antioxidants

Article Novel Characterization of Antioxidant , 3-Mercaptopyruvate Sulfurtransferase-Knockout Mice: Overexpression of the Evolutionarily-Related Enzyme

Noriyuki Nagahara 1,*, Mio Tanaka 2, Yukichi Tanaka 2 and Takaaki Ito 3

1 Isotope Research Laboratory, Nippon Medical School, Tokyo 113-8602, Japan 2 Department of Pathology, Kanagawa Children’s Medical Center, Yokohama 232-8555, Japan; [email protected] (M.T.); [email protected] (Y.T.) 3 Department of Pathology and Experimental Medicine, Graduate School of Medical Science, Kumamoto University, Kumamoto 860-8556, Japan; [email protected] * Correspondence: [email protected]

 Received: 25 March 2019; Accepted: 26 April 2019; Published: 1 May 2019 

Abstract: The antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) is localized in the cytosol and mitochondria, while the evolutionarily-related enzyme, rhodanese (thiosulfate sulfurtransferase, TST, EC 2.8.1.1) is localized in the mitochondria. Recently, both have been shown to produce hydrogen sulfide and polysulfide. Subcellular fractionation of liver mitochondria revealed that the TST activity ratio of MST-knockout (KO)/wild-type mice was approximately 2.5; MST activity was detected only in wild-type mice, as expected. The ratio of TST mRNA expression of KO/wild-type mice, as measured by real-time quantitative polymerase chain reaction analysis, was approximately 3.3. It is concluded that TST is overexpressed in MST-KO mice.

Keywords: knockout mouse; mercaptopyruvate sulfurtransferase; rhodanese; real-time quantitative polymerase chain reaction

1. Introduction Rhodanese (thiosulfate sulfurtransferase, TST) and 3-Mercaptopyruvate sulfurtransferase (MST, EC 2.8.1.2) and are ubiquitous, evolutionarily related enzymes distributed in both prokaryotes and eukaryotes [1,2]. Mouse MST gene aligns with TST one on chromosome 15 (Figure1) (NC_000081.6). The former enzyme is distributed in the cytosol and mitochondria [3] and the latter only in the mitochondria [4]. The 50 flanking promoter region of MST represents a classical housekeeping gene [5]. On the other hand, the 50 flanking promoter region of TST has not been clarified. It was reported that human congenital MST deficiency causes mercaptolactate-cysteine disulfiduria (MCDU) [6,7]. Most cases were complicated by mental retardation [8,9], however, in some cases, patients were mentally normal [10,11].

Antioxidants 2019, 8, 116; doi:10.3390/antiox8050116 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 116 2 of 9 Antioxidants 2019, 8, x FOR PEER REVIEW 2 of 9

Figure 1. Alignment of TST and MST genes on mouse chromosome 15. Each of two family genes Figure 1. Alignment of TST and MST genes on mouse chromosome 15. Each of two family genes contains two exons and an intron (NC_000081.6). The 50-Flanking region of the TST gene has not been contains two exons and an intron (NC_000081.6). The 5′-Flanking region of the TST gene has not been identified, however, a prompter region exists. Arrows represent transcriptional direction. Details are identified, however, a prompter region exists. Arrows represent transcriptional direction. Details are described in the text. described in the text. MST performs antioxidative function [12–17]. It has been also reported that hydrogen sulfide and polysulfideMST performs were antioxidative produced by function MST [18 –[12–17].22] and It TST has [19 been,23]. also Although reported these that products hydrogen reportedly sulfide andhave polysulfide important physiological were produced roles by [24 –MST27], MCDU [18–22 pathogenesis] and TST [19,23]. has not beenAlthough clarified. these To elucidateproducts reportedlythe underlying have pathogenesis, important physiological we produced roles MST-knockout [24–27], MCDU (KO) pathogenesis mice [15]. The has gene not targeting been clarified. ranged Tobetween elucidate 1 and the 3606 underlying bp from thepathogenesis, initiation codon we pr ATGoduced of mouse MST-knockout MST gene, (KO) which mice covered [15]. theThe exon gene 1, targetingintron, and ranged partial between exon 2 regions. 1 and 3606 The bp mice from exhibited the initiation significant codon anxiety-like ATG of mouse behaviors MST [ 15gene,], however which coveredthe result the could exon not 1, intron, be reconfirmed, and partial which exon resembled2 regions. The clinical mice features. exhibited To significant elucidate theanxiety-like etiology, behaviorswe measured [15], MST however and TSTthe activitiesresult could in KOnot micebe reconfirmed, using mitochondrial which resembled fractions clinical because features. rhodanese To elucidatewas distributed the etiology, only in mitochondriawe measured [3 ].MST We alsoand performedTST activities real-time in KO quantitative mice using polymerase mitochondrial chain fractionsreaction (PCR)because with rhodanese SYBR green was distributed I [28], which only was in useful mitochondria in measuring [3]. We the also transcription performed of real-time mRNA. quantitativeWe found that polymerase TST was overexpressed chain reaction and could(PCR) compensate with SYBR for green the eff ectI [28], of the which MST defectwas useful in the KOin measuringmice, but the the mechanism transcription has notof beenmRNA. clarified. We found that TST was overexpressed and could compensate for the effect of the MST defect in the KO mice, but the mechanism has not been clarified.2. Materials and Methods

2.2.1. Materials Ethics Statement and Methods for Animal Experiments The study was conducted in accordance with the Declaration of Helsinki, and the all experiments 2.1. Ethics Statement for Animal Experiments were performed in accordance with the guidelines and regulations for the Care and Use of Laboratory Animals,The study Nippon was Medical conducted School in (#28-008). accordance Littermate with the wild-type Declaration and KOof Helsinki, mice [15] wereand the deeply all experimentsanesthetized were by intra-abdominal performed in accordance pentobarbital with injection the guidelines (100 mg and/100 regulations g body weight), for the andCare sacrificed and Use ofby Laboratory exsanguination. Animals, Nippon Medical School (#28-008). Littermate wild-type and KO mice [15] were deeply anesthetized by intra-abdominal pentobarbital injection (100 mg/100 g body weight), and2.2. Subcellularsacrificed by Fractionation exsanguination. of Mitochondria and Cytosol Livers were excised from three littermate wild-type (#1–3) and three KO mice (#1–3) sacrificed 2.2.according Subcellular to the Fractionation method described of Mitochondria above. Mitochondrialand Cytosol and cytosolic fractionations were prepared usingLivers a Mitochondria were excised Isolation from three Kit for littermate Tissue (Thermo wild-type Fisher (#1–3) Scientific, and three Waltham, KO mice MA, (#1–3) USA). sacrificed After an accordingapproximately to the 100 method mg portion described of each above. liver Mitochondrial was washed and with cytosolic 4 mL of 20fractionations mM potassium were phosphate prepared usingbuffer a (pH Mitochondria 7.2 at 4 ◦C) twice,Isolation it was Kit cutfor into Tissue small (Thermo pieces inFisher 800 µ Scientific,L of the same Waltham, buffer. EachMA, USA). homogenate After anwas approximately prepared using 100 a Potter mg portion homogenizer of each (AR liver BROWN was washed Co. Ltd., with Tokyo. 4 mL Japan) of 20 with mM a loose-fittedpotassium phosphatepestle with buffer three strokes,(pH 7.2 thenat 4 each°C) twice, fractionation it was cut was into performed small pieces according in 800 to µL protocol of the 1 same described buffer. in Eachthe kit. homogenate Mitochondrial was fractionsprepared were using added a Potter to 50homogenizerµL of the same (AR bu BROWNffer, incubated Co. Ltd., on Tokyo. ice for 30Japan) min, and vortexed. After being frozen at 80 C for 30 min, they were incubated on ice for 30 min and with a loose-fitted pestle with three −strokes,◦ then each fractionation was performed according to vortexed. They were then frozen again at 80 C for 30 min, added to 5 µL of 10% triton X (Wako protocol 1 described in the kit. Mitochondrial− ◦fractions were added to 50 µL of the same buffer, incubated on ice for 30 min, and vortexed. After being frozen at −80 °C for 30 min, they were Antioxidants 2019, 8, 116 3 of 9

Pure Chemicals, Osaka, Japan), and incubated on ice for 30 min. After centrifuging at 20,000 g for × 10 min at 4 ◦C, the supernatant was used for TST and MST activity assays and western blot analyses for both enzymes.

2.3. TST and MST Activity Assays in Mitochondrial Fractions MST activity catalyzes the trans-sulfuration from 3-mercaptopyruvate to 2-mercaptoethanol and TST activity from thiosulfate to cyanide. The activities of both enzymes were measured for each mitochondrial fraction obtained according to the method described above. MST and TST activities were measured by a modification of the methods of Vachek and Wood [29] and Sörbo [30], respectively, as described previously [17]. One unit of MST activity was defined as 1 µmol of pyruvate formed per minute, and one unit of TST activity was defined as 1 µmol of thiocyanate formed per minute.

2.4. Preparation of mRNA, cDNA Synthesis, and Real-Time Quantitative PCR Analysis for TST Mouse livers were excised from littermate wild-type and KO mice, which were sacrificed by the method described above. Each mRNA was isolated using an mRNA Isolation Kit (Roche, Basel, Switzerland). The mRNA concentrations were measured using a NanoDrop Lite (Thermo Fisher Scientific). The cDNA samples were synthesized using a QuantiTect Reverse Transcription kit (QIAGEN, Venio, The Netherlands): Each mixture contained 1 µg of each RNA template, 2 µL of gDNA Wipeout Buffer, and RNase-free water for a final volume of 14 µL. Synthetic reactions were performed at 42 ◦C for 2 min. Further reactions were performed on the obtained 14-µL mixture, 1 µL of Quantiscript Reverse Transcriptase (RT), 4 µL of Quantiscript RT buffer, and 14 µL of RT Primer Mix at 42 ◦C for 15 min, and then 95 ◦C for 3 min. Mouse TST primers were designed based on the GenBank nucleotide database [31] of mouse TST as follows: F (forward) primer, GGAGCCCGGATATAGTAGGACTAGA; and R (reverse) primer, TTCGTCAGGAAGTCCATGAA. Mouse glyceraldehyde 3-phosphate dehydrogenase primer (GAPDH, QuantiTect Primer Assay, QIAGEN) and mouse β-actin (QuantiTect Primer Assay, QIAGEN) were used as controls. The SYBR Green real-time quantitative PCR assays were performed in three different PCR systems using a QuantiTect SYBR Green PCR kit (QIAGEN; triplicates). The mixture contained 1 µL of DNA template (50 ng), 1.5 µL of each F and R primer (10 ng), and 25 µL of 2 SYBR Green × PCR Master Mix for a final volume of 50 µL. The PCRs were performed using an Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific K.K., Tokyo, Japan). The following thermal cycling conditions were used: initial denaturation at 50 ◦C for 2 min and 95 ◦C for 15 min; followed by 45 cycles at 94 ◦C for 15 sec, 53 ◦C for 30 sec, and 72 ◦C for 35 sec; and by one cycle at 95 ◦C for 15 sec, 60 ◦C for 1 min, and 95 ◦C for 15 sec.

2.5. Western Blot Analysis of Mitochondrial Fractions for TST and MST Mitochondrial fractions (wild-type #1, 2.7 mg/mL; KO #1, 3.2 mg/mL) were used for western blot analysis—each 30-mg sample was loaded into a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 13.5 cm 15 cm) chamber with a stacking gel. Each sample was added to × 2% SDS, 100 mM Dithiothreitol (DTT), 0.1% bromophenol blue, and 10% glycerol in 50 mM Tris HCl buffer (pH 6.7; total volume, 22 µL), and then heated at 100 ◦C for 10 min. All reagents were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Separated proteins were transferred to Immuno-Blot polyvinylidene difluoride (PVDF) (BIO-RAD.com, Tokyo, Japan) with an electrotransfer apparatus (Nippon Eido Corp., Tokyo, Japan). Anti-rat MST rabbit polyclonal antibody was prepared and partially purified as described previously [1], and showed cross-reactivity with MST of other species [3] and also TST [3]. In this experiment, although the titer of the antibody for MST was present at more than 70 times the amount of that of TST [3], the anti-MST antibody was not treated with rat recombinant TST to reduce the cross-reacting with TST. On the other hand, anti-rat TST rabbit polyclonal antibody was newly produced using recombinant TST [1], and partial purification was performed using ammonium sulfate. Antioxidants 2019, 8, x FOR PEER REVIEW 4 of 9

