Department of Drug Metabolism and Toxicology, Faculty of Pharmaceutical Sciences, Kanazawa University, Kakuma-machi, Kanazawa, Japan.
J Biol Chem. 2012 Mar 16;287(12):9240-9. doi: 10.1074/jbc.M111.271288. Epub 2012 Jan 31.
Mycophenolic acid (MPA), the active metabolite of the immunosuppressant mycophenolate mofetil (MMF), is primarily metabolized by glucuronidation to a phenolic glucuronide (MPAG) and an acyl glucuronide (AcMPAG). It is known that AcMPAG, which may be an immunotoxic metabolite, is deglucuronidated in human liver. However, it has been reported that recombinant β-glucuronidase does not catalyze this reaction. AcMPAG deglucuronidation activity was detected in both human liver cytosol (HLC) and microsomes (HLM). In this study, the enzyme responsible for AcMPAG deglucuronidation was identified by purification from HLC with column chromatographic purification steps. The purified enzyme was identified as α/β hydrolase domain containing 10 (ABHD10) by amino acid sequence analysis. Recombinant ABHD10 expressed in Sf9 cells efficiently deglucuronidated AcMPAG with a K(m) value of 100.7 ± 10.2 μM, which was similar to those in HLM, HLC, and human liver homogenates (HLH). Immunoblot analysis revealed ABHD10 protein expression in both HLC and HLM. The AcMPAG deglucuronidation by recombinant ABHD10, HLC, and HLH were potently inhibited by AgNO(3), CdCl(2), CuCl(2), PMSF, bis-p-nitrophenylphosphate, and DTNB. The CL(int) value of AcMPAG formation from MPA, which was catalyzed by human UGT2B7, in HLH was increased by 1.8-fold in the presence of PMSF. Thus, human ABHD10 would affect the formation of AcMPAG, the immunotoxic metabolite.
霉酚酸（MPA）是免疫抑制剂霉酚酸酯（MMF）的活性代谢物，主要通过葡萄糖醛酸化代谢为酚基葡萄糖醛酸（MPAG）和酰基葡萄糖醛酸（AcMPAG）。已知可能具有免疫毒性的 AcMPAG 在人肝中发生去葡萄糖醛酸化。然而，据报道，重组β-葡萄糖醛酸酶不能催化该反应。在人肝胞质（HLC）和微粒体（HLM）中均检测到 AcMPAG 的去葡萄糖醛酸化活性。在本研究中，通过用柱色谱纯化步骤从 HLC 中纯化，鉴定出负责 AcMPAG 去葡萄糖醛酸化的酶。通过氨基酸序列分析，鉴定纯化的酶为含 10 个α/β水解酶结构域的蛋白（ABHD10）。在 Sf9 细胞中表达的重组 ABHD10 有效地使 AcMPAG 去葡萄糖醛酸化，K(m)值为 100.7±10.2 μM，与 HLM、HLC 和人肝匀浆（HLH）中的 K(m)值相似。免疫印迹分析显示 ABHD10 蛋白在 HLC 和 HLM 中均有表达。重组 ABHD10、HLC 和 HLH 的 AcMPAG 去葡萄糖醛酸化被 AgNO3、CdCl2、CuCl2、PMSF、双对硝基苯磷酸酯和 DTNB 强烈抑制。在 PMSF 存在的情况下，人 UGT2B7 催化的 MPA 生成 AcMPAG 的 CL(int)值在 HLH 中增加了 1.8 倍。因此，人 ABHD10 可能会影响 AcMPAG 的形成，而 AcMPAG 是一种具有免疫毒性的代谢物。
