Systematic characterization of glutathione S-transferases in common marmosets

Journal Pre-proofs Systematic characterization of glutathione S-transferases in common marmosets Yasuhiro Uno, Shotaro Uehara, Saki Tanaka, Norie Murayama, Hiroshi Yamazaki PII: DOI: Reference:

S0006-2952(20)30045-9 https://doi.org/10.1016/j.bcp.2020.113835 BCP 113835

To appear in:

Biochemical Pharmacology

Received Date: Accepted Date:

21 December 2019 31 January 2020

Please cite this article as: Y. Uno, S. Uehara, S. Tanaka, N. Murayama, H. Yamazaki, Systematic characterization of glutathione S-transferases in common marmosets, Biochemical Pharmacology (2020), doi: https://doi.org/ 10.1016/j.bcp.2020.113835

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© 2020 Published by Elsevier Inc.

Pharmacokinetics and Drug Metabolism

Systematic characterization of glutathione S-transferases in common marmosets

Yasuhiro Uno

a

a,b,#,* ,

c,#

c

c

Shotaro Uehara , Saki Tanaka , Norie Murayama , Hiroshi Yamazaki

c,*

Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima-city, Kagoshima b

890-8580, Japan, Shin Nippon Biomedical Laboratories, Ltd., Kainan, Wakayama 642-0017, c

Japan, and Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, Machida, Tokyo 194-8543, Japan

#

These authors contributed equally.

*Corresponding

author:

1) Yasuhiro Uno, D.V.M., Ph.D. Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima-city, Kagoshima 890-8580, Japan. Phone/Fax: +81-99-285-8715. E-mail address: [email protected] Or 2) Hiroshi Yamazaki, Ph.D. Laboratory of Drug Metabolism and Pharmacokinetics, Showa Pharmaceutical University, 3-3165 Higashi-Tamagawa Gakuen, Machida, Tokyo 194-8543, Japan. Phone: +81-42-721-1406. Fax: +81-42-721-1406. E-mail address: [email protected]

1

Abstract The common marmoset is an important primate species used in drug metabolism studies. However, glutathione S-transferases (GSTs), essential drug-metabolizing enzymes involved in the conjugation of various endogenous and exogenous substrates, have not been identified or characterized in this species. In this study, 20 GSTs [including 3 microsomal GSTs (MGSTs)] were identified and characterized in marmosets. Marmoset GSTs had amino acid sequences highly identical (86–99%) to human GSTs, except for GSTA4L, which had lower identities (59–62%) with human GSTAs. Phylogenetic analysis revealed that marmoset GSTs were closely clustered with their human counterparts. Marmoset GSTs had gene and genomic structures generally similar to their human counterparts, with some differences in GSTA, GSTM, and GSTT clusters. Marmoset GST mRNAs exhibited distinct tissue expression patterns: GSTA1, GSTA3, GSTA4L, GSTK1, GSTT1, GSTZ1, and MGST1 mRNAs were expressed most abundantly in liver. Other GST mRNAs were expressed most abundantly in small intestine, lung, brain, or kidney. Expression of GSTT4 and GSTT4L mRNAs was detected only in testis. Among all 20 marmoset GST mRNAs, the most abundant mRNAs were GSTA1 mRNA in liver, small intestine, and kidney; GSTM3 mRNA in testis; and MSGT3 mRNA in brain and lung. All 20 GSTs mediated the conjugation of GST substrates 1-chloro-2,4dinitrobenzene;

1,2-epoxy-3-(p-nitrophenoxy)propane;

styrene

7,8-oxide;

and/or

1-

iodohexane, but with different activity levels. Kinetic analyses showed that marmoset GSTM2/GSTM5 and GSTM5/GSTT1 effectively conjugated styrene 7,8-oxide and 1iodohexane, respectively, with the highest affinity. These results suggest that the 20 newly identified marmoset GSTs were functional drug-metabolizing enzymes able to conjugate typical GST substrates. Keywords: Common marmoset, GST, Tissue expression, Styrene 7,8-oxide, 1-Iodohexane 2

1. Introduction The glutathione S-transferase (GST) superfamily contains cytosolic, mitochondrial, and microsomal subfamilies. GSTs are important drug-metabolizing enzymes that catalyze the conjugation of reduced glutathione to electrophilic substrates such as halogenonitrobenzenes, arene oxides, and quinones [1]. In humans, cytosolic GSTs comprise seven classes of transferases, namely alpha (GSTA1–A5), mu (GSTM1–M5), omega (GSTO1/GSTO2), pi (GSTP1), sigma (GSTS1), theta (GSTT1/GSTT2), and zeta (GSTZ1). In contrast, mitochondrial GST comprises only the kappa class (GSTK1). Cytosolic and mitochondrial GSTs play roles in detoxifying potentially harmful, reactive, endogenously derived compounds and in metabolizing foreign chemicals, such as cancer chemotherapeutic drugs, carcinogens, and environmental pollutants [2]. In humans, the deletion of GSTM1 and/or GSTT1 leads to a deficiency of these important enzymes that is associated with a higher risk of head and neck, lung, breast, and brain cancer [3]. Impairments or deficiencies in GSTM1 and GSTT1 are also important in alcoholic liver disease [4] and drug-induced liver injury [5]. In humans, microsomal GSTs (MGST1–3) are part of the MAPEG (membrane-associated proteins in eicosanoid and glutathione metabolism) superfamily, the six members of which indirectly contribute to drug metabolism or detoxification. The other members of MAPEG are 5-lipoxygenase activating protein, leukotriene C4 synthase, and microsomal prostaglandin E2 synthase 1 [1]. Instead of contributing to drug metabolism or detoxification, these three nonGST enzymes largely contribute to the production of leukotriene C4 and prostaglandin E2, which play essential roles in fever, pain, and inflammation. In contrast, MGST1, MGST2, and MGST3 play roles in the detoxification of foreign compounds, although MGST2 and MGST3 can also synthesize leukotriene C4 [1]. Among the three human MGSTs, MGST1 is most 3

abundant in liver, where it metabolizes hydrophobic substrates, halogenated hydrocarbons, and phospholipid hydroperoxides [6]. The common marmoset (Callithrix jacchus) is an important nonhuman primate species used in drug metabolism studies. In this species, the cytochromes P450 (P450s), important drugmetabolizing enzymes, have been investigated to elucidate the similarities and differences between marmoset and human P450 enzymes [7, 8]. However, other drug-metabolizing enzymes, including GSTs, in common marmosets have not been fully investigated. Among nonhuman primates, systematic identification and characterization of GSTs in cynomolgus macaque were reported [9, 10]. The identification and characterization of GSTs are important to understand their molecular and enzymatic characteristics. In the present study, 20 GST cDNAs were isolated in marmosets, and the corresponding heterologously expressed proteins were characterized in terms of amino acid sequence and phylogenetic characteristics, tissue expression patterns, and enzymatic activities.

2. Materials and Methods 2.1. Materials Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA, USA) and FASMAC (Atsugi, Japan). 1-Chloro-2,4-dinitrobenzene (CDNB) and 1,2-epoxy-3-(pnitrophenoxy)propane (EPNPP) were purchased from Fujifilm Wako Pure Chemicals (Osaka, Japan). Marmoset testis cDNA was purchased from Genostaff (Tokyo, Japan). Anti-histidine tag antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG were purchased from BioDynamics Laboratory (Tokyo, Japan) and BD Gentest (Woburn, MA, USA), respectively. Ten human and 13 cynomolgus monkey liver cytosol samples were prepared as previously 4

described [11, 12] under the institutional approval (No. 30-13) . All other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA) or Fujifilm Wako Pure Chemicals, unless otherwise specified. 2.2. Preparation of RNA and cytosol Brain, lung, liver, kidney, and small intestine samples were collected from six marmosets (three males and three females, >2 years of age) at the Central Institution for Experimental Animals (Kawasaki, Japan), where this study was reviewed and approved (No. 1321) . Total RNA was extracted from these samples, as previously described [13], and was used for molecular cloning and quantitative polymerase chain reaction (qPCR). Cytosolic fractions were prepared, as described previously [14], from 11 marmoset liver samples. 2.3. Molecular cloning Reverse transcription (RT)-PCR was performed as previously described [13] using liver total RNA. Briefly, the RT reaction was carried out in a mixture containing 1 g of total RNA, oligo (dT), and SuperScript III RT reverse transcriptase (Invitrogen, Carlsbad, CA, USA) at 50C for 1 h. The RT product was subsequently used as the template for PCR reactions, performed using Phusion Hot Start Flex DNA Polymerase (New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocols. Testis cDNA (RT product) was used for GSTT4 and GSTT4L. The thermal cycler conditions were as follows: initial denaturation at 98C for 30 s and 35 cycles of 98C for 10 s, 65C for 20 s, and 72C for 20 s, followed by a final extension at 72C for 5 min. The primers used were designed based on the gene sequences found

in

the

marmoset

genome,

AATTCAGTTGCCGAGCCAAG-3 TGGTCTTGCATGTTCTTGGC-3

and for

including

cjGSTA1 GSTA1,

5

cjGSTA1

cjGSTA3

(5rt1) (3rt1) (5rt1)

555-

CCCGCTGGACTGATTAGAAACCAAGTA-3

and

cjGSTA3

(3rt1)

5-

GCAAAGCTTTTTTAAATATTGGCCTTGCATA-3 for GSTA3, cjGSTA4 (5rt1) 5AGCGGACTCCAGGAAGCC-3 and cjGSTA4 (3rt1) 5-ACGTTCTCAGTCACACATGG3 for GSTA4, cjGSTA4L (5rt1) 5-CCACAACTCACCCTATGTGTCTCTTCATC-3 and cjGSTA4L (3rt1) 5-GACTGGACCATAACGTCTGCCATCA-3 for GSTA4L, cjGSTK1 (5rt1)

