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Abstract
Common marmosets (Callithrix jacchus) are potentially primate models for preclinical drug metabolism studies because there are similarities in the molecular characteristics of cytochrome P450 enzymes between this species and humans. However, characterization of non–cytochrome P450 enzymes has not been clarified in marmosets. Here, we report characterization of flavin-containing monooxygenases FMO1–FMO5 identified in marmoset tissues. Marmoset FMO forms shared high amino acid sequence identities (93%–95%) and phylogenetic closeness with human homologous FMO forms. FMO1 and FMO3 mRNA were abundantly expressed in the liver and kidneys among five marmoset tissues examined, where FMO3 protein was detected by immunoblotting. FMO inhibition assays using preheated tissue microsomes indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver was mainly catalyzed by FMO3, the major hepatic FMO. Marmoset FMO3 protein heterologously expressed in Escherichia coli effectively catalyzed benzydamine N-oxygenation and sulindac sulfide S-oxygenation comparable to marmoset liver microsomes. These results indicate that the FMO3 enzyme expressed in marmoset livers mainly metabolizes benzydamine and sulindac sulfide (typical human FMO substrates), suggesting its importance for FMO-dependent drug metabolism in marmosets.
Introduction
Flavin-containing monooxygenases (FMOs; EC 1.14.13.8) are a family of xenobiotic-metabolizing enzymes involved in the oxygenation of a broad range of chemicals containing nitrogen, sulfur, or phosphorous (Krueger and Williams, 2005). In humans, functional genes FMO1–FMO5 (Lawton et al., 1994) and a nonfunctional pseudogene FMO6 (Hines et al., 2002) have been identified, and their mRNAs are expressed in various tissues. FMO3 is considered a major functional FMO enzyme in the human liver and contributes to the metabolism of the anti-inflammatory drugs benzydamine and sulindac sulfide and diet-derived trimethylamine (Shimizu et al., 2015).
The common marmoset (Callithrix jacchus) is a useful non–human primate species for pharmacokinetics studies because it has cytochrome P450 (EC 1.14.14.1) characteristic features that are significantly similar to humans (Uno et al., 2016). De novo transcriptome analysis indicate that FMO1-, FMO3-, FMO4-, and FMO5-like genes are expressed in the marmoset liver, kidneys, and intestines (Shimizu et al., 2014). FMO3 effectively catalyzes the N-oxygenation of potential proneurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the marmoset liver (Uehara et al., 2015). To date, only two FMO forms have been identified; however, little information on the molecular characteristics in marmosets is available (Uehara et al., 2015).
In this study, we isolated three marmoset FMO cDNAs based on FMO gene cluster organization and we analyzed them for their sequence identity, tissue expression, and catalytic activities using recombinant proteins heterologously expressed in Escherichia coli. This work is of importance for understanding the fundamental characteristics and functions of marmoset FMOs and the use of this species as a non–human primate model in preclinical drug development.
Materials and Methods
Detailed methods are provided separately in the Supplemental Material. FMO cDNAs were isolated by reverse transcription (RT)-polymerase chain reaction (PCR) with cDNA libraries transcribed from total RNA from marmoset tissues as described (Uehara et al., 2015). The structure of primate FMO gene clusters was determined by BLAT (UCSC Genome Bioinformatics; University of California, Santa Cruz, CA). Multiple alignment of amino acid sequences and phylogenetic analysis were performed with the GENETYX system (Software Development, Tokyo, Japan) and DNASIS Pro (Hitachi Software, Tokyo, Japan), respectively. The FMO amino acid sequences used were from GenBank (National Center for Biotechnology Information, Bethesda, MD). Pooled liver microsomes from marmosets (n = 5 males) were purchased from Corning Life Sciences (Woburn, MA). Pooled microsomes of the brain, lungs, liver, kidneys, and small intestine were prepared from tissue samples of 20 marmosets (10 males and 10 females, aged >2 years) at the Central Institution for Experimental Animals (Kawasaki, Japan) as described (Uehara et al., 2016a) and with approval from the Institutional Animal Care and Use Committee. FMO mRNA distribution in the brain, lungs, liver, kidneys, and small intestine, each pooled from six male and six female adult marmosets (aged >2 years), was analyzed by real-time RT-PCR as described (Uehara et al., 2016b). Recombinant marmoset FMO1 and FMO3 were heterologously expressed in E. coli using pET30 vectors (Novagen, Madison, WI) as described (Yamazaki et al., 2014). Tissue microsomes (10 μg), microsomes from five individual livers, and recombinant FMO3 protein (0.1 pmol) were separated on a 10% SDS-polyacrylamide gel by electrophoresis and were transferred to a polyvinylidene difluoride membrane (Uehara et al., 2016a). Benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities by recombinant FMO proteins and tissue microsomes were measured by high-performance liquid chromatography as described (Yamazaki et al., 2014). For FMO inactivation, liver or kidney microsomes were preheated at 45°C for 5 minutes without an NADPH-generating system (Taniguchi-Takizawa et al., 2015). All other reagents used were of the highest quality commercially available.
