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. 2021 Aug 9;85(10):2113–2120. doi: 10.1093/bbb/zbab141

Molecular cloning and characterization of common marmoset SULT1C subfamily members that catalyze the sulfation of thyroid hormones

Katsuhisa Kurogi 1,✉, Yoko Manabe 2, Ming-Cheh Liu 3, Masahito Suiko 4, Yoichi Sakakibara 5
PMCID: PMC8458394  PMID: 34370005

ABSTRACT

Cytosolic sulfotransferase SULT1C subfamily is one of the most flexible gene subfamilies during mammalian evolution. The physiological functions of SULT1C enzymes still remain to be fully understood. In this study, common marmoset (Callithrix jacchus), a promising primate animal model, was used to investigate the functional relevance of the SULT1C subfamily. Gene database search revealed 3 intact SULT1C genes and a pseudogene in its genome. These 4 genes were named SULT1C1, SULT1C2, SULT1C3P, and SULT1C5, according to the sequence homology and gene location. Since SULT1C5 is the orthologous gene for human SULT1C2P, we propose, here, to revisit the designation of human SULT1C2P to SULT1C5P. Purified recombinant SULT1C enzymes showed sulfating activities toward a variety of xenobiotic compounds and thyroid hormones. Kinetic analysis revealed high catalytic activities of SULT1C1 and SULT1C5 for 3,3′-T2. It appears therefore that SULT1C isoforms may play a role in the thyroid hormone metabolism in common marmoset.

Keywords: cytosolic sulfotransferase, SULT1C, thyroid hormones, common marmoset, primate

Graphical Abstract

Graphical Abstract.

Graphical Abstract

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Three SULT1C members may play an important role in the metabolism of thyroid hormones, especially inactive 3′,3′-T2, in common marmoset.

Cytosolic sulfotransferases (SULTs) play an important role in the metabolism of a wide array of xenobiotics such as drugs and polyphenols, as well as and endogenous compounds including steroids, and neurotransmitters (Strott 2005; Gamage et al.2006; Tibbs et al.2015). Sulfate conjugation, as mediated by the SULTs, involves the transfer of a hydrophilic sulfonate group to substrate compounds, leading to changes in their physiological activities and facilitation of their excretion from body. SULT enzymes have been detected in a broad range of organism from prokaryotes to eukaryotes (Suiko et al.2017). In vertebrates, the SULTs constitute a gene superfamily, and 6 gene families have been categorized based on their amino acid sequences (Blanchard et al.2004). Among the 6 SULT families, SULT1 gene family constitutes the largest cluster. In humans, 8 isoforms are found in the SULT1 gene family. SULT1A1 and SULT1A2 have been described as simple phenol SULTs (P-PSTs) and play an important role in the metabolism of, for example, simple phenolic drugs such as acetaminophen, minoxidil, and oxymorphone (Falany 1997; Sugahara et al.2003; Kurogi et al.2014). On the other hand, SULT1A3 has been described as the monoamine phenol SULT (M-PST), which plays a pivotal role in the metabolism of neurotransmitters including dopamine and serotonin (Falany 1997; Yasuda et al.2007). Similar to the SULT1A subfamily, the SULT1C subfamily isoforms, previously dubbed hydroxyarylamine sulfotransferases, have also been recognized to exhibit strong sulfating activities toward a variety of aromatic compounds including N-hydroxy-2-acetylaminofluorene, acetaminophen, and flavonoids (Sakakibara et al.1998; Hui et al.2015; Yamamoto et al.2015; Guidry et al.2017). Most of the SULT1C isoforms are expressed at fetal stage, with low level expression found in adult tissues (Sakakibara et al.1998; Stanley, Hume and Coughtrie 2005; Runge-Morris and Kocarek 2013; Duniec-Dmuchowski et al.2014). The physiological function of SULT1C enzymes has remains poorly understood, in part due to their predominantly fetal expressions.