5-GGAACCCGGGTGTCTCTG-3

AGTGCCTTATAGTGGAGCCG-3

for

CAGAATCCGCAGCAACCAG-3 GGATCAGGTAGGCAGGGATC-3

GSTM2,

and

for

GGCCTTATTTGCTGTTCCATGTAGCTGA-3

for

TACTTCCTGAATCGCCTGCA-3

and

ACATCAGACACAGCTTTATTGC-3

for

CGATGTCTCCCCAGGCTAAA-3

and

TCTGTGATGTAGACTTCCAACAA-3

for

CAGTCTGGTCTGTCGGTTTCC-3

6

(3rt1) (5rt1) (3rt1)

cjGSTS1

cjGSTS1

GSTS1, and

(5rt1)

cjGSTP1

GSTP1,

cjGSTT1

cjGSTT1

(5rt1)

(3rt1)

cjGSTP1

and for

(3rt1)

cjGSTO2

GSTO2,

for

(5rt1)

cjGSTO1

cjGSTO2

and

GAGACACAACACAGAATCGCA-3 ACTTGAAGGCAGCATGGATC-3

GSTM5, cjGSTO1

ATCTGCAACACCATGCCGCCCTACA-3 CCTCACTGTTTCCCATTGCCATTGAT-3

(3rt1)

cjGSTM5

(5rt1)

(3rt1) (5rt1) (3rt1)

55-

(5rt1)

cjGSTM5

GSTO1,

5-

(5rt1)

cjGSTM4

GSTM4, and

5-

(3rt1)

cjGSTM4

CTGTTTGCAGAACTCGCACAAACCA-3

(5rt1)

cjGSTM3

GSTM3, and

5-

(3rt1)

cjGSTM3

for

(3rt1)

cjGSTM2

cjGSTM2

for

CCCGGAGTAGGTCGCAGTTCA-3 GCATATGGGCTACTCACTCCA-3

cjGSTK1

GSTK1,

and

TACGGTTTCCTCAGTCCTCG-3 CAGCTCTGCAAGTCTTCCTG-3

and

55555555555555-

ACTTTGTGAACTGCTGAGGG-3

for

GSTT1,

CTCAACATGGCCCTGGAGCTCTACAT-3

cjGSTT4/4L

and

(5rt1)

cjGSTT4

(3rt1)

55-

GTCACTTGCTCTTCTTCAGCAACTCAGAAA-3 for GSTT4, cjGSTT4L (5rt1) and cjGSTT4L (3rt1) 5-ATCACAAGTACTTCTCCCGAAACTCACAGA-3 for GSTT4L, cjGSTZ1

(5rt1)

5-CGAAGTTTCTCGGCCTGGA-3

TTTCCAGGCCTCTTCAGTCC-3

for

GSTZ1,

ATTGTGTTTCTGTCCCGGTG-3

and

GGTCATGATTCCTCTGCATCTC-3

for

GGAAGGTCAGCACTCAAAGTC-3

cjMGST1

and for

CAGCCGTTCGTAGGTGATCT-3

and

cjGSTZ1

cjMGST1

MGST1,

TCTGCAAGCATTAAAGGGAAGAG-3

and

MGST2,

(5rt1) (3rt1)

cjMGST2

cjMGST2 and

cjMGST3

(3rt1)

(3rt1)

(3rt1)

55-

(5rt1)

cjMGST3

5-

(5rt1)

5555-

TGAGTGGTAAGAGTCAGGAGG-3 for MGST3. The PCR products were cloned into pCR4 vectors using a Zero Blunt TOPO PCR Cloning Kit for Sequencing (Invitrogen), into pMiniT 2.0 vectors using NEB PCR Cloning Kit (New England Biolabs), or into pGEM-T Easy Vectors using pGEM-T Easy Vector System (Promega, Madison, WI, USA), according to the manufacturers’ protocols. 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). 2.4. Bioinformatics Sequence data were analyzed using DNASIS Pro (Hitachi Software, Tokyo, Japan), Genetyx (Software Development, Tokyo, Japan), and Sequencher (Gene Codes Corporation, Ann Arbor, MI, USA). Multiple alignments were performed by the ClustalW program, and a phylogenetic tree was created using the neighbor-joining method. A homology search was conducted using 7

BLAST (National Center for Biotechnology Information). The gene and genomic structures of human and marmoset GSTs were analyzed using BLAT (UCSC Genome Bioinformatics) and SequenceViewer (National Center for Biotechnology Information). The amino acid and cDNA sequences used were either found in GenBank or identified in the present study. The 20 marmoset GST cDNAs identified in the current study were deposited in GenBank, i.e., GSTA1 (MF457755), GSTA3 (MH084969), GSTA4 (MF457756), GSTA4L (MH084970), GSTK1 (MF457757), GSTM2 (MF457759), GSTM3 (MF457760), GSTM4 (MF457758), GSTM5 (MH084971), GSTO1 (MF457761), GSTO2 (MH084972), GSTP1 (MF457762), GSTS1 (MF457763), GSTT1 (MF457764), GSTT4 (MH084973), GSTT4L (MH084974), GSTZ1 (MF457765), MGST1 (MF457766), MGST2 (MF457767), and MGST3 (MF457768).These marmoset GSTs were named based on the sequence homology to human GSTs and the gene arrangement in the marmoset and human genome. 2.5. qPCR analysis Expression levels of GST mRNAs were measured in marmoset brain, lung, liver, kidney, small intestine, and testis tissue samples using real-time RT-PCR, as reported previously [13]. Briefly, RT reactions were carried out as described above using random primers (Invitrogen); one twenty-fifth of the reaction mixture was subsequently used for the PCR. Testis cDNA was used for GSTT4 and GSTT4L. PCR amplification was carried out in a total volume of 20 µL using PowerUp SYBR Green Master Mix (Applied Biosystems) with the ABI PRISM 7500 fast sequence detection system (Applied Biosystems) according to the manufacturer’s protocols. The following primers were used at a final concentration of 400 nM: cjGSTA1 (5qrt2) 5ATCGCTATTTCCCTGCCTTT-3 and cjGSTA1 (3qrt2) 5-GGGTCAACCTCTTCCACGT3 for GSTA1, cjGSTA3 (5qrt1) 5-CACCAAGATTGCCTTGATCC-3 and cjGSTA3 (3qrt1) 5-GGGTCAAGCTCTTCCACAA-3

for

GSTA3, 8

cjGSTA4

(5qrt2)

5-

ACAAGTTGCAGGATGGTAACC-3 TGCCAAAGAGATTGTGCTTGT-3

and for

AAAGTTGCAGAAGGATGGATG-3 TTCAGGTCCTTCCCGTAGAA-3

for

and for

for

TTGTTCTTCCCATCCAGGAG-3

and for and

CCAGGAACTGTGAGAAGTGCTG-3

for

ATTTCCTCGCCTATGACGTT-3

and

ACAAAAGACCTGGGACGAAT-3

for

CAACCTTCTTTGGTGGCAGT-3 AGGAAACCTTGCCAGTCCTT-3

for

TGCATCAGGAGTTCTGCAAC-3 GCAGTCCGCTATCCCATACA-3

for

GACCTCACCCTGTACCAGTC-3 CCTGCCTCGTAGTTGGTGTA-3

for

TTGCATAAGATGAGGCGCAG-3

for

CAAGTCTTCAAAGGCCGACC-3 AGTACCCTGGGCATCAACTT-3

for

(3qrt2) (5qrt2) (3qrt2) (5qrt2) (3qrt2)

cjGSTM5

cjGSTO1

cjGSTO2

cjGSTP1

cjGSTP1 cjGSTS1

cjGSTS1 cjGSTT1

cjGSTT1 GSTT1,

9

(5qrt2)

cjGSTM4

cjGSTO2

GSTS1, and

(3qrt1)

cjGSTM3

cjGSTO1

GSTP1, and

(5qrt1)

cjGSTM2

cjGSTM5

GSTO2, and

ACTTACCCTTCACCAGAGCC-3

GSTM4,

GSTO1, and

cjGSTK1

cjGSTM4

GSTM5,

and

(3qrt1)

cjGSTM3 GSTM3,

GGAGAACCAGGCTATGGACG-3

(5qrt1)

cjGSTM2 GSTM2,

TCCTGGAGTTCACCGATACC-3

cjGSTA4L

cjGSTK1

GSTK1, and

(3qrt2)

cjGSTA4L

GSTA4L,

GCTGGCCAGACTCTGCTATG-3 TCTTGTCCCCAAGAAACCAG-3

GSTA4, and

AAGAACCAGCTCAGGGAGAC-3 CATCCACTTCTCTCCCAGCA-3

cjGSTA4

cjGSTT4

(5qrt1) (3qrt1) (5qrt2) (3qrt2) (5qrt2) (3qrt2) (5qrt1)

(3qrt1) (5qrt1) (3qrt1) (5qrt1) (3qrt1) (5qrt1)

555555555555555555555555-

AGCTGCCCATGAAGAGGATA-3

and

CCACCAAGTCAGCCAGTGAG-3

for

AGCTGCCCATGAAAAAGATG-3 TCTTCACCTCCGCCACTGT-3

cjGSTT4 GSTT4,

and for

GAAGTGATGGCATTCTGGGC-3

TCTCCCTTGCCAAATGTTGC-3

for

MGST1,

for

TAAGACCCCTGGACCATCAGCC-3

for

MGST2,

MGST3, and

(5qrt1) (3qrt1) (5qrt1) (3qrt1)

cjMGST2

cjMGST2

and

(3qrt1)

cjMGST1

cjMGST1

and

CTTGGCCTGGATTGTTGGAC-3 ATGGCAGCATTTGGATCCAC-3

GSTZ1,

for

(5qrt1)

cjGSTZ1 cjGSTZ1

and

GAGTATTTCGGGCGCAACAA-3 CAACAAGGCCAAAATCCCCA-3

cjGSTT4L

and

TTTCCCAGCTAACGGACGAT-3

cjGSTT4L

GSTT4L,

GCCTCAGGACCCAAAGAAGA-3

(3qrt1)

(5qrt1) (3qrt1)

cjMGST3

cjMGST3 and

(5qrt1) (3qrt1)

cjGAPDH

cjGAPDH

(5qrt1)

(3qrt1)