Results and Discussion
Analysis of common marmoset’s genome sequence showed that FMO1–FMO4 and FMO6 genes were localized in the gene cluster; the FMO5 gene was localized outside this cluster, in marmoset chromosome 18 (Supplemental Fig. 1). Marmoset FMO genes had one-to-one orthologous relationships with human FMO genes, even though FMO gene cluster organization is different between marmosets and humans. We successfully isolated FMO1–FMO5 cDNA in the marmoset liver by RT-PCR. Marmoset FMO1–FMO5 contained open reading frames of 532–556 amino acid residues, respectively (Supplemental Fig. 2), and had functionally important regions (FAD- and NADPH-pyrophosphate binding sites), and the two characteristic FMO pentapeptides (EGLEP and FATGY). Marmoset FMO1–FMO5 shared high amino acid sequence identities (93%–95%) with human FMO counterparts (Supplemental Table 1) and were phylogenetically more closely clustered with the corresponding primate orthologs than other species (Supplemental Fig. 3). Interestingly, in cynomolgus monkeys, FMO6 is a functional enzyme that is widely expressed in the kidneys, heart, testes, uterus, and liver (Uno et al., 2013); although we have tried, we failed to clone FMO6 cDNA from marmoset tissues.
To investigate the tissue distribution of FMO1–FMO5 mRNAs and proteins in marmosets, real-time RT-PCR was performed to measure expression levels of FMO1–FMO5 in pooled brains, lungs, livers, kidneys, and small intestine. FMO3 mRNA was the most abundant in the liver and kidneys, followed by the lungs (Fig. 1A), whereas FMO1 mRNA was also expressed abundantly in the liver and kidneys but not as much as FMO3 mRNA, the same as results previously reported by de novo transcriptome analysis (Shimizu et al., 2014). Indeed, FMO3 protein (approximately 50 kDa) was detected immunologically with anti-human FMO3 antibodies in pooled marmoset livers and kidneys (Fig. 1B), with a small nonspecific unknown band. No immunoreactive bands in these tissue microsomes were seen with commercial anti-human FMO1 antibodies (results not shown). Similar to marmosets, FMO3 is postnatally expressed in the kidneys of rabbits and rats (Ripp et al., 1999). FMO1 is reportedly expressed postnatally in the liver of dogs (Lattard et al., 2002), rabbits (Shehin-Johnson et al., 1995), rats (Novick et al., 2009), and mice (Itoh et al., 1997). Human FMO1, FMO2, and FMO3 are predominantly expressed in the kidneys, lungs, and liver, respectively, whereas FMO4 and FMO5 are widely expressed in various tissues (Zhang and Cashman, 2006; Uno et al., 2013). In humans, FMO1 is expressed in the fetal liver, but its expression is rapidly extinguished after birth (Koukouritaki et al., 2002). Apparent sex differences in FMO3 protein expression levels in the liver (Ripp et al., 1999) were found in mice, but not in marmosets (Fig. 1A). Marmoset FMO2 and FMO5 mRNA were dominantly expressed in the lungs and liver, whereas FMO4 mRNA was expressed in the liver, kidneys, and small intestine at very low levels, similar to human and cynomolgus monkey FMO forms (Zhang and Cashman, 2006; Uno et al., 2013). These results suggested that tissue distribution of FMO3 and FMO1 was partially different between marmosets and humans.