Compared with other SULT1 subfamilies, more variations are found among SULT1C members. Among mammals, 2 mouse SULT1C genes, 3 rat SULT1C genes, and 4 human SULT1C genes have been identified (Freimuth et al.2004). The divergence of the SULT1C genes has raised some controversy with regard to the orthology of SULT1C subfamily members in mammals (Freimuth et al.2004). To date, 4 SULT1C isoforms, SULT1C1, SULT1C2, SULT1C3 and SULT1C4, and one SULT1C pseudogene have been classified into SULT1C subfamily across mammalian species. Human SULT1C gene cluster consists of SULT1C2, SULT1C3, and SULT1C4, whereas mouse SULT1C gene cluster consists of SULT1C1 and SULT1C2. Of SULT1C genes, only SULT1C2 is conserved across mammalian species and SULT1C3 seems to appear only in primates (Freimuth et al.2004; Runge-Morris and Kocarek 2013). A recent study has revealed that a SULT1C3 variant with alternative C-terminal exons, SULT1C3d, is generated as a chimera enzyme from the SULT1C1 gene remnant and SULT1C3 (Kurogi et al.2017). The bulk of the putative SULT1C1 gene, except exons 7/8, is absent in the human genome. In recent years, several primate SULT1C members have been identified in cynomolgus macaques (Macaca fascicularis) and common marmoset (Callithrix jacchus) genome (Uno, Murayama and Yamazaki 2019, 2020). In cynomolgus macaques, 4 SULT1C genes, designated SULT1C3-like, SULT1C2v1, SULT1C2v2, and SULT1C4 have been identified, and sulfating activities of 3 of these isoforms with phenolic compounds like p-nitrophenol were detected (Uno, Murayama and Yamazaki 2019). In contrast, 4 SULT1C genes, designated SULT1C2, 2 SULT1C3-like isoforms, and SULT1C4, have been identified in common marmoset, and of the 4, SULT1C2 has been shown to display sulfating activity toward p-nitrophenol (Uno, Murayama and Yamazaki 2019). Nevertheless, the orthology of these primate SULT1C genes and the enzymatic characteristics of these isoforms remain to be fully elucidated.

We report here the classification, cloning, and enzymatic characterization (substrate specificity, pH-profile and kinetic parameters) of 3 common marmoset SULT1Cs, with SULT1C2v2 (SULT1C2P) renamed to SULT1C5. Sulfating activity toward 59 potential substrate compounds was analyzed using recombinant SULT1C enzymes. The pH-dependence and kinetic parameters of these enzymes in catalyzing the sulfation of thyroid hormones were determined.

Materials and methods

Materials

Bithionol, capsaicin, daidzein, eugenol, genistein, p-nitrophenol, and α-tocopherol were purchased from Wako Pure Chemical Industries, Ltd. 1-Naphthol was obtained from Katayama Chemical, Ltd., and 2-naphthol was a product of Nacaraitesque, Inc. Caffeic acid, ellagic acid, hesperidin, 1 -naphthylamine (NA), 4-ocylphenol, pentachlorophenol, rutin, l-thyroxine (T4), 3,3′,5-triiodo-l-thyronine (T3), 3,3′,5′-triiodo-l-thyronine (reverse T3, rT3), 3,5-diiodo-l-thyronine (3,5-T2), 3,3′-diiodo-l-thyronine (3,3′-T2), vaniline, adenosine 5′-triphosphate (ATP), sodium dodecyl sulfate (SDS), sodium acetate, 2-morpholinoethanesulfonic acid (MES), N-2-hydroxylpiperazine-N′-2-ethanesulfonic acid (HEPES), 3-[N-tris-(hydroxymethyl)methylamino]-propanesulfonic acid (TAPS), N-Cyclohexyl-2-aminoethanesulfonic acid (CHES), Trizma base, dithiothreitol (DTT), were products of Sigma-Aldrich Co. LLC. γ-CEHC and equol were from Cayman Chemicals. Isopropyl β-d-thiogalactopyranoside (IPTG) was from Takara. Common marmoset 1st strand cDNA mix (CMDM-01) was a product of Genostaff. KOD-Plus-Neo DNA polymerase was purchased from TOYOBO. BamHI, EcoRI, and NdeI restriction endonucleases were from New England Biolabs. SULT1C1 cDNA nucleotide and oligonucleotide primers were synthesized by Integrated DNA Technologies. Escherichia coli XL1-Blue MRF′ and BL21 competent cells were from Stratagene. BigDye Terminator v 3.1 Cycle Sequencing Kit was purchased from Applied Biosystems. Silica gel thin-layer chromatography (TLC) plates were products of Millipore. Carrier-free sodium [35S]sulfate was a product of American Radiolabeled Chemicals, Inc. [35S]-PAPS was synthesized from ATP and carrier-free [35S]sulfate using the bifunctional human ATP sulfurylase/APS kinase and its purity determined as previously described (Yanagisawa et al.1998).

Molecular cloning of common marmoset SULT1C members

Coding region of SULT1C1 cDNA was chemically synthesized based on the nucleotide sequence (GenBank Accession no. XM_017964121) deposited in NCBI gene database. The restriction sites were embedded at 5′- and 3′-ends of the coding region by PCR using oligonucleotide primers (Table S1). Coding regions of SULT1C2 (XM_002757476) and SULT1C5 cDNAs (XM_017964032) were amplified from common marmoset 1st strand cDNA mix by PCR using gene specific oligonucleotide primers with restriction sites incorporated at the end (Table S1). Amplification conditions for SULT1C1 and SULT1C2 were 2 min at 94 °C and 30 cycles of 98 °C for 10 s and 68 °C for 30 s under the action of KOD -Plus- Neo. For SULT1C5, amplification conditions were 2 min at 94 °C and 35 cycles of 98 °C for 10 s, 55 °C for 30 s, and 68 °C for 30 s. Reaction mixtures were treated with the corresponding restriction enzymes, BamHI, EcoRI, and NdeI. Digested PCR products were extracted from agarose gel and subcloned into pGEX4T1 prokaryotic expression vector, and transformed into E. coli XL 1-Blue MRF′. Full-length cDNA sequences of inserts were validated using Applied Biosystems 3500xL Genetic Analyzer with BigDye Terminator v3.1 Cycle Sequencing Kit.

Purification of recombinant common marmoset SULT1C enzymes

To express the recombinant common marmoset SULT enzymes, pGEX4T1 constructs harboring respective SULT cDNAs were individually transformed into BL21 (DE3) cells. After the cells were grown to the density of 0.6 OD at 600 nm, isopropyl β-d-thiogalactopyranoside (IPTG) was added at 0.1 mm to the growth LB medium supplemented with 0.1 mg/mL ampicillin. After a 4-h induction at 37 °C, the cells were collected by centrifugation. Cells were then homogenized in Lysis buffer (50 mm Tris-HCl at pH 8.0, 150 mm NaCl, 1 mm EDTA) using an Ohtake French Press. After the insoluble fraction was cleared by centrifugation (20 400 g for 20 min), recombinant GST-fusion proteins were fractionated from supernatants using glutathione–Sepharose resin. Purified recombinant SULT1C enzymes were then released from the resin by thrombin digestion for 3 h at 4 °C. Purified enzymes were analyzed by SDS-PAGE on a 12% polyacrylamide gel using method of Laemmli (Laemmli 1970). Protein concentration of purified SULT1C enzymes was determined using Lowry's method, with bovine serum albumin as the standard (Lowry et al.1951).

Enzymatic assay

The sulfating activity of purified recombinant common marmoset SULTs was assayed using radioactive [35S]-PAPS as the sulfonate donor. The standard assay mixture, with a final volume of 25 µL, contained 50 mm HEPES buffer at pH 7.0, 0.2 µm [35S]-PAPS (15 Ci/mmol), and 10 µm substrate dissolved in DMSO. Control with DMSO, in place of substrate, was also prepared. The reaction was started by the addition of 0.1 µg enzyme (for SULT1C1), allowed to proceed for 30 min at 37 °C. For SULT1C2 and SULT1C5, 1.0 and 0.01 µg amounts of enzymes were used, respectively. After the reaction was terminated with 3 min heat at 98 °C, the reaction mixture was applied on to a silica gel TLC plate, and subjected to ascending TLC with n-butanol/isopropanol/formic acid/water (3:1:1:1, by volume) as the solvent system. Autoradiograph was taken from the TLC plate using a fluorescent image analyzer (Typhoon FLA 9500 BGR) and the radioactive spots corresponding to [35S]-sulfated products of compounds tested were quantified by comparison with a standard spot. The results obtained were calculated and expressed in nmol of sulfated product formed/min/mg purified enzyme. For the pH-dependent experiments, different buffers (50 mm MES at pH 5.5-7.0; HEPES at pH 7.0-8.0; TAPS at pH 8.0-9.0; and CHES at pH 9.0-10.0 were used in the reactions with 10 µm 3, 3′-T2 as substrate. For the kinetic studies, enzyme assay was performed with varying concentrations of thyroid hormones (3,3′-T2, T3, rT3, T4) under the standard assay condition. Data obtained were analyzed by Eadie–Hofstee plots and the subsequent nonlinear regression analysis was carried out based on Michaelis–Menten kinetics using GraphPad Prism5 software.

Results

Sequence analysis and nomenclature

Previous studies have shown that rhesus monkey and common marmoset contain several SULT1C genes (Uno, Murayama and Yamazaki 2019, 2020). In order to clarify the orthology of SULT1C members, SULT1C gene search in NCBI database and sequence alignment analysis were performed in addition to the chromosomal localization analysis. Database search revealed the presence of 3 SULT1C genes and a SULT1C pseudogene, SULT1C3, in common marmoset genome and 5 SULT1C genes were identified in rhesus monkey genome (Figure 1). Sequence homology and gene location analysis have pinpointed the orthology and designation for common marmoset and rhesus monkey SULT1C genes (Figure 1 and Table 1). Common marmoset SULT1C1 showed the highest amino acid sequence identities, 95.7% and 89.1%, to rhesus monkey and mouse SULT1C1, respectively, among rhesus monkey and human SULT1C members. Similar to SULT1C1, common marmoset SULT1C2 showed the highest amino acid sequence identities (84.1%-97.0%) to human, rhesus monkey and mouse SULT1C2. In the case of SULT1C3, only amino acid sequences derived from exons 1-4 were analyzed due to the absence of exons 5-8 in common marmoset SULT1C3P gene. Sequence analysis revealed the highest amino acid sequence identities, 73% and 72%, to the corresponding regions of rhesus monkey SULT1C3 and human SULT1C3a. Thus, SULT1C3 gene appears to be a pseudogene in common marmoset. Human SULT1C2P, alternatively named SULT1C1P (Gen ID:151 234), has been reported as a pseudogene that has undergone exons rearrangement (Freimuth et al.2004). Sequence analysis of the nucleotide sequence of exons 2-5 indicated that human SULT1C2P share 56%, 62%, 90%, and 87% sequence identities with human SULT1C2, human SULT1C4, rhesus monkey SULT1C5, and common marmoset SULT1C5, respectively (Table S2). Therefore, common marmoset SULT1C5 appears to be the orthologous gene for human SULT1C2P, which thus is renamed SULT1C5P.

Figure 1.

Figure 1.

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Gene structure of SULT1C gene subfamily. SULT1C genes of human, rhesus monkey, common marmoset, and mouse were aligned according to the order of each gene in the chromosome. Alternative exons (7b and 8b) of human SULT1C3 were aligned to corresponding sequences of SULT1C1. Human SULT1C5P and common marmoset SULT1C3P correspond to orthologous pseudogenes.

Table 1.

Amino acid sequence homology of SULT1C subfamily members in human (hum), rhesus monkey (rhe), common marmoset (com), and mouse (mou)

1 2 3 4 5 6 7 8 9 10 11 12 13 14
1. mouSULT1C1 100
2. comSULT1C1 89.1 100
3. rheSULT1C1 89.8 95.7 100
4. mouSULT1C2 59.5 59.8 59.8 100
5. comSULT1C2 62.8 62.8 62.5 84.1 100
6. rheSULT1C2 62.5 62.5 62.2 83.1 97.0 100
7. humSULT1C2 61.8 62.2 61.8 83.1 95.6 97.3 100
8. rheSULT1C3 59.9 60.2 60.5 55.1 55.7 55.7 55.4 100
9. humSULT1C3a 65.1 67.4 68.8 51.7 54.7 54.1 53.7 77.6 100
10. humSULT1C3d 58.2 58.9 59.5 52.0 53.7 53.7 53.7 83.9 89.8 100
11. rheSULT1C4 64.3 65.3 64.7 60.6 64.0 63.0 62.7 57.6 56.2 56.2 100
12. humSULT1C4 63.3 63.9 63.6 61.4 64.4 63.4 63.1 57.0 56.0 56.0 96.3 100
13. comSULT1C5 60.0 61.7 61.3 57.3 60.4 60.4 59.4 52.3 52.3 50.7 62.2 61.9 100
14. rheSULT1C5 61.7 63.3 63.7 59.0 61.8 61.8 60.8 53.3 54.0 51.3 65.0 64.6 89.7 100
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Molecular cloning of common marmoset SULT1C isoforms

Based on available sequence information, 5′- and 3′-primer were designed in order to express the full-length common marmoset SULT1C enzymes in E. coli. Using first-strand cDNA mix derived from 23 tissues/organs as a template, CDS regions encoding 3 SULT1C isoforms were amplified using respective gene specific 5′- and 3′-primer pair. SULT1C2 and SULT1C5 cDNAs were successfully amplified from common marmoset cDNA mix, whereas no amplification of SULT1C1 cDNA was detected from the cDNA mix (data not shown). Therefore, SULT1C1 cDNA was chemically synthesized based on the sequence (GenBank Accession no. XM_017964121) deposited at NCBI. Cloned SULT1C2 and SULT1C5 cDNAs matched 100% with the reported sequences, XM_002757476 and XM_035273499, respectively (Figure 2). All 3 SULT1C isoforms contain 3 so-called signature sequences; 5′-phosphosulfate binding (5′-PSB) loop, 3′-phosphate binding (3′-PB) motif and p-loop related motif, as shown in Figure 2. Since these 3 motifs are important in the binding of the sulfonate donor, PAPS (Weinshilboum et al.1997; Negishi et al.2001), 3 SULT1C members cloned are likely functional SULT enzymes.

Figure 2.

Figure 2.

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Amino acid sequence alignment of common marmoset SULT1C subfamily members. The alignment was performed using Clustal Omega program in EMBL-EBI. The underlines represent the signature sequences, 5′-PSB, 3′-PB, and p-loop.

Bacterial expression, purification, and substrate specificity of recombinant common marmoset SULT1C isoforms

pGEX-4T1 harboring common marmoset SULT1C cDNA was transformed into BL21 (DE3) E. coli cells for the expression of recombinant enzymes. Recombinant common marmoset SULT1C enzymes were purified from E. coli cells by GST affinity chromatography followed by the thrombin digestion. As shown in Figure 3, purified SULT1C enzymes all migrated at approximately 35 000 molecular weight position upon SDS-PAGE. These results were consistent with the predicted molecular weight (35 900, 34 900, and 35 800, respectively) of SULT1C1, SULT1C2, and SULT1C5 plus 144 of N-terminal additional sequences, Gly-Ser. Purity of the 3 recombinant enzymes was greater than 95%. To examine the substrate specificity of purified SULT1C enzymes, a total of 59 endogenous and xenobiotic compounds were tested as substrates. For endogenous compounds, all 3 SULT1C isoforms showed significant sulfating activity toward thyroid hormones, but not other endogenous substrates including catecholamines, steroid hormones, and bile acids (cf. Table 2 and Table S3). SULT1C1 and SULT1C5 exhibited the strongest activity specifically toward 3,3′-T2. On the other hand, SULT1C2 showed weaker but broader activity toward all 4 thyroid hormones tested, including 3,3′-T2. In contrast to endogenous compounds, all 3 SULT1C enzymes displayed a variety of sulfating activities toward xenobiotic compounds tested. Of the 3 enzymes, SULT1C1 showed the broadest activity toward xenobiotic compounds, including naphthols and polyphenols. These results indicated that common marmoset SULT1C enzymes may play an important role in the metabolism of thyroid hormones, especially 3,3′-T2, and, additionally, also contribute to the metabolism of phenolic xenobiotic compounds.

Figure 3.

Figure 3.

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SDS gel electrophoretic pattern of purified common marmoset SULT1C enzymes. SDS-PAGE was performed on a 12.5% gel, followed by Coomassie blue staining. Lanes 1-5 showed the samples from each step of SULT1C1 purification. Cells before induction (lane 1), cells after induction (lane 2), supernatant of cell lysates (lane 3), purified GST-fusion enzyme (lane 4), purified SULT1C1 enzyme after digestion by thrombin (lane 5). Lanes 6 and 7 showed purified SULT1C2 and SULT1C5 enzymes after digestion by thrombin, respectively. Protein molecular weight markers were co-electrophoresed (lane M).

Table 2.

