Article Cite This: Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Molecular and Functional Characterization of N‑Acetyltransferases NAT1 and NAT2 in Cynomolgus Macaque Yasuhiro Uno,*,† Norie Murayama,‡ and Hiroshi Yamazaki*,‡ †
Shin Nippon Biomedical Laboratories, Ltd., Kainan 642-0017, Japan Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-0042, Japan
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‡
ABSTRACT: Arylamine N-acetyltransferases (NATs) are drug-metabolizing enzymes essential for the metabolism of endogenous substrates and xenobiotics, and their molecular characteristics have been extensively investigated in humans, but not in cynomolgus macaques, nonhuman primate species important for drug metabolism studies. In this study, cynomolgus NAT1 and NAT2 cDNAs were isolated from livers. NAT1 and NAT2 were characterized by molecular analyses and drug-metabolizing assays. A total of 9 transcript variants were found for cynomolgus NAT1, similar to human NAT1, and contained 1−4 exons with the coding region largely conserved with human NAT1. Genomic organization was similar between cynomolgus macaques and humans. Cynomolgus NAT1 and NAT2 amino acid sequences showed high sequence homology (95% and 89%, respectively) and showed close relationships with human NAT1 and NAT2 in a phylogenetic tree. Cynomolgus NAT2 mRNA was predominantly expressed in liver among the 10 different tissues analyzed, followed by kidney and jejunum. In contrast, cynomolgus NAT1 mRNA showed more ubiquitous expression with relatively more abundant expression in liver, kidney, and jejunum, along with testis. Metabolic assays using recombinant proteins showed that cynomolgus NAT1 and NAT2 metabolized human NAT substrates, including paminobenzoic acid, sulfamethazine, isoniazid, and 2-aminofluorene. Interestingly, p-aminobenzoic acid and isoniazid were largely metabolized by NAT1 and NAT2, respectively, in cynomolgus macaques and humans; sulfamethazine, a human NAT2 substrate, was metabolized by both NAT enzymes in cynomolgus macaques. These results suggest molecular and enzymatic similarities of NAT1 and NAT2 between cynomolgus macaques and humans, despite some small differences in substrate specificity of the enzymes.
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INTRODUCTION Arylamine N-acetyltransferase (NAT), including NAT1 and NAT2 in humans, is the enzyme important for acetylation of xenobiotics including environmental carcinogens.1−3 Human NAT1 catalyzes the acetylation of p-aminosalicylate, while human NAT2 predominantly acetylates hydralazine, procainamide, and isoniazid.1,2 However, some substrates such as 5aminosalicylic acid, 4-bromoaniline, and 4-iodoaniline are acetylated by both human NAT enzymes,1,2 indicating different but overlapping substrate specificity. In humans, NAT1 and NAT2 are adjacent genes in chromosome 8p22.4 Human NAT1 and NAT2 mRNAs show distinct tissue expression patterns; NAT2 mRNA is expressed mainly in liver and intestine, while NAT1 mRNA is expressed in various tissues including liver and intestine.5−7 Human NAT1 and NAT2 genes contain an intronless coding region, preceded by 5′UTR, and in human NAT1, 5′UTR is variable due to two alternative promoters and alternative splicing, resulting in several different transcript variants.5,8 Numerous genetic variants have been identified in human NAT1 and NAT2 genes, including functionally relevant mutations.9−11 For example, the R64W mutation of NAT1 and NAT2 results in unfolded protein and reduced activity of the enzyme. The D122N mutation of NAT2 results in the © XXXX American Chemical Society
folded but inactive protein because Asp122 is an important part of the catalytic triad.9,11 Moreover, several NAT alleles are associated with the susceptibility to urinary bladder cancer in smokers and in workers exposed to aromatic amines.12,13 Therefore, NAT alleles are toxicologically important for the enzyme activities and susceptibility to cancer. Cynomolgus macaque (Macaca fascicularis) is an important primate species widely used in drug metabolism studies. Molecular and functional characteristics of drug-metabolizing enzymes such as cytochromes P450 are generally similar between cynomolgus macaques and humans; however, some differences are noted. 14 For example, CYP2C76, not orthologous to any human P450, is partly responsible for differences in pitavastatin metabolism between cynomolgus macaques and humans.15,16 Moreover, CYP1D1 and CYP2G2 are pseudogenes in humans, but are expressed as functional genes in cynomolgus macaques17,18 and thus might account for species differences in drug metabolism. Therefore, characterization of drug-metabolizing enzymes is essential to understand the similarities and differences of drug metabolism between animals and humans. Received: August 22, 2018 Published: October 17, 2018 A