Determination of FMO mRNA (A) and FMO protein (B and C) levels in marmoset tissues. (A) Expression levels of marmoset FMO1–FMO5 mRNAs in five marmoset tissues (each pool of six male and six female marmosets) were measured by real-time RT-PCR. Raw values of target gene expression were normalized with the 18S rRNA level. Each data point represents the average and S.D. of triplicate determinations from three representative experiments. (B and C) Pooled tissue microsomes from marmosets (20 μg/lane) (B) and recombinant marmoset FMO3 protein (0.1 pmol FMO3/lane), individual liver microsomes from marmosets (male, lanes 1–2; female, lanes 3–5) (20 μg/lane) (C) were analyzed by immunoblotting using anti-human FMO3 antibodies. Protein disulfide isomerase (PDI) expression was assessed as a loading control.
To assess the importance of FMO forms in drug oxidation in the marmoset liver, drug oxygenation activities by liver microsomes preheated for FMO inhibition were measured. Preheat-sensitive benzydamine N-oxygenation and sulindac sulfide S-oxygenation activities in marmoset liver and kidney microsomes was found (Supplemental Table 2). Recombinant FMO3-mediated benzydamine N-oxygenation activity and its Vmax/Km value were higher than those for recombinant FMO1, suggesting that FMO3 abundantly expressed in the liver plays a role in the N-oxygenation of benzydamine in the marmoset liver (Table 1), similar to the human liver (Taniguchi-Takizawa et al., 2015). Kinetic analyses also indicated that marmoset liver microsomes effectively catalyzed sulindac sulfide S-oxygenation (Vmax/Km, 0.12 ml/min per milligram protein) (Table 1), compared with those of humans (Vmax/Km, 0.02 ml/min per milligram protein), as previously reported (Yamazaki et al., 2014). Marmoset FMO3 was catalytically efficient (Vmax/Km, 0.80 ml/min per nanomole) for sulindac sulfide S-oxygenation and showed a low Km value (38 μM), comparable to marmoset liver microsomes (23 μM), similar to human FMO3 (Yamazaki et al., 2014). Marmoset FMO1 showed low Km (43 μM) and high Vmax/Km (0.53 ml/min per nanomole), compared with human FMO1 (Km, 280 μM; Vmax/Km, 0.01 ml/min per nanomole). Considering this together with tissue distribution, FMO1 might account for the potential species differences in sulindac sulfide S-oxygenation rates between marmoset and human livers. These results indicated that benzydamine N-oxygenation and sulindac sulfide S-oxygenation in the marmoset liver were mainly catalyzed by FMO3.
Kinetic parameters of benzydamine N-oxygenation and sulindac sulfide S-oxygenation by recombinant FMO proteins and liver microsomes from marmosets
Kinetic parameters were calculated from fitted curves by nonlinear regression and are presented as means ± S.E.
In summary, marmoset FMO1–FMO5 had amino acid sequences that were highly identical (>93%) to human FMO1–FMO5, as well as a phylogenetically close relationship with human FMO1–FMO5. In contrast with humans, FMO3 and FMO1 mRNA was abundant in the marmoset liver and kidneys among five FMO forms. Recombinant marmoset FMO3 enzymes heterologously expressed in E. coli effectively metabolized typical human FMO substrates, benzydamine and sulindac sulfide. These results indicated that marmoset FMO1–FMO5 has sequences that are similar to those of humans but are partially different from humans in terms of tissue expression. Importantly, marmoset and human FMO3, a major hepatic FMO in both species, had similar enzymatic properties, suggesting the similarity of FMO-dependent drug metabolism for marmosets and humans.
Authorship Contributions
Participated in research design: Uehara, Shimizu, Uno, Yamazaki.
Conducted experiments: Uehara, Shimizu.
Contributed new reagents or analytic tools: Inoue, Sasaki.
Performed data analysis: Uehara, Shimizu, Uno, Yamazaki.
Wrote or contributed to the writing of the manuscript: Uehara, Shimizu, Uno, Yamazaki.
Footnotes
- Received January 20, 2017.
- Accepted March 1, 2017.
S.U., M.S., and Y.U. contributed equally to this work.
This research was supported partly by the Japan Society for the Promotion of Science [Grant-in-Aid for Young Scientists B 15K18934 (to S.U.)]. This work resulted from “Construction of System for Spread of Primate Model Animals” under the Strategic Research Program for Brain Sciences of the Japan Agency for Medical Research and Development.
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This article has supplemental material available at dmd.aspetjournals.org.
Abbreviations
- FMO
- flavin-containing monooxygenase
- PCR
- polymerase chain reaction
- RT
- reverse transcription
- Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics