT1A4^L48V, which were overexpressed in HEK293 cell lines. The kinetic profiles and kinetic parameters (K_m, V_max and CL_int) obtained with either UGT1A4^P24T or UGT1A4^L48V were compared with those obtained with the wild-type enzyme. The interaction of TAM on UG1A4-catalyzed DHT glucuronidation was also investigated with the three UGT1A4 polymorphic enzymes. Results: UGT1A4^L48V had higher enzyme efficiency (CL_int) compared with wild-type UGT1A4 on DHT glucuronidation; UGT1A4^P24T and UGT1A4^L48V had lower CL_int than wild-type UGT1A4 for t-AND and LTG glucuronidation. The TAM CL_int with UGT1A4^P24T and UGT1A4^L48V glucuronidation and the UGT1A4^P24T-catalyzed DHT glucuronidation were, on the other hand, similar to those of the wild-type enzyme. With all three enzymes, TAM activated UGT1A4-catalyzed DHT glucuronidation in a concentration-dependent fashion. Conclusion: Decreased CL_int of UGT1A4^P24T and UGT1A4^L48V on LTG glucuronidation may lead to interindividual variations in LTG metabolism in vivo. However, it is less likely that these polymorphisms would have impact on DHT and t-AND metabolism in vivo because these compounds are glucuronidated by multiple enzymes.

Original submitted 31 May 2011; Revision submitted 19 July 2011

KEYWORDS::

• dihydrotestosterone
• enzyme kinetic
• glucuronidation
• heteroactivation
• lamotrigine
• polymorphism
• tamoxifen
• transandrosterone
• UGT1A4

Acknowledgements

The authors thank Philip Lazarus (Penn State University, PA, USA) for his generous gift of the three HEK293 cell lines expressing the three UGT1A4 enzymes. The authors also thank Swati Nagar (Temple University, PA, USA) for helping with the HEK293 cell culture.

Financial & competing interests disclosure

The authors thank Bristol-Myers-Squibb for partial support of this project. Additional support was by NIH/NINDS program project grant 3P50 NS16308-15A1. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined in the Declaration of Helsinki for all human or animal experimental investigations. In addition, for investigations involving human subjects, informed consent has been obtained from the participants involved.

Additional information

Funding

The authors thank Bristol-Myers-Squibb for partial support of this project. Additional support was by NIH/NINDS program project grant 3P50 NS16308-15A1. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed. No writing assistance was utilized in the production of this manuscript.