5555555555555-

GGGGCAATTCGGTGTGGTGA-3 for GAPDH. Standard curves were created by plotting Ct values from the logarithm of a serial ten-fold dilution (102–107 copies) versus the absolute amounts of purified marmoset GST cDNAs. The copy numbers of target marmoset GST cDNAs were calculated by comparing their Ct values with the corresponding standard curve. To determine the relative expression levels, the raw data were normalized to the GAPDH mRNA levels based on three independent amplifications. Expression levels below 100 copies were considered to be below the limit of quantitation. 2.6. Preparation of recombinant GST proteins For the 20 identified marmoset GSTs, recombinant proteins were expressed in bacterial fractions using generated expression plasmids. To prepare expression plasmids, PCR was 10

performed as described above using each GST cDNA as a template. The PCR products were then subcloned into vectors using reverse primers containing restriction enzyme sites (underlined in the primer sequences below) of EcoRI for GSTT1 and of XhoI for the remaining GSTs. It should be noted that some of the primers were previously designed for cynomolgus monkey (mf) GSTs and contained one or two bases different from those of marmoset GSTs; however, these differences did not change the corresponding amino acid residues. The primers used were: mfGSTA1/2/5 (5exp1) 5-ATGGCAGAGAAACCCAAGCTC-3 and mfGSTA1/2 (3exp1) 5-CCGCTCGAGCTGCTTTACTAAAACCTGAAAATCTTCCTTG-3 for GSTA1, mfGSTA3 (5exp1) 5-ATGGCGGGGAAGCCCAAG-3 and cjGSTA3 (3exp1) 5CCGCTCGAGCATGGCTGCTTTATTAAAACTTGAAAATCT-3 for GSTA3, mfGSTA4 (5exp1)

5-ATGGCAGCAAGGCCCAAG-3

and

cjGSTA4

(3exp1)

CCGCTCGAGTTACGGCCTAAAGATGTTGTAGACGGTTCTC-3

for

5-

GSTA4,

cjGSTA4L (5exp1) 5-ATGGCAGCCAAACCCAAGC-3 and cjGSTA4L (3exp1) 5CCGCTCGAGCAACCTGCTGCCATTAGAACTG-3 for GSTA4L, cjGSTK1 (5exp1) 5ATGGGGCCCGTGCCGCG-3

and

mfGSTK1

(3exp1)

5-

CCGCTCGAGGCAATCTTAAAGTCTGGCATTCAC-3 for GSTK1, cjGSTM2 (5exp1) 5ATGCCTATGATACTGGGATACTGGGACATC-3

and

mfGSTM2

(3exp1a)

5-

CCGCTCGAGTTACTTGTTGCCCCAGACAGCCATC-3 for GSTM2, cjGSTM3 (5exp1) 5-ATGTCGTGCCAGTCGTCTATGGTT-3

and

cjGSTM3

(3exp1)

5-

CCGCTCGAGTCAGCATATAGCCTTGTTGCCCCATT-3 for GSTM3, cjGSTM4 (5exp1) 5-ATGCCCATGACACTGGGGTACTG-3 and mfGSTM4 (3exp1a) 5-CCGCTCGAGCCTTCAAGGCATTACTTGTTGC-3

for

ATGCCCATGACACTGGGGTACTG-3

GSTM4, and

cjGSTM5

cjGSTM5

(5exp1a) (3exp1)

55-

CCGCTCGAGCCTTATTTGCTGTTCCATGTAGCTGA-3 for GSTM5, cjGSTO1 (5exp1) 11

5-ATGTCCGGGGAGTCAGCCA-3

and

cjGSTO1

(3exp1)

5-

CCGCTCGAGCCCTTCAGAGCCCATAATCACA-3 for GSTO1, cjGSTO2 (5exp1) 5ATGCTCAGACGCCTGGAGACC-3

and

cjGSTO2

(3exp1)

5-

CCGCTCGAGCTCAGTCCAGCCCAAAGTCAAAG-3 for GSTO2, mfGSTP1 (5exp1) 5ATGCCGCCCTACACCGTGGTC-3

and

mfGSTP1

(3exp1)

5-

CCGCTCGAGTCACTGTTTCCCGTTGCCATT-3 for GSTP1, mfGSTS1 (5exp1) 5ATGCCAAACTACAAACTCACTTATTTTA-3

and

mfGSTS1

(3exp1)

5-

CCGCTCGAGCTAGAGTTTGGTTTGGGGTCTTC-3 for GSTS1, mfGSTT1 (5exp1) 5ATGGGCCTGGAGCTGTACCTG-3

and

cjGSTT1

(3exp1)

5-

CGGAATTCGAGGTTTCCCAGCTCACTGGATCA-3 for GSTT1, cjGSTT4/4L (5exp1) 5ATGGCCCTGGAGCTCTACAT-3

and

cjGSTT4

(3exp1)

5-

CCGCTCGAGTCACTTGCTCTTCTTCAGCAACTC-3 for GSTT4, cjGSTT4/4L (5exp1) and cjGSTT4L (3exp1) 5-CCGCTCGAGATCACAAGTACTTCTCCCGAAACTCA-3 for GSTT4L, and cjGSTZ1 (5exp1) 5-ATGCAGGCGGGGAAGCCTGT-3 and cjGSTZ1 (3exp1) 5-CCGCTCGAGCTAGGCCCTCAGCTCGGTGGGTGT-3 for GSTZ1. The amplified DNAs were digested using restriction enzymes and were subcloned into a pET30a vector (Novagen, Madison, WI), providing a 6His-tag at the N-terminus. Marmoset GST proteins were expressed in Escherichia coli BL21 using expression plasmids with N-terminus 6 x histidine tags in a similar manner as cynomolgus GSTs described previously [9-12]. Briefly, recombinant His-tagged GST proteins in bacterial fractions [15] were quantified by gel electrophoresis using sodium dodecyl sulfate polyacrylamide gels and immunoblotting using anti-histidine tag antibody, as described previously [11, 12].

12

2.7. Enzyme assays Conjugation activities of the 20 recombinant marmoset GST proteins were evaluated using the same concentrations of typical GST substrates CDNB (1.0 mM); EPNPP (0.50 mM); styrene 7,8-oxide (0.80 and 1.6 mM); and 1-iodohexane (0.050 and 0.10 mM), as cynomolgus GST proteins described previously [9-12] . Similarly, the catalytic activities of GSTs were measured in cytosolic fractions from 11 marmoset, 14 cynomolgus macaque, and 10 human livers. Briefly, each reaction mixture (1.0 mL) contained either bacterial cytosolic fractions expressing recombinant GST proteins or liver cytosolic fractions (0.1 mg protein), 5.0 mM glutathione, and substrate (1.0 mM CDNB or 0.50 mM EPNPP) in 0.10 mM potassium phosphate buffer (pH 7.4). CDNB and EPNPP conjugate formation was recorded at 340 and 360 nm, respectively, at 25°C for 5 min in a spectrophotometer (U-3000, Hitachi), and rates of conjugate formation were calculated using the coefficients 9.6 and 0.5 mM-1cm-1, respectively. Kinetic analyses were performed by nonlinear regression data analysis employing Michaelis-Menten equations using the Prism program (GraphPad Software, La Jolla, CA, USA) for styrene 7,8oxide (0.050–1.6 mM) and 1-iodohexane (3.1–100 µM) conjugation reactions in 250 mM TrisHCl (pH 7.2) and 100 mM sodium phosphate (pH 7.4), respectively, and conjugate formation rates were measured at 234 and 226 nm. The statistical significance for conjugation activities of liver cytosolic fractions was calculated using unpaired Student’s t test with p < 0.05.

3. Results 3.1. Identification of marmoset GST cDNAs Twenty GST cDNAs were isolated in marmosets by RT-PCR, namely GSTA1, GSTA3, GSTA4, GSTA4L, GSTK1, GSTM2, GSTM3, GSTM4, GSTM5, GSTO1, GSTO2, GSTP1, 13

GSTS1, GSTT1, GSTT4, GSTT4L, GSTZ1, MGST1, MGST2, and MGST3. Except for GSTT4 and GSTT4L (which were isolated from testis), these cDNAs were isolated from liver. Nineteen of these marmoset GST cDNAs had open reading frames of 199–250 amino acid residues that were highly identical (87–99%) with human GSTs. The exception was GSTA4L, which had low sequence identity (59–62%) to all human GSTAs (Table 1). The deduced amino acid sequences of marmoset MGSTs were also highly identical (90–96%) to those of human MGSTs (Table 1). Marmoset GSTs had similar primary sequence structures to human GSTs (Fig. 1). Phylogenetic analysis revealed that these marmoset GSTs were more closely clustered with human GSTs than with their dog or rat homologs; however, cynomolgus macaque GSTs generally showed a closer relationship with humans than marmoset GSTs did (Fig. 2). Unlike cynomolgus and marmoset GSTs, many rat GSTs did not have one-to-one relationships with human GSTs; however rat MGSTs did exhibit one-to-one relationships with human MGSTs, just as cynomolgus and marmoset MGSTs did (Fig. 2). 3.2. Gene structures of marmoset GSTs The gene structures of GSTs were analyzed for human and marmoset genome data using BLAT (Fig. 3). Of the 20 marmoset GSTs, the largest was GSTO2 (>23 kb) and the smallest was GSTP1 (2.4 kb). Among human GSTs, the largest was GSTS1 (>44 kb) and the smallest was GSTP1 (3.1 kb). The size of marmoset GSTS1 was much smaller than human GSTS1 due to a shorter exon 1. The sizes of marmoset MGST1, MGST2, and MGST3 were 9.4, 40, and 6.2 kb, respectively. The larger size of MGST2, compared with MGST1 and MGST3, in marmosets was similar to that in humans. Precise sizes were not determined for the first and last exons because the full-length cDNA sequences were not available. Except for exon 6 of marmoset GSTZ1 and exon 2 of MGST3, the sizes of all the exons determined for the corresponding GSTs were similar between marmosets and humans. There were no data for exon 3 of marmoset 14

