Abstract
We have reported that the protein-protein interaction between UDP-glucuronosyltransferase (UGT) 2B7 and cytochrome P450 3A4 (CYP3A4) alters UGT2B7 function. However, the domain(s) involved in the interaction are largely unknown. To address this issue, we examined in more detail the CYP3A4-UGT2B7 association by means of immunoprecipitation, overlay assay, and cross-linking involving 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. Purified CYP3A4 or glutathione transferase (GST)-tagged CYP3A4 was cross-linked to UGT2B7 in solubilized baculosomes. The formation of the cross-linked complex was detected by immunoblotting using both antibodies against CYP3A4 and UGTs. Although the GST-tagged CYP3A4 containing the region ranging from Tyr25 to Ala503 was cross-linked to UGT2B7, the same did not occur when another construct containing Met145 to His267 was used. This observation was consistent with the result of the overlay assay indicating that CYP3A4 lacking the N-terminal hydrophobic segment retains the ability to associate with UGT2B7, whereas the Met145-to-His267 region loses this capacity. Although the Met145-to-His267 peptide was recognized by one anti-CYP3A4 antibody that has the ability to coimmunoprecipitate UGT2B7, it was not recognized by another antibody incapable of coimmunoprecipitating UGT2B7. The epitope of the latter antibody was mapped to the Leu331-to-Lys342 region, which is located on the J-helix of CYP3A4. Taken together, the results obtained suggest that 1) CYP3A4 and UGT2B7 are a pair of enzymes in proximity to each other and 2) either the Leu331-to-Lys342 domain or the surrounding region plays a role in the interaction with UGT2B7, whereas the hydrophobic Met145-to-His267 region does not contribute to this interaction.
Footnotes
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This work was supported in part by the Japan Society for Promotion of Science [Grant-in-Aid for Scientific Research (C) 19590147] and by the National Health and Medical Research Council of Australia.
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This work was presented, in part, at the Forum 2005: Hygienic Pharmaceutical Science and Environmental Toxicology; Tokushima, Japan; 28 October 2005), J Health Sci51(Suppl):S-100 (abstract).
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ABBREVIATIONS: P450, cytochrome P450; UGT, UDP-glucuronosyltransferase; ER, endoplasmic reticulum; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; GST, glutathione transferase; NHS, N-hydroxysuccinimide; PNGaseF, protein N-glycosidase; PAGE, polyacrylamide gel electrophoresis; PVDF, polyvinylidene diflouride; CNX, calnexin; MES, 2-(N-morpholino)ethanesulfonic acid.
- Received September 12, 2008.
- Accepted January 21, 2009.
- The American Society for Pharmacology and Experimental Therapeutics
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