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
(NP_032700). Cynomolgus NAT1 and NAT2 amino acid sequences were deduced from the cDNAs identified in this study. For analysis of gene structure, we used cynomolgus NAT cDNA sequences identified in this study and human NAT cDNA sequences from GenBank, including human NAT1v1 (NM_001160170), v2 (NM_001160171), v3 (NM_001160172), v4 (NM_001160173), v5 (NM_000662), v6 (NM_001160174), v7 (NM_001160175), v8 (NM_001160176), v9 (NM_001160179), and v10 (NM_001291962); and NAT2 (NM_000015). Measurement of mRNA Expression. Real-time RT-PCR was performed as reported previously15 to measure expression levels of cynomolgus NAT1 and NAT2 mRNAs in brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus, except that fast PCR was performed in this study. Briefly, one twenty-fifth of the RT products obtained as described above using random primers (Invitrogen) was used for PCR carried out using Fast SYBR Green Master Mix (Applied Biosystems) with the ABI PRISM 7500 fast sequence detection system (Applied Biosystems) according to the manufacturer’s protocols. Primers were used at a final concentration of 200 nM, including mfNAT1 (5qrt2) 5′-CTAGAAGACAGCAAATACCGAAAAATC-3′ and mfNAT1 (3qrt2) 5′-AATGATTTGCTAGTAAACACAGATGCT-3′ for cynomolgus NAT1 and mfNAT2 (5qrt1) 5′-CTTTATTACAGACCTTGGAAGCAAGA-3′ and mfNAT2 (3qrt1) 5′-CAGTTAATGTTTCCAAGTCCAGCTT-3′ for cynomolgus NAT2. The raw data were normalized by the βactin level to determine the relative expression level as described previously,20 based on three independent amplifications. Preparation of Recombinant Protein. Expression plasmids were prepared and were used to express cynomolgus NAT1 and NAT2 proteins as described previously.19,21 PCR was performed as described earlier using KOD-Plus-Neo DNA polymerase with each NAT cDNA as a template to generate expression plasmids. The primers used were mfNAT1 (5exp1) 5′-ATGGACATTGAAGCATATCTTGAAAGAATT-3′ and mfNAT1 (3exp1) 5′CCGCTCGAG-CGCCCAGCCTACTCCTTATTCTAAA-3′ for cynomolgus NAT1 and mfNAT2 (5exp1) 5′-ATGGACATTGAAGCATATTTTGAAAGAATTGG-3′ and mfNAT2 (3exp1) 5′CCGCTCGAG-CTAAATAGTGAAGGATCCATTACCAGGTTT3′ for cynomolgus NAT2. Reverse primers contained restriction enzyme site sequences XhoI to facilitate subcloning of the PCR products into vectors. After restriction enzyme digestion, the amplified DNAs were subcloned into a pET30a vector (Novagen, Madison, WI) to generate protein with a 6xHis-tag at the N-terminus. The expression plasmids prepared were used for protein expression in Escherichia coli (E. coli), and cytosolic preparations were carried out as described previously.19,21 Immunoblotting. For cynomolgus macaques and humans, liver cytosolic fractions and NAT proteins heterologously expressed in E. coli were analyzed by immunoblotting. The proteins separated on 12.0% SDS polyacrylamide gels were transferred to nitrocellulose membranes (0.45 μm, Bio-Rad, Munich, Germany) and then incubated with antihuman NAT2 rabbit polyclonal antibodies (Lifespan Bioscience, Seattle, WA). Quantification of NAT proteins in liver cytosolic fractions was performed in comparison with the band intensities using standards (recombinant NAT proteins). Anti-6 x histidine-tag mouse monoclonal antibodies (BioDynamics Laboratory, Tokyo, Japan) were also used to measure cynomolgus NAT proteins heterologously expressed in E. coli, in comparison with the band intensity of the histidine-tagged human flavin-containing monooxygenase 3 (FMO3) used as a standard. This FMO3 protein was previously immunoquantified using other antihuman FMO3 antibodies as previously described.19,22 After washing, the membranes were further incubated with secondary antibodies, antirabbit IgG goat antibodies (Sigma-Aldrich), or horseradish peroxidase-conjugated antimouse IgG goat antibodies. An ECL Western blotting detection reagent (GE Healthcare, Buckinghamshire, United Kingdom) was used to detect the signals according to the manufacturer’s instructions. Enzyme Assays. AcCoA-dependent acetylation of p-aminobenzoic acid, sulfamethazine, 2-aminofluorene, and isoniazid was performed. Typical reaction mixtures of 250 μL containing NAT1 and
In this study, we isolated cynomolgus NAT1 and NAT2 cDNAs from liver. Cynomolgus NAT1 and NAT2 were characterized by sequence and phylogenetic analyses, tissue expression pattern, and enzymatic assays using recombinant proteins.