GSTK1 because it was located in a gap in the genome assembly. The exon sizes in marmosets were 52–142 bp for GSTAs, 36–117 bp for GSTK1, 65–111 bp for GSTMs, 99–223 bp for GSTO1, 102–223 bp for GSTO2, 36–108 bp for GSTP1, 93–110 bp for GSTS1, 88–177 bp for GSTTs, 50–100 bp for GSTZ1, and 58–122 bp for MGSTs. The sizes of corresponding exons were similar among the GSTs of the same subfamily: for example, exons 3, 4, 5, and 6 of GSTAs were, respectively, 52, 133, 142, and 132 bp; exons 2, 3, 4, 5, and 6 of GSTMs were, respectively, 76, 65, 82, 101, 96, and 111 bp; and exons 2, 3, and 4 of GSTTs were, respectively, 88, 151, and 177 bp in both marmosets and humans. These results indicate that GST genes have similar structures in both marmosets and humans. 3.3. Genomic organization of marmoset GSTs GSTA, GSTM, GSTO, and GSTT formed polycistronic gene clusters in the marmoset genome, whereas GSTK1, GSTP1, GSTS1, GSTZ1, MGST1, MGST2, and MGST3 were monocistronic (Fig. 4). The arrangement and direction of the GSTA, GSTM, GSTO, and GSTT genes in the gene clusters were generally similar in humans and macaques, but some differences were noted. In the marmoset GSTA gene cluster, there were no orthologs of human GSTA2 or GSTA5; however, GSTA4L, which is unique to marmosets, was present. In the GSTM gene cluster, the orthologs of all GSTMs, except for GSTM1, were present, but the positions of GSTM2 and GSTM5 were transposed. In the GSTT gene cluster, GSTT1 and GSTT4 genes were present, but GSTT2-like genes were absent. Moreover, GSTT4L, which is unique to marmosets, was present. Furthermore, the direction and position of GSTT4 were different from those of human GSTT4, possibly due to inversion of this genomic segment. These results suggest general similarities in GST genomic organization between marmosets and humans, although some differences were noted.

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3.4. Tissue expression of marmoset GST mRNAs To measure the expression of marmoset GST mRNAs, qPCR analysis was performed in brain, lung, liver, kidney, and small intestine tissue samples. Some GST mRNAs showed preferential expression in specific tissues, whereas others showed more universal expression patterns. GSTA1, GSTA3, GSTA4L, GSTK1, GSTT1, GSTZ1, and MGST1 mRNAs were most abundantly expressed in liver; GSTM2, GSTM3, GSTM5, GSTS1, MGST2, and MGST3 mRNAs in small intestine; GSTM4, GSTO1, and GSTP1 mRNAs in lung; GSTA4 mRNA in brain; and GSTO2 mRNA in kidney (Fig. 5). Expression of GSTT4 and GSTT4L mRNAs was detected only in testis. A sex difference (more than three-fold) in expression was observed only for marmoset GSTA3 mRNA in liver and lung (Fig. 5). By comparing GST mRNA expression levels across tissue types, we established that marmoset GSTA1 mRNA was the most abundant GST in liver, kidney, and small intestine (Fig. 6). In liver, the second most abundant was GSTT1 mRNA, followed by MGST1, GSTZ1, GSTM5, GSTK1, and MGST3 mRNAs (Fig. 6). In small intestine and kidney, the second and third most abundant were MGST3 and GSTM5 mRNAs; GSTK1 mRNA was also relatively abundant in kidney. MGST3 mRNA was the most abundant in brain, lung, and testis, and GSTM5 mRNA was the second most abundant in brain and lung (Fig. 6). 3.5. Catalytic activity of GST proteins To assess the catalytic functionality of marmoset GSTs, their conjugation activities with respect to CDNB; EPNPP; styrene 7,8-oxide; and 1-iodohexane were measured. Marmoset liver cytosolic fractions catalyzed the conjugations of CDNB; EPNPP; styrene 7,8-oxide; and 1iodohexane, just as human and cynomolgus monkey liver cytosolic fractions do (Fig. 7). Under the present conditions, the apparent activities from liver cytosolic fractions from humans, 16

cynomolgus monkeys, and marmosets were not significantly different. Marmoset GSTs heterologously expressed in E. coli substantially catalyzed the conjugation of CDNB (GSTA1, GSTA3, GSTK1, GSTM2, GSTM5, and GSTP1); EPNPP (GSTK1, GSTO2, GSTS1, and GSTZ1); styrene 7,8-oxide (GSTM2, GSTM3, GSTM4, GSTM5, GSTT1, and GSTT4); and 1-iodohexane (GSTM2, GSTM3, and GSTT1). In contrast, marmoset MGST1, MGST2, and MGST3 catalyzed the conjugation of CDNB and EPNPP at much lower levels (Table 2). Kinetic analyses revealed that marmoset GSTM2 and GSTM5 effectively catalyzed the conjugation of styrene 7,8-oxide (kcat/Km, 2.2 and 2.7 μM-1･min-1, respectively) with the highest affinities (Km, 64 and 90 μM, respectively), whereas marmoset GSTM5 and GSTT1 effectively catalyzed the conjugation of 1-iodohexane (kcat/Km, 5.3 and 7.3 μM-1･min-1, respectively) with the highest affinities (Km, 9 and 9 μM, respectively) (Table 3). Marmoset GSTA4, GSTA4L, GSTO1, and GSTT4L catalyzed the conjugation of CDNB; EPNPP; styrene 7,8-oxide; and 1iodohexane with low activity levels (Fig. 8). These results suggest that the 20 newly described marmoset GSTs were functional drug-metabolizing enzymes, some of which efficiently catalyzed the conjugation of typical GST substrates. 4. Discussion GSTs are important enzymes that catalyze the conjugation of reduced glutathione with electrophilic substrates; currently, GSTs remain to be investigated in marmosets. In the present study, 20 GSTs, including 3 MGSTs, were newly identified in marmosets and were characterized in terms of sequence and phylogenetic data, tissue expression patterns, gene and genomic structure, and enzymatic function. These marmoset GSTs had high amino acid sequence identities with the corresponding human GSTs (Table 1). Phylogenetic analysis revealed that marmoset GSTs were closely clustered with their corresponding human GSTs 17

(Fig. 2). In contrast, several rat GSTs, especially those of the GSTA and GSTM subfamilies, were found not to be orthologous to human GSTs (Fig. 2). Moreover, the gene structures of orthologous GSTs were mostly similar in marmosets and humans, despite some differences in the GSTA, GSTM, and GSTT gene clusters (Fig. 4). Therefore, the molecular characteristics of these 20 GSTs are generally similar in marmosets and humans. Marmoset GSTA1, GSTA3, and GSTA4 had 88–99% amino acid sequence identities with human GSTAs, whereas marmoset GSTA4L did not have an ortholog in humans (Table 1). No orthologs of human GSTA2 or GSTA5 were found in the marmoset genome (Fig. 4). Marmoset GSTA1 mRNA, which was expressed preferentially in liver and small intestine (Fig. 5), was the most abundant mRNA in liver, kidney, and small intestine (Fig. 6). In humans, GSTA1 protein is expressed abundantly in liver, kidney, and small intestine [16], with decreasing expression levels from the proximal to the distal portions of the small intestine [17]. Marmoset GSTA1 showed a higher conjugation activity toward CDNB than the other GSTAs did (Fig. 8), similar to human GSTA1 [18]. Human GSTA1 also more efficiently catalyzed the isomerization of androgen steroid hormone D5-androstene-3,17-dione (kcat/Km, 0.5 μM-1s-1) than other human GSTAs did [19, 20], suggesting the physiological importance of GSTA1 for the biosynthesis of hormones. Phe220 and Phe222 in the C-terminal segment of human GSTA1, which provides part of the glutathione and hydrophobic ligand binding sites, are key residues for catalytic function [21], and these two key residues are conserved in humans and marmosets (Fig. 1A). These results suggest that marmoset and human GSTA1 have similar functional characteristics. Among the five tissue types analyzed, marmoset GSTA3, GSTA4, and GSTA4L mRNAs were most abundantly expressed in liver (Fig. 5); however, their absolute expression levels in liver were minimal (Fig. 6). Although marmoset GSTA3, GSTA4, and GSTA4L exhibited 18

conjugation activities (Fig. 8), their contribution to overall GST activities in liver is most likely limited. Similarly, human GSTA5 catalyzes the conjugation of the lipid peroxidation product 4-hydroxynonenal, but no human GSTA5 transcript has been found in any human tissue [22]. Marmoset GSTK1 was highly homologous to human GSTK1 in terms of amino acid sequences (Table 1). Moreover, marmoset GSTK1 mRNA showed ubiquitous expression, with more abundant expression in liver and kidney (Fig. 5), similar to the expression profile of human GSTK1 mRNA [23]. Marmoset GSTK1 substantially catalyzed the conjugation reactions of CDNB and EPNPP. In humans, GSTK1 conjugates CDNB, but not EPNPP [24]. Human GSTK1 is mainly localized in mitochondria and peroxisomes [23]. The C-terminus sequences (Ala–Arg–Leu) of GSTK1 are reportedly essential for localization of the protein to peroxisomes [23] and were conserved in humans and marmosets (Uno, unpublished data). The respiratory chain in mitochondria and lipid metabolism in both mitochondria and peroxisomes produce reactive oxygen species such as oxygen free radicals and hydrogen peroxides [25]. GSTK1-dependent peroxidase activity might be important for the detoxication of lipid peroxides generated in peroxisomes and/or mitochondria in marmosets. Marmoset GSTM2, GSTM3, GSTM4, and GSTM5 were highly identical (88–96%) to their human orthologs (Table 1). Among all the GST mRNAs analyzed, marmoset GSTM5 mRNA exhibited relatively high expressions in brain, lung, liver, kidney, and small intestine (Fig. 6). In contrast, human GSTM5 is not detected in liver as mRNA or protein [26]. Marmoset GSTM3 mRNA was relatively more abundantly expressed in testis (Fig. 5), as is also true for human GSTM3 protein [16]. All marmoset GSTMs catalyzed the conjugation of the human GSTM1 substrate styrene 7,8-oxide (Fig. 8), but marmoset GSTM5 was the most efficient of the marmoset GSTMs (Table 3). Human GSTM1 protein is abundant in liver [27], but GSTM1 was not found in the marmoset genome (Fig. 4), although it could be located in a gap in the current 19

genome assembly. Alternatively, marmoset GSTM5, which efficiently conjugates human GSTM1 substrates, might play the same role as human GSTM1, just as cynomolgus GSTM5 does [28]. Marmoset GSTO1, GSTO2, GSTP1, GSTS1, and GSTZ1 had high amino acid sequence identities with their human orthologs (Table 1). Marmoset GSTP1, GSTS1, and GSTZ1 mRNAs showed preferential expressions in lung/small intestine, small intestine, and liver, respectively, whereas GSTO1 and GSTO2 mRNAs were more ubiquitously expressed (Fig. 5). Marmoset GSTO2, GSTS1, and GSTZ1 exhibited high conjugation activities toward EPNPP, whereas marmoset GSTP1 showed high conjugation activity toward CDNB (Fig. 8). Marmoset GSTZ1 had a characteristic motif (SSCSWRVRIAL) in the N-terminal region, including the first three residues (Ser14–Ser15–Cys16) (Uno, unpublished data); these three residues play a role in the characteristic binding of glutathione in human GSTZ1 [29]. The marmoset GSTT1 amino acid sequence was highly identical to that of human GSTT1 (Table 1). Marmoset GSTT1 mRNA was most abundantly expressed in liver (Fig. 5), just as human GSTT1 protein is [30]. Marmoset GSTT1 protein purified by affinity chromatography immunoreactivity was closely associated with activity toward theta-specific substrate methyl chloride [31]. Marmoset GSTT1 also conjugated EPNPP (Fig. 8), just as human GSTT1 does [30]. Moreover, marmoset GSTT1 efficiently catalyzed the conjugation of human GSTT substrate 1-iodohexane (Table 3), suggesting the importance of GSTT1 in xenobiotic metabolism. Marmoset GSTT4 and GSTT4L were highly homologous to human GSTT4 (Table 1), which has not been characterized, and their mRNA expression was detected only in testis. These GSTTs conjugated the four substrates tested in the current study, suggesting that they are functional enzymes. It would be of great interest to investigate the role of these enzymes in marmoset testis. 20

Marmoset MGST1, MGST2, and MGST3 had high amino sequence identities (90–96%) with their human orthologs (Table 1). Among the tissues analyzed, marmoset MGST1 mRNA was preferentially expressed in liver and small intestine, whereas marmoset MGST2 and MGST3 mRNAs were more widely expressed (Fig. 5). In humans, the predominant expression of MGST1 mRNA in liver and pancreas and wider expression patterns of MGST2 and MGST3 mRNAs have been noted [32, 33]. Marmoset MGST1 mRNA was relatively more abundant in liver, whereas MGST3 mRNA was the first or second most abundant mRNA in brain, lung, and small intestine (Fig. 6). Marmoset MGST1–3 catalyzed the conjugation reactions of CDNB and EPNPP, but their activities were substantially lower than those of other GSTs (Table 2), indicating that MGST1–3 might not contribute much to overall GST activities in marmosets. However, these enzymes might have physiological significance, because human MGST2 and MGST3 catalyze the production of proinflammatory mediator leukotriene-C4 from leukotrieneA4 [33, 34]. All 20 newly identified marmoset GSTs catalyzed the conjugation reactions of CDNB, EPNPP, styrene 7,8-oxide, and/or 1-iodohexane, but their activities differed depending on the substrate (Fig. 8; Table 2), possibly as a result of substrate specificities. Although GSTs have overlapping substrate specificities in general, some GSTs show unique catalytic characteristics to a certain degree [35]. Under the present conditions, the apparent GST activities from liver cytosolic fractions from humans, cynomolgus monkeys, and marmosets were not significantly different (Fig. 7). Styrene 7,8-oxide and 1-iodohexane are most efficiently conjugated by GSTM5 and GSTT1/2, respectively, in humans and cynomolgus macaque [9-12], but were not selective for these reactions in marmosets (Fig. 8). Analysis of enzyme properties using various GST substrates might elucidate the distinct substrate specificities of marmoset GSTs. Marmoset GSTA4L and GSTT4L were not orthologous to any human GST and, if these enzymes have specific substrates, they might account for species differences of GST-dependent 21

metabolism between marmosets and humans. In summary, we identified in five marmoset tissues the cDNAs of 20 GSTs (including 3 MGSTs), and their deduced amino acid sequences were highly identical to their orthologous human GSTs. Most of the mRNAs of these GSTs showed tissue expression patterns similar to their human counterparts, although some differences were found, particularly for GSTA3 and GSTM5 mRNAs. Moreover, the 20 marmoset GST enzymes conjugated typical GST substrates CDNB; EPNPP; styrene 7,8-oxide; and/or 1-iodohexane. Therefore, these 20 GSTs were expressed at the mRNA level and encoded functional drug-metabolizing enzymes in marmosets.

Acknowledgements This work resulted from the "Construction of System for Spread of Primate Model Animals" initiative under the Strategic Research Program for Brain Sciences of the Japan Agency for Medical Research and Development. We thank Drs. Erika Sasaki, Takashi Inoue, Yusuke Kamiya, and Makiko Shimizu for their support with the experiments. The authors also greatly thank David Smallbones for copyediting a draft of this article. Authorship (1) Study conception and design: YU, SU, and HY. (2) Acquisition, analysis and/or interpretation of data: YU, SU, ST, NM, and HY. (3) Drafting/revision of the work for intellectual content and context: YU, SU, and HY. (4) Final approval and overall responsibility for the published work: YU, SU, and HY. Declaration of interest The authors report no conflicts of interest. 22

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[28] Y. Uno, N. Murayama, H. Yamazaki, Molecular and functional characterization of cytosolic sulfotransferases in cynomolgus macaque, Biochem Pharmacol 166 (2019) 153-162. [29] P.G. Board, R.T. Baker, G. Chelvanayagam, L.S. Jermiin, Zeta, a novel class of glutathione transferases in a range of species from plants to humans, Biochem J 328 ( Pt 3 (1997) 929-935. [30] P.J. Sherratt, D.J. Pulford, D.J. Harrison, T. Green, J.D. Hayes, Evidence that human class Theta glutathione S-transferase T1-1 can catalyse the activation of dichloromethane, a liver and lung carcinogen in the mouse. Comparison of the tissue distribution of GST T1-1 with that of classes Alpha, Mu and Pi GST in human, Biochem J 326 ( Pt 3) (1997) 837-46. [31] T.G. Schulz, F.A. Wiebel, R. Thier, D. Neubert, D.S. Davies, R.J. Edwards, Identification of thetaclass glutathione S-transferase in liver cytosol of the marmoset monkey, Arch. Toxicol. 74(3) (2000) 133-8. [32] M. Estonius, L. Forsberg, O. Danielsson, R. Weinander, M.J. Kelner, R. Morgenstern, Distribution of microsomal glutathione transferase 1 in mammalian tissues. A predominant alternate first exon in human tissues, Eur J Biochem 260(2) (1999) 409-13. [33] P.J. Jakobsson, J.A. Mancini, D. Riendeau, A.W. Ford-Hutchinson, Identification and characterization of a novel microsomal enzyme with glutathione-dependent transferase and peroxidase activities, J Biol Chem 272(36) (1997) 22934-9. [34] P.J. Jakobsson, J.A. Mancini, A.W. Ford-Hutchinson, Identification and characterization of a novel human microsomal glutathione S-transferase with leukotriene C4 synthase activity and significant sequence identity to 5-lipoxygenase-activating protein and leukotriene C4 synthase, J Biol Chem 271(36) (1996) 22203-10. [35] P.J. Sherratt, J.D. Hayes, Glutathione S-transferases, in: C. Ioannides (Ed.), Enzyme systems that metabolise drugs and other xenobiotics, J. Wiley, Chichester, 2002, pp. 319-352.

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Figure captions Fig. 1. Multiple alignments of marmoset and human GST amino acid sequences. Amino acid sequences of human and marmoset GSTAs (A), GSTMs (B), GSTTs (C), and MGST1 (D) were aligned using the ClustalW program. Asterisks and dots under the sequences indicate identical and conserved residues, respectively. Fig. 2. Phylogenetic analysis of marmoset GSTs. Phylogenetic trees were created by the neighbor-joining method based on cytosolic/mitochondrial (A) and microsomal (B) GST amino acid sequences from humans (h), cynomolgus macaques (mf), marmosets (cj), dogs (d), and rats (r). Human FLAP and GSTA1 were used as outgroups. The scale bar indicates 0.1 amino acid substitutions per site. Fig. 3. Exon–intron structures of GSTs. Gene structures were determined in marmosets (cj) and humans (h) by aligning each GST cDNA sequence with the marmoset and human genomes for cytosolic/mitochondrial (A) and microsomal (B) GSTs using BLAT. Arrows indicate introns containing short gaps of unsequenced regions in the genome data. Exon 8 of marmoset GSTM2 is not shown because the location could not be specified in the current genome assembly. Fig. 4. Genomic organization of GSTs. The genomic organization was established for marmosets and humans using SequenceViewer. GSTAs (A), GSTMs (B), and GSTTs (C) formed gene clusters in the genome, and these gene clusters and MGST1 (D) were located in genomic regions that corresponded well in marmosets and humans. Black arrows indicate expressed GSTs or MGST1, whereas the gray arrow indicates a pseudogene. This figure is not drawn to scale.

26

Fig. 5. Tissue expression patterns of marmoset GST mRNAs. Expression levels of the 20 marmoset GSTs mRNAs were measured using real-time RT-PCR in brain, lung, liver, kidney, and small intestine tissue samples. In brain, lung, liver, kidney, and intestine, GSTT4 and GSTT4L mRNAs were below the limit of quantitation (<102 copies). The expression level of each GST mRNA was normalized to the GAPDH mRNA level. Values represent the average  S.D. from three independent amplifications. Fig. 6. Expression levels of marmoset GST mRNAs in brain, lung, liver, kidney, and small intestine. Expression levels of the 20 GST mRNAs measured using real-time RT-PCR were compared in brain, lung, liver, kidney, and small intestine. GSTT4 and GSTT4L mRNAs in brain, lung, liver, kidney, and intestine were below the limit of quantitation, and GSTA4L mRNA in testis was below the limit of quantitation (<102 copies). The expression level for each GST mRNA was normalized to the GAPDH mRNA level and represents the average of three independent amplifications. Fig. 7. Glutathione conjugation activities of liver cytosolic fractions from individual humans (n=6–10), marmosets (n=7–11), and cynomolgus monkeys (n=8–13). Conjugation activities were evaluated in liver cytosol samples by measuring conjugate formation rates with a spectrophotometer. Long lines represent mean activities. Short lines indicate 25h and 75th percentiles. Fig. 8. Glutathione conjugation activities of recombinant marmoset GSTs. The conjugation activities of 17 marmoset cytosolic/mitochondrial GSTs were measured with respect to CDNB at a substrate concentration of 1.0 mM (A); EPNPP at a substrate concentration of 0.5 mM (B); styrene 7,8-oxide at substrate concentrations of 0.8 and 1.6 mM (C); and 1-iodohexane at substrate concentrations of 0.05 and 1.0 mM (D).

27

28

Table 1 Sequence identities of GST amino acids between marmosets and humans.

Marmoset

Human

GSTA1

GSTA1 GSTA2 GSTA3

Sequence identity, % 92 90 89

GSTA3

GSTA1 GSTA2 GSTA3

87 85 88

GSTA4

GSTA4

99

GSTA4L

GSTA1 GSTA2 GSTA3 GSTA4 GSTA5

62 61 62 59 62

GSTK1

GSTK1

87

GSTM2 GSTM3 GSTM4

GSTM2 GSTM3 GSTM4

91 94 96

GSTM5

GSTM1 GSTM5

88 90

GSTO1 GSTO2

GSTO1 GSTO2

96 89

GSTP1

GSTP1

91

GSTS1

GSTS1

95

GSTT1 GSTT4 GSTT4L

GSTT1 GSTT4 GSTT4

93 88 86

GSTZ1

GSTZ1

90

MGST1 MGST1 90 MGST2 MGST2 96 MGST3 MGST3 96 The amino acid sequences of marmoset and human GSTs were compared using BLAST. 29

Table 2 Catalytic activities of marmoset microsomal GSTs recombinantly expressed in E. coli. Marmoset GST

CDNB

EPNPP

nmol/min/nmol GST MGST1

120

210

MGST2

50

390

MGST3

20

40

Enzymatic assays were performed with the bacterial membrane fraction (expressing recombinant GST proteins) using CDNB and EPNPP as substrates at 1.0 and 0.5 µM, respectively. Results are presented as the means of triplicate determinations.

30

Table 3. Kinetic parameters for conjugation activities of marmoset GSTs toward styrene 7,8oxide and 1-iodohexane. Km, µM

kcat, min-1

kcat/Km (µM・min)-1

Glutathione conjugation with styrene 7,8-oxide M2

64 ± 40

142 ± 14

2.2 ± 0.6

M3

110 ± 46

142 ± 11

1.3 ± 0.4

M4

365 ± 204

188 ± 34

0.5 ± 0.5

M5

90 ± 79

247 ± 36

2.7 ± 0.9

T1

160 ± 78

133 ± 15

0.8 ± 0.5

T4

183 ± 86

107 ± 12

0.6 ± 0.5

Glutathione conjugation with 1-iodohexane M2

22 ± 9

30 ± 4

1.4 ± 0.4

M3

26 ± 13

37 ± 7

1.4 ± 0.5

M4

21 ± 10

33 ± 6

1.6 ± 0.5

M5

9±5

48 ± 6

5.3 ± 0.6

T1

9±3

66 ± 6

7.3 ± 0.3

Kinetic parameters were calculated by non-linear regression analysis [mean ± standard error, n = 6-7 points of the substrate concentrations of styrene 7,8-oxide (50–1600 μM) and 1iodohexane (3.1–100 μM)] with the bacterial cytosolic fraction (expressing recombinant GST proteins).

31

Fig. 1

A hGSTA1 hGSTA2 hGSTA3 hGSTA4 hGSTA5 cjGSTA1 cjGSTA3 cjGSTA4L cjGSTA4

1:MAEKPKLHYFNARGRMESTRWLLAAAGVEFEEKFIKSAEDLDKLRNDGYLMFQQVPMVEIDGMKLVQTRAILNYIASKYNLYGKDIKERALIDMYIEGIA 1:MAEKPKLHYSNIRGRMESIRWLLAAAGVEFEEKFIKSAEDLDKLRNDGYLMFQQVPMVEIDGMKLVQTRAILNYIASKYNLYGKDIKEKALIDMYIEGIA 1:MAGKPKLHYFNGRGRMEPIRWLLAAAGVEFEEKFIGSAEDLGKLRNDGSLMFQQVPMVEIDGMKLVQTRAILNYIASKYNLYGKDIKERALIDMYTEGMA 1:MAARPKLHYPNGRGRMESVRWVLAAAGVEFDEEFLETKEQLYKLQDGNHLLFQQVPMVEIDGMKLVQTRSILHYIADKHNLFGKNLKERTLIDMYVEGTL 1:MAEKPKLHYSNARGSMESIRWLLAAAGVELEEKFLESAEDLDKLRNDGSLLFQQVPMVEIDGMKLVQTRAILNYIASKYNLYGKDMKERALIDMYTEGIV 1:MAEKPKLHYFNARGRMESTRWLLAAAGVEFEEKLIKSPEELDKLRNDGYLMFQQVPMVEIDGMKLVQSRAILNYIASKYNLYGKDIKERALIDMYIEGMA 1:MAGKPKLHYFNARGRMEPIRWLLAAAGVEFEEQFLESAEDLEKLKNDGYLMFQQVPMVEIDGMKLVQSRAILNYIASKYDLYGKDIKERALIDMYTEGMA 1:MAAKPKLYYFHGRGRMESIRWLLAAAGVEFEEEFLETREQYEKLQKDGCLLFGQVPLVEVDGMMLTQTRAILSYLAAKFNFYGKDLKERVRIDMYVDGTL 1:MAARPKLHYPNGRGRMESVRWVLAAAGVEFDEEFLETKEQLHKLQDGNHLLFQQVPMVEIDGMKLVQTRSILHYIADKHNLFGKDLKERTLIDMYVEGTL ** .***.*.. **.**..**.*******..*......*.. **.... *.*.***.**.***.*.*.*.**.*.*.*....**..**...**** .* .

100 100 100 100 100 100 100 100 100

hGSTA1 hGSTA2 hGSTA3 hGSTA4 hGSTA5 cjGSTA1 cjGSTA3 cjGSTA4L cjGSTA4

101:DLGEMILLLPVCPPEEKDAKLALIKEKIKNRYFPAFEKVLKSHGQDYLVGNKLSRADIHLVELLYYVEELDSSLISSFPLLKALKTRISNLPTVKKFLQP 101:DLGEMILLLPFTQPEEQDAKLALIQEKTKNRYFPAFEKVLKSHGQDYLVGNKLSRADIHLVELLYYVEELDSSLISSFPLLKALKTRISNLPTVKKFLQP 101:DLNEMILLLPLCRPEEKDAKIALIKEKTKSRYFPAFEKVLQSHGQDYLVGNKLSRADISLVELLYYVEELDSSLISNFPLLKALKTRISNLPTVKKFLQP 101:DLLELLIMHPFLKPDDQQKEVVNMAQKAIIRYFPVFEKILRGHGQSFLVGNQLSLADVILLQTILALEEKIPNILSAFPFLQEYTVKLSNIPTIKRFLEP 101:DLTEMILLLLICQPEERDAKTALVKEKIKNRYFPAFEKVLKSHRQDYLVGNKLSWADIHLVELFYYVEELDSSLISSFPLLKALKTRISNLPTVKKFLQP 101:DLYEMILLLPFCKPEEKDTKIAMIKEKTKNRYFPAFEKVLKSHGQDYLVGNKLSRADIHLVELLYYVEEVDPSLISSFPLLKALKTRISNLPTVKKFLQP 101:DLYEMILLLPLCRPEEKDTKIALIQEKTKNRYFPAFEKVLKSHEQDYLVGNKLSRADIQLVELLYFVEELDPSLISSFPLLQALKTRISNLPTLKKFLQP 101:DLMAMIMLAPFQRPEEKQETLASVIQKAKARYFPVFEKILKDHGEDFLVGNKFSWADIQMLEAILMVEELNASVLSDFPLLKAFKTRISNIPTIKKFLQP 101:DLLELLIMHPFLKPDDQQKEVVNMAQKAIIRYFPVFEKILRGHGQNFLVGNQLSLADVILLQTILALEEKIPNILSAFPFLQEYTVKLSNIPTIKRFLEP ** ......... *.... . ... .* ..****.***.*..*....****..*.**. ........**.. ...*.**.*.......**.**.*.**.*

200 200 200 200 200 200 200 200 200

hGSTA1 hGSTA2 hGSTA3 hGSTA4 hGSTA5 cjGSTA1 cjGSTA3 cjGSTA4L cjGSTA4

201:GSPRKPPMDEKSLEEARKIFRF 201:GSPRKPPMDEKSLEESRKIFRF 201:GSPRKPPADAKALEEARKIFRF 201:GSKKKPPPDEIYVRTVYNIFRP 201:GSQRKPPMDEKSLEEARKIFRF 201:GSPRKPPTDAKTLEEARKIFRF 201:GSPRKPPTNAKSVEEARKIFKF 201:GSQRKPPPDAHYVDLVSNILQF 201:GSKKKPPPDEIYVRTVYNIFRP **..*** ... ......*...