Specific activities of common marmoset SULT1C isoforms with endogenous and xenobiotic compounds as substratesa

Specific activity (nmol/min/mg)
Substrate SULT1C1 SULT1C2 SULT1C5
Endogenous substrates
 3,3′-T2 17.77 ± 1.28 0.07 ± 0.01 33.56 ± 3.53
 T3 ND 0.07 ± 0.01 ND
 rT3 ND 0.04 ± 0.01 0.60 ± 0.08
 T4 ND 0.32 ± 0.01 ND
Xenobiotic substrates
 pNP 0.87 ± 0.04 0.01 ± 0.01 0.31 ± 0.08
 1-Naphthol 0.67 ± 0.08 ND 0.58 ± 0.08
 2-Naphthol 0.56 ± 0.06 ND 0.59 ± 0.09
 Eugenol 0.80 ± 0.06 ND 0.05 ± 0.02
 Vanillin 0.27 ± 0.04 ND 0.86 ± 0.10
 Caffeic acid 0.07 ± 0.03 0.01 ± 0.01 ND
 Ellagic acid ND 0.02 ± 0.01 ND
 Daidzein ND 0.02 ± 0.01 ND
 Equol 0.74 ± 0.10 ND ND
 Genistein 0.62 ± 0.12 0.02 ± 0.01 ND
 Hesperidin 0.10 ± 0.03 ND ND
 Rutin 0.05 ± 0.02 ND ND
 Capsaicin 0.57 ± 0.11 ND 1.58 ± 0.34
 γ-CEHC 0.23 ± 0.08 0.01 ± 0.01 ND
 Pentachlorophenol ND 0.02 ± 0.01 ND
 Bithionol ND 0.11 ± 0.01 ND
 NA 0.44 ± 0.05 ND ND
 4-Octylphenol 0.44 ± 0.13 ND ND
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a

Specific activity refers to nmol substrate sulfated/min/mg purified enzyme. Data present mean ± SD derived from 3 determinations. The concentration of substrates used in reaction was 10 µm.

Characterization of the sulfation of thyroid hormones by common marmoset SULT1C isoforms

To characterize the sulfation of thyroid hormones by common marmoset SULT1C isoforms, the pH-dependence of their sulfating activity was first examined. As shown in Figure 4, with 3,3′-T2 as the substrate, all 3 SULT1C isoforms showed comparable pH-profile and were active over pH 6.5-9.0, with a pH optimum at pH 7.0 for SULT1C1 and SULT1C5 and pH 8.5 for SULT1C2. The kinetics of the sulfation of thyroid hormones were subsequently examined and analyzed based on Eadie–Hofstee plots and the nonlinear regression analysis. As shown in Figure 5, the sulfation of thyroid hormones by all 3 SULT1C isoforms appeared to follow Michaelis–Menten saturation kinetics. Based on the results from these experiments, the kinetic constants, Km and Vmax, were calculated. Table 3 shows the kinetic constants determined for the sulfation of individual thyroid hormones by common marmoset SULT1C isoforms. SULT1C1 displayed a much lower Km value and a higher Vmax/Km value with 3,3′-T2, compared with the other 2 common marmoset SULT1C isoforms, with thyroid hormones as substrates. SULT1C2 showed the comparable Km and Vmax values toward all 4 thyroid hormones tested. Although SULT1C5 exhibited the highest Vmax value with 3,3′-T2, the Km value was much higher than that of SULT1C1. Based on the kinetic data, SULT1C1 exhibited the highest catalytic efficiency toward 3,3′-T2 among 3 common marmoset SULT1C isoforms.

Figure 4.

Figure 4.

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pH dependency of the sulfating activity of common marmoset SULT1C enzymes. The pH-profiles of the sulfating activity of common marmoset SULT1C1 (a), SULT1C2 (b), and SULT1C5 (c) with 3,3′-diiodothyronine (3,3′-T2) were shown. Data shown represent calculated mean ± SD derived from 3 experiments.

Figure 5.

Figure 5.

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Kinetic analyses of the sulfation of thyroid hormones by common marmoset SULT1C enzymes. The fitting curves were generated using Michaelis–Menten for the sulfation of 3,3′-T2 by SULT1C1 (a), the sulfation of 3,3′-T2, T3, rT3, T4 by SULT1C2 (b), and the sulfation of 3,3′-T2 by SULT1C5 (c). Eadie–Hofstee plots are inserted under each fitting curve. Data shown represent calculated means ± SD derived from 3 experiments.

Table 3.