produced using recombinant TST [1], and partial purification was performed using ammonium Antioxidantssulfate. 2019, 8, 116 4 of 9 Western blot analyses with anti-MST and anti-TST polyclonal antibodies (1:1500 and 1:500, respectively)Western blotwere analyses performed. with Alkaline anti-MST phosphatase-conjugated and anti-TST polyclonal goat antibodies anti-rabbit (1:1500 IgG and (1:1500, 1:500, respectively)Jackson ImmunoResearch were performed. Laboratories, Alkaline phosphatase-conjugated Inc., West Grove, PA, goat USA) anti-rabbit was used IgG (1:1500,as a secondary Jackson ImmunoResearchantibody. A 5-Bromo-4-chloro-3-indolyl-phospha Laboratories, Inc., West Grove,te/nitro PA, USA) blue was tetrazolium used as a secondary(BCIP/NBT) antibody. Color ADevelopment 5-Bromo-4-chloro-3-indolyl-phosphate Substrate (Promega, Madison,/nitro blue WI, tetrazolium USA) was (BCIP used/NBT) for Color staining Development alkaline Substratephosphatase-conjugated (Promega, Madison, probes. WI, USA) was used for staining alkaline phosphatase-conjugated probes.

2.6.2.6. Protein Determination ProteinProtein concentrationsconcentrations were were determined determined using aus Coomassieing a Coomassie protein assay protein kit (Pierce assay Biotechnology, kit (Pierce Inc.,Biotechnology, Rockford, IL,Inc., USA) Rockford, with crystalline IL, USA) bovine with serum crystalline albumin bovine (BSA, MPserum Biochemicals, albumin Irvine,(BSA, CA,MP USA)Biochemicals, as the standard. Irvine, CA, USA) as the standard.

2.7.2.7. Statistical Analysis TheThe significancesignificance ofof didifferencefference betweenbetween valuesvalues waswas estimatedestimated withwith thethe Student’sStudent’s tt-test,-test, andand pp valuesvalues lessless thanthan 0.050.05 werewere deemeddeemed significant.significant. All results havehave beenbeen roundedrounded toto nono moremore thanthan threethree significantsignificant figures.figures.

3.3. Results 3.1. TST and MST Activity Assays in Mitochondrial Fractions 3.1. TST and MST Activity Assays in Mitochondrial Fractions TST activities in liver mitochondrial fractions of wild-type and KO mice (triplicate) were TST activities in liver mitochondrial fractions of wild-type and KO mice (triplicate) were 2.65 ± 2.65 0.0331 10 1 and 6.61 0.563 10 1 unit/mg, respectively (p = 8.99 10 6; Figure2). 0.0331± × 10−1 ×and −6.61 ± 0.563 ×± 10−1 unit/mg,× − respectively (p = 8.99 × 10−6; Figure ×2). MST− activity of MST activity of wild-type mice was 3.44 0.0415 10 1 unit/mg (MST activity was not detected in KO wild-type mice was 3.44 ± 0.0415 × 10−1± unit/mg× (MST− activity was not detected in KO mice; Figure mice; Figure2). This experiment revealed that TST activity in KO mice was significantly increased 2). This experiment revealed that TST activity in KO mice was significantly increased compared to compared to that in wild-type mice. MST activity was detected only in wild-type mice. that in wild-type mice. MST activity was detected only in wild-type mice.