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EXPERIMENTAL PROCEDURES
Materials. Human liver total RNA was purchased from Takara (Otsu, Japan). Human NAT1 and NAT2 recombinant proteins were obtained from Corning Life Sciences (Woburn, MA). Greiner (Tokyo, Japan) synthesized oligonucleotides. All other reagents were purchased from Sigma-Aldrich (St. Louis, MO) or Wako Pure Chemicals (Osaka, Japan), unless otherwise specified. Tissues, RNA, and Liver Cytosolic Fraction. Total RNA was extracted as described previously15 from brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus which were collected from six cynomolgus macaques (three males and three females, 4−5 years of age) and were used for analysis of tissue expression patterns. Liver samples were collected from cynomolgus macaques (7−15 years of age, 3−5 kg), including 4 males and 4 females bred in Cambodia, 4 males bred in Indonesia, 4 males bred in China, and 3 male rhesus macaques bred in China (7 years of age, 3− 5 kg), and snap-frozen immediately in liquid nitrogen. From these liver samples, cytosolic fractions were prepared as described previously.19 Liver samples and the liver cytosols were stored at −80 °C until use. This study was reviewed and approved by the Institutional Animal Care and Use Committee of Shin Nippon Biomedical Laboratories, Ltd. (Kainan, Japan). Molecular Cloning. Cynomolgus NAT1 and NAT2 cDNAs were isolated by reverse transcription (RT)-polymerase chain reaction (PCR) using liver total RNA as described previously.15 Briefly, the first-strand cDNA was synthesized using total RNA (1 μg), SuperScript III RT reverse transcriptase (Invitrogen, Tokyo, Japan), and oligo (dT) at 50 °C for 1 h and was used as the template for PCR reactions performed using KOD-Plus-Neo DNA polymerase (Toyobo, Osaka, Japan) with a thermal cycler (Applied Biosystems, Foster City, CA) according to the manufacturer’s protocols. Thermal cycling conditions were as follows: an initial denaturation at 94 °C for 2 min; 35 cycles at 98 °C for 10 s, 65 °C for 30 s, and 68 °C for 2.5 min; and a final extension at 68 °C for 7 min. Similarly, human NAT1 and NAT2 cDNAs were isolated using human total liver RNA, except that Prime STAR HS DNA Polymerase (Takara) was used for PCR. The primers used were mmNAT1 (5rt2) 5′-GAGAGCACTTCCTCATAGACGTTGGATGT-3′ and mmNAT1 (3rt1) 5′GGTGAGCTGGGTGACAAATACACAAGAT-3′ for cynomolgus NAT1v1, mmNAT1 (5rt3) 5′-GCCTGCATCAGAAGATGTTTGTAAGCCTAT-3′ and mmNAT1 (3rt1) for cynomolgus NAT1v2, and mfNAT2 (5rt1) 5′-GAGATCACTTCCCTTGCAGACTTTGG3′ and mfNAT2 (3rt1) 5′-GGGTGATACATACACAAGGGTTTATTTTGTTC-3′ for cynomolgus NAT2. The PCR products were cloned using a Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen), and the inserts were sequenced using an ABI PRISM BigDye Terminator v3.0 Ready Reaction Cycle Sequencing Kit (Applied Biosystems) with an ABI PRISM 3730 DNA Analyzer (Applied Biosystems) according to the manufacturer’s protocols. Sequence Analysis. Sequencher (Gene Codes Corporation, Ann Arbor, MI), the Genetyx system (Software Development, Tokyo, Japan), or DNASIS Pro (Hitachi Software, Tokyo, Japan) was used to analyze raw data. Multiple alignment was carried out with the ClustalW program, and phylogenetic analysis was performed by the neighbor-joining method. BLAST (National Center for Biotechnology Information) and BLAT (UCSC Genome Bioinformatics) were, respectively, used for a homology search and analysis of the genome data. Amino acid sequences used were human NAT1 (NP_001153642) and NAT2 (NP_000006); pig NAT1 (ACR78281) and NAT2 (ACR78285); rat NAT1 (NP_446305), NAT2 (NP_446306), and NAT3 (NP_001013070); and mouse NAT1 (NP_032699), NAT2 (NP_035004), and NAT3 B