222 222 222 222 222 222 222 222 222

B hGSTM1v1 hGSTM2 hGSTM3 hGSTM4v1 hGSTM5 cjGSTM2 cjGSTM3 cjGSTM4 cjGSTM5

1:----MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDFPNLPYLIDGAHKITQSNAILCYIARKHNLCGETEEEKIR 1:----MPMTLGYWNIRGLAHSIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDFPNLPYLIDGTHKITQSNAILRYIARKHNLCGESEKEQIR 1:MSCESSMVLGYWDIRGLAHAIRLLLEFTDTSYEEKRYTCGEAPDYDRSQWLDVKFKLDLDFPNLPYLLDGKNKITQSNAILRYIARKHNMCGETEEEKIR 1:----MSMTLGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLNEKFKLGLDFPNLPYLIDGAHKITQSNAILCYIARKHNLCGETEEEKIR 1:----MPMTLGYWDIRGLAHAIRLLLEYTDSSYVEKKYTLGDAPDYDRSQWLNEKFKLGLDFPNLPYLIDGAHKITQSNAILRYIARKHNLCGETEEEKIR 1:----MPMILGYWDIRGLAHAIRLLLEYTDSSYEEKMYTMGDAPDYDRSQWLKEKFKLGLDFPNLPYLIDGAHKITQSNAILRYIARKHNLCGETEEEKIW 1:MSCQSSMVLGYWDIRGLAHAIRLLLEFTDTSYEEKRYTCGEAPDYDRSQWLDVKFKLNLDFPNLPYLLDGKNKITQSNAILRYIARKHNMCGETEEEKIR 1:----MPMTLGYWDIRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLKEKFKLGLDFPNLPYLIDGAHKITQSNAILRYIARKHNLCGETEEEKIR 1:----MPMTLGYWDLRGLAHAIRLLLEYTDSSYEEKKYTMGDAPDYDRSQWLKEKFKLGLDFPNLPYLIDGAHKITQSNAILRYIARKHSLCGETEEEKIR ..*.****..*****.******.**.**.**.**.*.********** .****.*********.**..*********.******..***.*.*.*.

96 96 100 96 96 96 100 96 96

hGSTM1v1 97:VDILENQTMDNHMQLGMICYNPEFEKLKPKYLEELPEKLKLYSEFLGKRPWFAGNKITFVDFLVYDVLDLHRIFEPKCLDAFPNLKDFISRFEGLEKISA 196 hGSTM2 97:EDILENQFMDSRMQLAKLCYDPDFEKLKPEYLQALPEMLKLYSQFLGKQPWFLGDKITFVDFIAYDVLERNQVFEPSCLDAFPNLKDFISRFEGLEKISA 196 hGSTM3 101:VDIIENQVMDFRTQLIRLCYSSDHEKLKPQYLEELPGQLKQFSMFLGKFSWFAGEKLTFVDFLTYDILDQNRIFDPKCLDEFPNLKAFMCRFEALEKIAA 200 hGSTM4v1 97:VDILENQAMDVSNQLARVCYSPDFEKLKPEYLEELPTMMQHFSQFLGKRPWFVGDKITFVDFLAYDVLDLHRIFEPNCLDAFPNLKDFISRFEGLEKISA 196 hGSTM5 97:VDILENQVMDNHMELVRLCYDPDFEKLKPKYLEELPEKLKLYSEFLGKRPWFAGDKITFVDFLAYDVLDMKRIFEPKCLDAFLNLKDFISRFEGLKKISA 196 cjGSTM2 97:EDILENQLMDNRMQLARLCYDPDFERLKLEYLEGLPEMLKLYSQFLGKRPWFLGDKITFVDFIAYDVLERNQVFEPTCLDAFPNLKDFISRFEGLEKISA 196 cjGSTM3 101:VDIVENQVMDFRIQLIKLCYSSDHEKLKPQYLEQLPGQLKQFSMFLGKFSWFAGEKLTYVDFLTYDILDQNRIFEPKCLDEFPNLKAFMGRFEALEKIAA 200 cjGSTM4 97:VDILENQAMDVSNQLARVCYSPDFEKLKPEYLEGIPTMMQHFSQFLGKGPWFVGDKITFVDFLAYDILDLHRIFEPTCLDAFPNLKDFISRFEGLEKISA 196 cjGSTM5 97:VDILENQTMDTRMQLAMLCYNPEFEKLKPKYLEELPEKLKLYSQFLGKRPWFAGDKITFVDFLAYDVLDQNRIFEPSCLDAFPNLKDFMSRFEGLKKISA 196 .**.*** ** ...*...** ...*.**. **...*. ....*.****..**.*.*.*.***..**.*. ...*.* ***.*.***.*..***.*.**.* hGSTM1v1 hGSTM2 hGSTM3 hGSTM4v1 hGSTM5 cjGSTM2 cjGSTM3 cjGSTM4 cjGSTM5

197:YMKSSRFLPRPVFSKMAVWGNK--197:YMKSSRFLPRPVFTKMAVWGNK--201:YLQSDQFCKMPINNKMAQWGNKPVC 197:YMKSSRFLPKPLYTRVAVWGNK--197:YMKSSQFLRGLLFGKSATWNSK--197:YMKSSRFLPRPVFTKMAVWGNK--201:YIQSDNFFKMPINNKMAQWGNKAIC 197:YMKSSRFLPRPLYTRVAVWGNK--197:YMKSSRFVPGLLFGKSATWNSK--*..*..*.. . . ..*.*..*

218 218 225 218 218 218 225 218 218

32

C hGSTT1 hGSTT2 hGSTT2B cjGSTT1 cjGSTT4 cjGSTT4L

1:MGLELYLDLLSQPCRAVYIFAKKNDIPFELRIVDLIKGQHLSDAFAQVNPLKKVPALKDGDFTLTESVAILLYLTRKYKVPDYWYPQDLQARARVDEYLA 1:MGLELFLDLVSQPSRAVYIFAKKNGIPLELRTVDLVKGQHKSKEFLQINSLGKLPTLKDGDFILTESSAILIYLSCKYQTPDHWYPSDLQARARVHEYLG 1:MGLELFLDLVSQPSRAVYIFAKKNGIPLELRTVDLVKGQHKSKEFLQINSLGKLPTLKDGDFILTESSAILIYLSCKYQTPDHWYPSDLQARARVHEYLG 1:MGLELYLDLLSQPCRAIYIFAKKNGIPFELRTVDLIKGQHLSDAFSQVNPLKKVPALKDGDFTLSESVAILLYLTHKYKVPDHWYPQDLQARARVDEYLA 1:MALELYMDLLSAPCRAVYIFSKKHDISFNFQFVDLLKGHHHSKEYIDINPLRKLPSLKDGKFVLTESVSILSYLCRKYSAPLHWYPPDLHTRARVEEFMA 1:MALELYMDLLSAPCRAVYIFSKKHDIPFNFQFVDLLKGHPYNKEYIKINPLRKLPSLKDGKFVLTESVAILYYLCRKYSTPSHWYPPDLHIRARVDEFMA *.***..**.*.*.**.***.**. *..... *** **.. .... ..*.* *.* ****.* *.**..** ** ** *..*** **..**** *...

100 100 100 100 100 100

hGSTT1 hGSTT2 hGSTT2B cjGSTT1 cjGSTT4 cjGSTT4L

101:WQHTTLRRSCLRALWHKVMFPVFLGEPVSPQTLAATLAELDVTLQLLEDKFLQNKAFLTGPHISLADLVAITELMHPVGAGCQVFEGRPKLATWRQRVEA 101:WHADCIRGTFGIPLWVQVLGPLI-GVQVPEEKVERNRTAMDQALQWLEDKFLGDRPFLAGQQVTLADLMALEELMQPVALGYELFEGRPRLAAWRGRVEA 101:WHADCIRGTFGIPLWVQVLGPLI-GVQVPEEKVERNRTAMDQALQWLEDKFLGDRPFLAGQQVTLADLMALEELMQPVALGYELFEGRPRLAAWRGRVEA 101:WQHTTLRRSCLRALWHKVMFPVFLGEPVSPQTLAATLAELDVNVQLLEDKFLQNKAFLAGPHISLADLVAITELMHPVGAGCQVFKGRPKLAAWRNRMEA 101:WQHTAFQLPMKRIVWLKLMIPKITGEEVSAEKIDHAVAEVKNSLKLFEEYFLQDKMFITGDQISLADLVAVVEIMQPMAANYNAFLNSSKLAEWRKRVEL 101:WQHVAFQLPMKKMVWLKLLIPKITGEEVSAEKMDHTVAEVKNSLKLFEEYFLQDKMFITGDQISLADLVAVVEMMQPMAANYNAFLNSSKVAEWRMRVEL *.. . .* .. * . *. *. .. .. . ....*..**... *. * ...****.* *.*.*..... * .....* ** *.*.

200 199 199 200 200 200

hGSTT1 hGSTT2 hGSTT2B cjGSTT1 cjGSTT4 cjGSTT4L

201:AVGEDLFQEAHEVILK-AK-DFPPADPTIKQKLMPWVLAMIR--200:FLGAELCQEAHSIILSILEQAAKKTLPTPSPEAYQAMLLRIARIP 200:FLGAELCQEAHSIILSILEQAAKKTLPTPSPEAYQAMLLRIARIP 201:AVGEDLFQEAHEIILK-AK-DFPPADPTTKQKLMPRVLAMIQ--201:SIGSGLFMEAHSRLMQLADWDFSTLDPVVKENISELLKKSK---201:SIGSGLFREAHNRLMQLADWDFSTLDPVVKEKICEFREKYL---. . .*. . * *..*** .. . ..