Kinetic constants of common marmoset SULT1C isoformsa

Substrate V max K m (µm) V max/Km
(nmol/min/mg)
SULT1C1
 3,3′-T 20.45 ± 0.70 0.48 ± 0.08 42.60
SULT1C2
 3,3′-T2 0.18 ± 0.01 15.49 ± 2.91 0.01
 T3 0.24 ± 0.02 28.21 ± 4.68 0.01
 rT3 0.12 ± 0.02 52.49 ± 16.51 0.01
 T4 0.82 ± 0.02 16.19 ± 0.86 0.05
SULT1C5
 3,3′-T2 52.47 ± 7.42 8.55 ± 2.58 6.14
 rT3 0.24 ± 0.02 28.21 ± 4.68 0.01
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a

Results represent means ± SD derived from 3 determinations. Kinetic parameters were determined based on the equation for the Michaelis–Menten kinetics.

Discussion

In primates, 5 isoforms of SULT1C genes have been identified to date (Uno, Murayama and Yamazaki 2019, 2020). In this study, 4 common marmoset SULT1C genes, including a pseudogene SULT1C3P, were found and designated according to the proposed nomenclature system (Freimuth et al.2004). For common marmoset SULT1C3P, only exons 1-4 were found, implying that exons 5-8 were deleted after the generation of SULT1C3 gene during the evolution. Since the orthologous genes for human SULT1C2P were identified in rhesus monkey and common marmoset and designated as SULT1C5, we propose that human SULT1C2P be renamed as SULT1C5P.

In a previous study, common marmoset SULT1C2 was shown to display significant sulfating activity toward dopamine, naphthol, p-nitrophenol, E2, and DHEA using a phosphate (by-product of sulfation reaction) detection kit (Uno et al.2020). The current study showed that common marmoset SULT1C2 displayed sulfating activity toward thyroid hormones and some xenobiotic phenolic compounds, but not dopamine, E2, and DHEA. The discrepancy may be due to the different assay procedures adopted in the 2 studies. While the method employed in this study is capable of detecting sulfated product directly, the phosphate detection kit relies on the detection of a reaction by-product, phosphate. It is possible that the results obtained using the reaction by-product detection kit may yield nonspecific activity. Previous studies have shown that human SULT1C2 exhibited weak but significant activity toward thyroid hormones among endogenous compounds (Li, Clemens and Anderson 2000; Allali-Hassani et al.2007). Catalytic properties of common marmoset and human SULT1C2 with thyroid hormones as substrates are comparable (Li, Clemens and Anderson 2000), implying that enzymatic functions of SULT1C2 may be conserved in primate.

Common marmoset SULT1C1 and SULT1C5 showed specific and stronger activity toward 3,3′-T2 among substrates tested than did common marmoset SULT1C2. SULT1C1 and SULT1C5 have been identified as pseudogenes in human, while functional SULT1C1 enzymes have been characterized in rat, mouse, and chicken (Nagata et al.1993; Matsui et al.1998; Tamura et al.1998; Wilson et al.2004). Among these orthologous SULT1C1 isoforms, only chicken SULT1C1 has been reported to show the sulfating activity toward endogenous compounds, particularly thyroid hormones (Visser 1994). Like common marmoset SULT1C1, chicken SULT1C1 exhibited the strongest catalytic efficiency (Vmax/Km) with 3,3′-T2. These observations indicated that SULT1C1 may play an important role in the metabolism of 3,3′-T2. In contrast to SULT1C1, much less is known about SULT1C5. A recent study showed that cynomolgus macaques SULT1C5 (SULT1C2v2) exhibited nonspecific sulfating activity toward dopamine, naphthol, p-nitrophenol, E2, and DHEA using a phosphate (by-product of sulfation reaction) detection kit (Uno, Murayama and Yamazaki 2019). In this study, common marmoset SULT1C5 was also investigated. The 3,3′-T2-sulfating activity detected suggested that SULT1C5, together with SULT1C1, may play a role in the metabolism of 3,3′-T2. 3,3′-T2 is an inactive metabolite of the active form of thyroid hormones, T3, which is produced under the action of iodothyronine deiodinase. Since 3,3′-T2 sulfate, but not 3,3′-T2, rapidly undergoes deiodination, 3,3′-T2 sulfation may serve to accelerate the degradation of 3,3′-T2 and excretion of iodine (Visser 1994; van der Spek, Fliers and Boelen 2017).