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology NAT2 (40 pmol equivalents/mL), substrates (0.10 or 0.30 mM), and 1 mM DTT in 20 mM sodium phosphate buffer (pH 8.0) were initiated by addition of AcCoA (1.0 mM). After incubation at 37 °C for 10 min, the reaction was terminated by adding 25 μL of ice-cold 1.0 M acetic acid. The reaction rates were determined at 280 nm in the spectrometry.19,21 Catalytic activity of NAT1 and NAT2 was expressed as the nanomoles of substrates acetylated per minute per milligram of protein or nmol NAT. Data are mean and SD values in triplicate determinations unless specified.
NAT1 and NAT2, respectively, in amino acids (Table 1). Interestingly, the most homologous sequence to cynomolgus NAT1 and NAT2 was NAT1 in pigs and NAT2 in rats and mice. By phylogenetic analysis, cynomolgus NAT1 and NAT2 were most closely clustered with human NAT1 and NAT2, respectively, unlike pig, rat, or mouse NATs (Figure 3). These results suggest that cynomolgus NAT1 and NAT2 are closely related to human NAT1 and NAT2, respectively. Gene Structure of Cynomolgus NATs. Because several NAT1 transcript variants found in humans result from three alternative promoters, three forward primers of cynomolgus NAT1 were designed at the putative first exons corresponding to the human transcript variants. Only two of these primers showed amplification, resulting in the two transcript variants (NAT1v1 and v2) comprising a total of nine different transcripts, of which three transcripts (v1−3, v1−5, and v2− 22) contained a complete open reading frame (Figure 4). Based on the numbers of the clones isolated, these three transcripts represented the majority of the transcripts identified; 17 (57%) of 30 clones for NAT1v1 and 19 (79%) of 24 clones for NAT1v2. The other six transcripts contained variable exons likely due to alternative splicing, and indels, disrupting an open reading frame (Figure 4). In contrast, cynomolgus NAT2 did not express transcript variants and contained the intronless coding region, preceded by noncoding exon 1, similar to human NAT2 (Figure 4). The cDNA sequences of cynomolgus NAT1 (transcript v2−22, KX025147) and NAT2 (KX025148) identified have been deposited to GenBank. Expression of Cynomolgus NATs in Tissues. By realtime RT-PCR, expression of cynomolgus NAT mRNAs was measured in brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus. Among these tissues, cynomolgus NAT1 mRNA was most abundantly expressed in liver, followed by kidney and jejunum (Figure 5). In contrast, cynomolgus NAT1 mRNA was expressed in most tissues analyzed with relatively more abundant expression in liver, kidney, jejunum, and testis. In liver, cynomolgus NAT2 mRNA was more abundant (9.0-fold) than NAT1 mRNA, similar to kidney (6.2-fold) and jejunum (2.5-fold). Expression of cynomolgus NAT1 and NAT2 proteins in liver cytosolic fractions was analyzed by immunoblotting using antihuman NAT2 antibodies (Figure 6A). Apparent single
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RESULTS Identification and Analysis of Cynomolgus NAT cDNAs. Analysis of (rhesus) macaque genome data found the gene sequences highly homologous to human NAT1 and NAT2. Macaque NAT1 and NAT2 were located adjacently in the gene cluster, and their location and direction were similar to the human orthologs (Figure 1). Based on these NAT1 and
Figure 1. Genomic organization of NATs. The macaque and human genomes were analyzed using BLAT as described in Experimental Procedures. NAT1 and NAT2 were located in a gene cluster in the macaque genome, and their location and directions corresponded to the human orthologs. Sizes of the genes and distances between the genes are not proportional to actual measurements.