240 244 244 240 241 241

D hMGST1a cjMGST1

1:MVDLTQVMDDEVFMAFASYATIILSKMMLMSTATAFYRLTRKVFANPEDCVAFGKGENAKKYLRTDDRVERVRRAHLNDLENIIPFLGIGLLYSLSGPDP 100 1:MVDLSQLTDDEVFRAFASYATIILSKMMLMSAATAFYRMTRKVFANPEDCATFGKGENAKKFLRTDDRVERVRRAHLNDLENIVPFLGIGLLYSLSGPDL 100 **** * ***** ***************** ****** *********** ********* ********************* ***************

hMGST1a 101:STAILHFRLFVGARIYHTIAYLTPLPQPNRALSFFVGYGVTLSMAYRLLKSKLYL cjMGST1 101:STAVLHFRIFVGARIYHTIAYLTPLPQPNRALGFFVGYGVTLSMAYRLLKSRLYL *** **** *********************** ****************** ***

33

155 155

Fig. 2

A hGSTA1 mfGSTA1 hGSTA2 mfGSTA2 cjGSTA1 hGSTA3 mfGSTA3 cjGSTA3 hGSTA5 mfGSTA5 dGSTA3 rGSTA1 rGSTA2 rGSTA3 rGSTA5 rGSTA6 hGSTA4 cjGSTA4 mfGSTA4 dGSTA4 cjGSTA4L rGSTA4 hGSTM2 mfGSTM2 cjGSTM2 rGST Yb3 cjGSTM5 hGSTM5 mfGSTM5 hGSTM1v1 hGSTM4v1 mfGSTM4 cjGSTM4 rGSTM4 rGSTM1 rGSTM3 rGSTM2 rGSTM6L rGSTM6 hGSTM3 mfGSTM3 cjGSTM3 rGSTM5 hGSTP1 mfGSTP1 cjGSTP1 dGSTP1 rGSTP1 hGSTS1 mfGSTS1 cjGSTS1 dGSTS1 rGSTS1 hGSTK1v1 mfGSTK1 cjGSTK1 rGSTK1 hGSTO1v1 mfGSTO1 cjGSTO1 rGSTO1 hGSTO2v1 mfGSTO2 cjGSTO2 rGSTO2 hGSTZ1 mfGSTZ1 cjGSTZ1 hGSTT1 mfGSTT1 cjGSTT1 rGSTT1 rGSTT3 hGSTT2 hGSTT2B mfGSTT2 rGSTT2 hGSTT4 cjGSTT4 cjGSTT4L rGSTT4 hFLAP 0.1

34

35

B hMGST1a mfMGST1 cjMGST1 rMGST1 hMGST2v1 mfMGST2 cjMGST2 rMGST2 hMGST3 mfMGST3 cjMGST3 rMGST3 hGSTA1 0.1

36

Fig. 3

37

A

0

5

10

15

20

cjGSTA1 hGSTA1 hGSTA2 

cjGSTA3 hGSTA3 

cjGSTA4



cjGSTA4L hGSTA4 hGSTA5 cjGSTK1



hGSTK1 hGSTM1 

cjGSTM2 hGSTM2 cjGSTM3 hGSTM3 cjGSTM4 hGSTM4 cjGSTM5 hGSTM5 cjGSTO1 hGSTO1 

cjGSTO2



hGSTO2 cjGSTP1 hGSTP1 cjGSTS1







hGSTS1 cjGSTT1 hGSTT1 hGSTT2 hGSTT2B cjGSTT4 cjGSTT4L hGSTT4 cjGSTZ1 hGSTZ1

38

25

30

35

40

45

B 0

5

10

15

20

25

30

35

40

cjMGST1 hMGST1v1a cjMGST2





hMGST2 cjMGST3 hMGST3

39



(kb) 45

Fig. 4

A

Human chromosome 6 TMEM14A GSTA2

GSTA1

GSTA5

GSTA3

GSTA4

ICK

GSTA1

GSTA3

GSTA4L

GSTA4

ICK

Marmoset chromosome 4 TMEM14A

B Human chromosome 1 AMPD2

GSTM4

GSTM2

GSTM1

GSTM5

GSTM3

EPS8L3

GSTM5

GSTM2

Gap

GSTM3

EPS8L3

GSTT4

GSTT1

Marmoset chromosome 1 AMPD2

GSTM4

C Human chromosome 22 SLC2A11

GSTT2B

DDT-L

DDT

GSTT2

GSTT2P CABIN1

Marmoset chromosome 7 PIWIL3

Gap

GSTT4

DDT

DDT-L

GSTT1

GSTT4L

CABIN1

E

D Human chromosome 12 Marmoset chromosome 9 CFAP43

GSTO1

GSTO2

Human chromosome 12 Marmoset chromosome 9 ITPRTP

SLC15A5

40

MGST1

LMO3

41

Fig. 5

1.E-04

1.E-04

5.E-05

0

0

0

0

GSTK1

GSTM2

2.E-03

GSTM3

3.E-04

7.E-04

1.E-03

2.E-04

4.E-04

0

0

0

0

GSTM5

4.E-03

GSTO1

8.E-04

GSTO2

3.E-04

4.E-04

2.E-04

8.E-04

0

0

0

0

GSTS1

GSTT1

3.E-03

GSTZ1

2.E-03

0

0

Small intestine

Kidney

Liver

Lung

Brain

Small intestine

0

Kidney

0

Liver

3.E-03

Lung

3.E-04

Brain

MGST3

6.E-03

42

Small intestine

0

Kidney

0

Liver

2.E-03

Lung

1.E-03

Brain

2.E-03

MGST2

MGST1

3.E-03

2.E-04

6.E-04

GSTP1

2.E-03

2.E-03

5.E-04

GSTM4

8.E-04

Kidney

2.E-02

1.E-03

GSTA4L

1.E-04

Small intestine

GSTA4

2.E-04

Liver

GSTA3

2.E-04

Lung

GSTA1

3.E-02

Relative expression level (normalized with GAPDH mRNA level, arbitrary units)

Female

Brain

Male

43

0

GSTK1

GSTS1

MGST1

MGST2

GSTO1

GSTT4L

GSTZ1

GSTM4

Testis (Male)

4.E-03

44 GSTO1

GSTS1

GSTA1

GSTA4L

GSTA4L

GSTA1

GSTA3 GSTO2

GSTA4L

GSTA3

GSTA3

GSTA3

MGST2

GSTS1 GSTA4L

GSTS1

GSTM3

GSTA4L

GSTM3

GSTA4

GSTZ1

GSTO2

GSTO2

GSTZ1

GSTM3

GSTT1

GSTK1

GSTO1

GSTM4

GSTP1

GSTA4

GSTM2

GSTS1

GSTM3

MGST2

GSTZ1

MGST1

GSTM4

GSTK1

GSTO1

GSTM2

GSTT1

GSTP1

GSTM5

MGST3

GSTA1

GSTA4L

GSTA3

MGST2

GSTO2

GSTS1

GSTZ1

MGST1

GSTM3

GSTT1

GSTK1

GSTO1

MGST1

Small intestine (Female)

GSTA4

Kidney (Female)

GSTO2

GSTA3

GSTM4

Liver (Female)

GSTP1

GSTA4

MGST2

GSTA4

GSTO1

MGST2

GSTM2

GSTK1

GSTM5

MGST1

MGST3 GSTM5

Lung (Female)

MGST1

GSTS1

GSTM3

GSTT1

GSTM4

GSTO2

3.E-03

GSTM2

GSTZ1 GSTT1

3.E-02

GSTO1

2.E-02 GSTP1

GSTZ1

GSTT1

GSTK1

GSTM5

MGST3

GSTA3

GSTS1

GSTA4L

GSTA1

GSTM3

GSTA4

GSTO2

GSTZ1

MGST2

MGST1

2.E-03

MGST2

Small intestine (Male) GSTA1

GSTS1

GSTO1 GSTM4

GSTM4

GSTP1

GSTM2

GSTA4

GSTM5

1.E-03

GSTK1

0

GSTM4

0 MGST3

Kidney (Male)

GSTA1

GSTA3

GSTO2 GSTM3

Liver (Male)

MGST1

GSTS1 GSTA4L

GSTA3

GSTA4L

GSTT1 GSTK1

Lung (Male)

GSTP1

GSTA4 GSTM3

GSTA4

0.E+00

GSTP1

0

GSTM2

2.E-03

MGST1

GSTM4

GSTP1

GSTO1

MGST2

GSTM2

MGST3

GSTK1

GSTM5

0 GSTM2

0

GSTM5

GSTO1 MGST2

0

GSTZ1

6.E-04

MGST3

6.E-04

MGST3

2.E-03

GSTM4

GSTO2

GSTP1

GSTM2

0 MGST1

1.E-03

MGST3

1.E-03

GSTM5

Brain (Male)

GSTA1

GSTA4L

GSTA3

GSTO2

GSTA4

GSTZ1

GSTM3

GSTM4

GSTT1

GSTO1

GSTT1 GSTZ1

3.E-03

GSTS1

MGST2

GSTK1

GSTP1

MGST1

GSTK1

GSTM5

3.E-02

GSTA4

0

GSTM5

1.E-02

GSTA1

1.E-02

GSTT1

2.E-03

GSTT1

0

GSTM2

GSTA1 MGST3

1.E-03

GSTT4

8.E-03

GSTA3

GSTM5

8.E-03

GSTA1

8.E-03

MGST3

2.E-02

GSTA1

GSTO2

MGST3

GSTM2

GSTP1

GSTM3

Relative expression level (normalized with GAPDH mRNA level , arbitrary units)

Fig. 6 Brain (Female)

Fig. 7

1200

(A) CDNB

[S], 1.0 mM

600

0

Human

Conjugation activity, nmol/min/mg protein

400

Marmoset (B) EPNPP

Monkey [S], 0.5 mM

200

0

Human

40

Marmoset

Monkey

(C) Styrene 7,8-oxide [S], 1.6 mM

20

0

Human

8

Marmoset

Monkey

(D) 1-Iodohexane [S], 0.1 mM

4

0

Human

Marmoset Liver cytosol 45

Monkey

Fig. 8

18000

(A) CDNB

1.0 mM

(B) EPNPP

0.5 mM

(C) Styrene 7,8-oxide

0.8 mM 1.6mM

(D) 1-Iodohexane

0.05 mM 0.1 mM

9000

0

Conjugation activity, nmol/min/nmol GST

3600

1800

0 250

125

0 80

40

0 Recombinant marmoset GSTs 46

Author Contributions (1) Study conception and design; YU, SU, and HY. (2) Acquisition, analysis and/or interpretation of data; YU, SU, ST, NM, and HY. (3) Drafting/revision of the work for intellectual content and context; YU, SU, and HY. (4) Final approval and overall responsibility for the published work. YU, SU, and HY.

47