It should be noted that SULT1C1 migrated slower than other 2 SULT1C isoforms by the denaturing SDS-PAGE, while the predicted molecular weight of SULT1C1 is almost same as SULT1C5 (Figure 3). It is possible that SULT1C5 could be truncated during the purification procedure as an artificial form. It is also worth mentioning that both native and recombinant human SULT1 subfamily enzymes differentially migrated by denaturing SDS-PAGE (Teubner et al.2007), even though the molecular weight of those enzymes are almost same (32 000-34 000). Therefore, the different migration among 3 recombinant SULT1C enzymes may reflect the structural aspect other than the artificial proteolysis. Further studies will be warranted to clarify the stability and native form of common marmoset SULT1C enzymes.

Tissue distribution of SULT1C isoforms remains to be fully elucidated, although some previous studies demonstrated the expression of some SULT1C isoforms in liver, intestines, and kidney. Mouse SULT1C1 has been reported to be strongly expressed in olfactory, brain, and fetal liver and kidney (Wilson et al.2004; Alnouti and Klaassen 2006; 2011) while liver-specific expression of rat SULT1C1 has been reported (Dunn and Klaassen 1998; Klaassen, Liu and Dunn II 1998; Matsui et al.1998). While no expression data of primate SULT1C1 isoform is available, the hepatic, olfactory, neuronal, and renal expressions of common marmoset SULT1C1 are possible. In humans, SULT1C2 has been shown to be expressed in kidney, stomach, and thyroid gland (Her et al.1997; Dooley et al.2000; Teubner et al.2007). In agreement with the expression profile in humans, the strong renal and stomach expression of SULT1C2 have been confirmed in mouse, common marmoset, and cynomolgus macaque (Alnouti and Klaassen 2006; Uno, Murayama and Yamazaki 2019, 2020). Therefore, in addition to substrate specificity, tissue expression of SULT1C2 may be highly conserved across species. Expressions of SULT1C5 in liver, testis, and uterus has been reported in cynomolgus macaque and its expression pattern differed from that of SULT1C4 (Uno, Murayama and Yamazaki 2019). Detailed expression pattern of SULT1C5 will need to be clarified in order to better understand the physiological relevance of primate specific SULT1C5 isoform.

To summarize, 3 common marmoset SULT1C isoforms were cloned, purified, and characterized in the current study. Gene synteny analysis revealed that SULT1C subfamily has been diversified in primate. Enzymatic characterization showed that SULT1C1 and SULT1C5 exhibited the sulfating activity, with high catalytic efficiency, toward 3,3′-T2 and a broad range of xenobiotic compounds. SULT1C2 showed weak sulfating activity toward thyroid hormones, which is in agreement with the characteristics of human SULT1C2. These finding implied that thyroid hormones sulfation by SULT1C2 may play an important role across the species. Taken together, this study suggested that evolution of SULT1C subfamily may play an important role the thyroid hormones metabolism. Further studies are warranted in order to clarify the physiological relevance of isoforms of the SULT1C subfamily.

Supplementary Material

zbab141_Supplemental_File
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Contributor Information

Katsuhisa Kurogi, Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki, Japan.

Yoko Manabe, Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki, Japan.

Ming-Cheh Liu, Department of Pharmacology, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, Toledo, OH, USA.

Masahito Suiko, Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki, Japan.

Yoichi Sakakibara, Department of Biochemistry and Applied Biosciences, University of Miyazaki, Miyazaki, Japan.

Data availability

The data underlying this article will be shared on reasonable request to the corresponding author.

Author contribution

K.K., L.M.C., S.M., and S.Y. participated in research design. K.K. and M.Y. conducted experiments and performed data analysis. K.K., L.M.C., S.Y. wrote or contributed to the writing of the manuscript.

Funding

This work was supported by JSPS KAKENHI under Grant Numbers 26450130 (M.S.), 15H04502 (Y.S.) and 17H05028 (K.K.), and a grant from National Institutes of Health (Grant Number R03HD071146; to M.C.L).

Disclosure statement

No potential conflict of interest was reported by the authors.

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Associated Data

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Supplementary Materials

zbab141_Supplemental_File
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Data Availability Statement

The data underlying this article will be shared on reasonable request to the corresponding author.

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