NAT2 sequences, gene-specific primers were designed, and NAT1 and NAT2 cDNAs were successfully isolated from cynomolgus liver by RT-PCR. Cynomolgus NAT1 and NAT2 amino acid sequences deduced from the identified cDNAs contained open reading frames of 290 amino acid residues with primary sequence structures highly conserved in NAT proteins, including I/VPFENLxx, RGGfCdrxxxf, and DaGdjj23 (Figure 2). A catalytic triad (Cys68, His107, Arg122) was also conserved in cynomolgus and human NATs. Cynomolgus NAT1 and NAT2, highly identical (89%) to each other, shared high sequence homologous (95% and 89%) with human
Figure 2. Multiple alignment of cynomolgus and human NAT amino acid sequences. Cynomolgus NAT1 and NAT2 were aligned with human NAT1 and NAT2 as described in Experimental Procedures. The solid lines and arrows above the sequences indicate characteristic motif of NAT proteins (I/VPFENLxx, RGGfCdrxxxf, and DaGdjj) and catalytic triad residues, respectively. Asterisks and dots under the sequences indicate identical and conserved residues, respectively. C
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology Table 1. Sequence Identity of NAT Amino Acids between Cynomolgus Macaques and Other Speciesa human human cynomolgus pig rat
mouse
NAT1 NAT2 NAT1 NAT2 NAT1 NAT2 NAT1 NAT2 NAT3 NAT1 NAT2 NAT3
cynomolgus
pig
rat
mouse
NAT1
NAT2
NAT1
NAT2
NAT1
NAT2
NAT1
NAT2
NAT3
NAT1
NAT2
NAT3
100
81 100
95 81 100
82 89 83 100
85 77 86 79 100
76 74 76 74 80 100
76 74 78 73 75 70 100
81 73 83 75 80 72 83 100
69 67 68 68 69 67 71 72 100
75 72 76 72 75 70 92 81 70 100
82 75 84 76 82 74 83 94 73 82 100
68 66 69 67 70 65 69 73 88 68 73 100
a
Cynomolgus NAT amino acid sequences were compared using BLAST as described in Experimental Procedures. Highest sequence identities in each species are shown in bold for cynomolgus and human NATs.
some interanimal variability and distinct but overlapping substrate specificity.
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DISCUSSION NATs play important roles for metabolizing and detoxifying various compounds such as drugs and environmental carcinogens; however, their characteristics have not been fully elucidated in cynomolgus macaques. In this study, cynomolgus NAT1 and NAT2 were identified and characterized by sequence and phylogenetic analyses, tissue expression patterns, gene and genome structure, and enzymatic assays. Cynomolgus NAT1 and NAT2 had characteristic motifs of NAT proteins (Figure 2), were highly homologous to human NAT1 and NAT2 (Table 1), and were closely clustered with human NAT1 and NAT2 in a phylogenetic tree (Figure 3). Moreover, genomic organization and gene structure of NAT1 and NAT2, respectively, were also similar in macaques and humans (Figures 1 and 4). Therefore, cynomolgus and human NAT1 and NAT2 are orthologs with similar molecular characteristics. Several NAT1 transcript variants were found in cynomolgus macaques, similar to humans (Figure 4). Because human NAT1 mRNAs are transcribed from two or more promoters,8,24,25 three forward primers were designed at the macaque genomic region to amplify the transcripts potentially transcribed from the three corresponding promoters (Figure 4). Of the three forward primers, two gave rise to nine transcript variants, indicating at least two alternative promoters are used to transcribe NAT1 in cynomolgus macaques. No NAT1 transcripts were found with the remaining primer, possibly indicating that this third promoter might not be functional in cynomolgus macaques. Most of the NAT1 transcripts and NAT2 transcript contained the complete coding region. These results indicated that NAT1 and NAT2 were intact genes in cynomolgus macaques and cynomolgus NAT1 transcripts were transcribed from different promoters, similar to humans. Cynomolgus NAT1 and NAT2 showed distinct tissue expression patterns. Cynomolgus NAT1 mRNA was expressed in various tissues, whereas cynomolgus NAT2 mRNA was preferentially expressed in liver, kidney, and jejunum (Figure 5). In liver, cynomolgus NAT1/NAT2 proteins were also detected by immunoblotting (Figure 6A). In humans, NAT2 protein is relatively more abundant in liver and intestine, and
Figure 3. Phylogeny of NAT amino acid sequences. The phylogenetic tree was created by the neighbor-joining method using NAT amino acid sequences from humans (h), cynomolgus macaques (mf), pigs (p), rats (r), and mice (m). The scale bar indicates 0.1 amino acid substitutions per site for distance measurement.
bands were detected for cynomolgus and rhesus NAT1/2 proteins corresponding to human NAT1/2 (Figure 6A). Drug-Metabolizing Activities of NAT Proteins. Cynomolgus NAT1 and NAT2 proteins heterologously expressed in E. coli and human NAT1 and NAT2 commercially available were assessed for their drug-metabolizing activities toward NAT substrates, p-aminobenzoic acid, sulfamethazine, 2aminofluorene, and isoniazid (Table 2). Cynomolgus NAT1 and NAT2 efficiently metabolized p-aminobenzoic acid and isoniazid, respectively, similar to humans. On the other hand, sulfamethazine, the human NAT2 substrate, was metabolized by both NAT1 and NAT2 in cynomolgus macaques. Both cynomolgus and human NAT1 and NAT2 commonly acetylated 2-aminofluorene (Table 2). N-Acetyltransferase activities of liver cytosolic fractions from cynomolgus macaques (4 males and 4 females bred in Cambodia, 4 males bred in Indonesia, and 4 males bred in China) and 3 male rhesus macaques were determined at the substrate concentrations of 300 μM (Figure 6B). NAT1- and NAT2-dependent N-acetylation activities did not significantly differ among the three groups of cynomolgus macaques or between males and females. The results indicated that cynomolgus NAT1 and NAT2 are functional enzymes with D
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
Figure 4. Cynomolgus NAT gene structures. NAT1 and NAT2 cDNA sequences of cynomolgus macaques (mf, A) and humans (h, B) were aligned with the macaque and human genome, respectively, using BLAT to determine gene structure as described in Experimental Procedures. Arrows above the exons show the location of three primers (Primer 1 to 3) used to amplify NAT1 transcripts in cynomolgus macaques and humans (the corresponding locations). Arrows below the exons show the insertion (ins) and deletion (del) found in the coding region. Coding region is presented by black color.
in addition to these tissues, NAT2 mRNA is also expressed in kidney.5,26 Similar tissue expression patterns of NAT mRNAs suggest that cynomolgus macaques and humans might have similar regulatory mechanisms for gene expression. In humans, an active Sp1 box, known to drive constitutive gene expression, is located in the upstream regulatory element of NAT1,27 but is not present in that of NAT2,26 possibly indicating the importance of this regulatory element for distinct expression of NAT1 and NAT2 mRNAs. The similar regulatory element might control ubiquitous expression of cynomolgus NAT1 mRNA. In liver, cynomolgus NAT2 mRNA was much more abundant than cynomolgus NAT1 mRNA (Figure 5), indicating that cynomolgus NAT2 is the major hepatic NAT
and is important for NAT-dependent drug-metabolizing activity in liver. In this study, cynomolgus NAT1 and NAT2 heterologously expressed in E. coli metabolized one or more human NAT substrates including p-aminobenzoic acid, sulfamethazine, isoniazid, and 2-aminofluorene (Table 2), indicating that these proteins are functional drug-metabolizing enzymes. pAminobenzoic acid and isoniazid were, respectively, largely metabolized by NAT1 and NAT2 in cynomolgus macaques and humans, but sulfamethazine, a human NAT2 substrate, was metabolized by both NAT enzymes in cynomolgus macaques (Table 2). These results suggest enzymatic similarities of NAT1 and NAT2 between cynomolgus E
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology
Figure 5. Tissue expression pattern of cynomolgus NAT mRNAs. Real-time RT-PCR was performed to measure expression levels of cynomolgus NAT1 (solid bar) and NAT2 (open bar) mRNAs in brain, lung, heart, liver, kidney, adrenal gland, jejunum, testis, ovary, and uterus as described in Experimental Procedures. Expression levels of each NAT mRNA were normalized to β-actin level, representing the average ± SD from three independent amplifications. The expression level of NAT2 mRNA in liver was arbitrarily adjusted to 1, with which all other expression levels were compared.
macaques and humans with some differences in substrate specificity. In rhesus macaques, a primate species closely related to cynomolgus macaques, p-aminobenzoic acid and isoniazid are metabolized by NAT enzymes, and p-aminobenzoic acid is highly metabolized by NAT1,28 similar to cynomolgus macaques. Rhesus NAT1 also metabolizes NAT substrates, p-aminobenzoic acid, 5-aminosalicylate, and procainamide among others and is a highly active enzyme showing excessive activities with NAT substrates compared with nine other primates species including humans.28 It is of great interest to investigate cynomolgus NAT enzymes using various NAT substrates. Amino acid changes of NAT proteins between cynomolgus macaques and humans were found in the region important for NAT function. For example, residue 93, involved in substrate specificity of the enzyme, is Phe in human NAT2,3 but was Val in cynomolgus NAT2, and human and cynomolgus NAT1 (Figure 2). In human NAT2, Val93 introduces a bump in the van der Waals surface of substrate pocket, resulting in selective binding of sulfonamide class substrates to the enzyme.29 Residue 125, one of the key residues in the substrate pocket, is Ser in human NAT2, but was Phe in cynomolgus NAT2, and human and cynomolgus NAT1 (Figure 2). By mutagenesis, F125S substitution abolishes substrate preference of human NAT1 for p-aminosalicylic acid over sulfamethazine,30 because structural analysis indicated Phe125 to be important for binding to p-aminosalicylic acid.29 The differences in substrate preference of cynomolgus NAT2 compared to human NAT2 (Table 2) were possibly accounted for by the substitution of residues 93 and 125. By random mutagenesis, human NAT2 activity is reduced with mutation at residues 54, 146, 185, 198, and 23831 where cynomolgus NAT2 contained different residues (Figure 2). Residues of E276Q and M280V in cynomolgus NAT1 and K278N, N285D, and F288L in cynomolgus NAT2 were located in the C-terminal region, which is important for enzyme activity and substrate specificity of NAT enzyme.32 In human NATs, the C-terminal region extends into the interior of the folded enzyme close to the buried catalytic triad and restricts its accessibility,29 and mutations in this region might
Figure 6. N-Acetyltransferase activities of liver cytosolic fractions from cynomolgus and rhesus macaques. (A) Immunochemical determination of NATs with antihuman NAT2 antibodies in liver cytosolic fractions (0.1 mg protein) of cynomolgus macaques bred in Cambodia (lanes 1, 2), Indonesia (lanes 3, 4), and China (lanes 5, 6), rhesus macaques (lanes 7, 8), and humans (lane 9). (B) NAcetyltransferase activities of liver cytosolic fractions from cynomolgus macaques (4 males and 4 females bred in Cambodia, 4 males bred in Indonesia, and 4 males bred in China) and 3 male rhesus macaques at the substrate concentrations of 300 μM. Data with bars are average and SD values for individual animals.
influence active site access and substrate specificity of the enzyme. Several amino acid changes were found at the residues in the regions important for the enzyme function of cynomolgus NAT1 and NAT2. The residues of F103V, V106I, S216A, and I218T in cynomolgus NAT2 and S214A in cynomolgus NAT1 were located in the regions important for substrate binding (residues 93−106 and 212−222).33 The residue of S125F in cynomolgus NAT2 is located in the flexible loop important for substrate specificity (residues 122−131).33 Moreover, several amino acid changes were found at the residues of the functional polymorphisms that influence enzyme characteristics, including V149I, E167D, and S214A in cynomolgus NAT110 and R268K in cynomolgus NAT2.11 Of these residues, residues 103, 214, and 216 among others form hydrogen bonds, and residues 214 and 288 are involved in van der Waals contacts, with the substrate CoA.29 Residue 106 among others makes hydrophobic interactions with the F
DOI: 10.1021/acs.chemrestox.8b00236 Chem. Res. Toxicol. XXXX, XXX, XXX−XXX
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Chemical Research in Toxicology Table 2. N-Acetyltransferase Activities of Liver Cytosolic Fractions and Recombinant NAT1 and NAT2 Proteins of Cynomolgus Macaques and Humansa monkey NAT1 substrate
μM
p-aminobenzoic acid
100 300 100 300 100 300 100 300
sulfamethazine 2-aminofluorene isoniazid
nmol/min/nmol NAT 460 1400 25 110 440 510