FigureFigure 2.2. TST and MSTMST activityactivity assaysassays inin mitochondrialmitochondrial fractions.fractions. TST activity in KO micemice waswas approximately 2.5 times more than the activity in wild-type mice (p = 8.99 10 5, n = 3). MST activity approximately 2.5 times more than the activity in wild-type mice (p = 8.99× × 10−−5, n = 3). MST activity was not detected in KO mice. KO: MST-knockout mice; ND: not detected; Wild: wild-type mice. was not detected in KO mice. KO: MST-knockout mice; ND: not detected; Wild: wild-type mice. 3.2. Preparation of mRNA, cDNA Synthesis, and Real-Time Quantitative PCR Analysis for TST 3.2. Preparation of mRNA, cDNA Synthesis, and Real-Time Quantitative PCR Analysis for TST 2 Isolated mRNAs of wild-type and KO mice were measured to be 5.36 10 ng/µL(A260/A280 = 2.02) Isolated2 mRNAs of wild-type and KO mice were measured to be× 5.36 × 102 ng/µL (A260/A280 = and 6.11 10 ng/µL(A260/A280 = 1.99), respectively. The cDNA synthesis yields for wild-type and KO 2.02) and× 6.11 × 1023 ng/µL (A260/A280 = 1.99), respectively. The3 cDNA synthesis yields for wild-type mice were 1.13 10 µg/µL(A260/A280 = 1.85) and 1.15 10 µg/µL(A260/A280 = 1.85), respectively. × 3 × 3 and TargetKO mice genes were for 1.13 TST × 10 of µg/µL wild-type (A260 and/A280 KO= 1.85) mice and were 1.15 measured× 10 µg/µL as (A4.42260/A2801.65 = 1.85),101 ± × andrespectively.1.03 0.158 102 bp, respectively (triplicate; p = 1.15 10 2). Internal control genes for ± × × − glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were 1.82 0.119 102 and 1.28 0.150 102 bp, ± × ± × Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 9

Target genes for TST of wild-type and KO mice were measured as 4.42 ± 1.65 × 101 and 1.03 ± 0.158 × 102 bp, respectively (triplicate; p = 1.15 × 10−2). Internal control genes for Antioxidantsglyceraldehyde-3-phosphate2019, 8, 116 dehydrogenase (GAPDH) were 1.82 ± 0.119 × 102 and 1.28 ± 0.1505 × of 10 9 2 bp, respectively. Corrected values of TST mRNAs GAPDH (each target gene value/mean GAPDH gene value) for wild-type and KO mice were 2.43 ± 0.911 × 10−1 and 8.01 ± 1.23 × 10−1, respectively. respectively. Corrected values of TST mRNAs GAPDH (each target gene value/mean GAPDH gene The results of the revaluation were 3.03 ± 1.14 × 10−1 and 1.00 ± 0.154, respectively (Figure 3). On the value) for wild-type and KO mice were 2.43 0.911 10 1 and 8.01 1.23 10 1, respectively. The other hand, control genes for β-actin were 5.04± ± 0.340× −× 102 and 1.29± ± 0.186× ×− 102 bp, respectively. results of the revaluation were 3.03 1.14 10 1 and 1.00 0.154, respectively (Figure3). On the Corrected values of TST mRNAs (each± target× gene− value/mean± β-actin gene value) for wild-type and other hand, control genes for β-actin were 5.04 0.340 102 and 1.29 0.186 102 bp, respectively. KO mouse were 8.75 ± 3.28 × 10−2 and 7.94 ± 0.122± × 10−×1, respectively.± The results× of the revaluation Corrected values of TST mRNAs (each target gene value/mean β-actin gene value) for wild-type and were 1.10 ± 0.413 × 10−1 and 1.00 ± 0.0153, respectively (Figure 3). In conclusion, mRNA expression of KO mouse were 8.75 3.28 10 2 and 7.94 0.122 10 1, respectively. The results of the revaluation TST in KO mice was± significantly× − greater ±than in× wild-type− mice. This result is consistent with the were 1.10 0.413 10 1 and 1.00 0.0153, respectively (Figure3). In conclusion, mRNA expression results of± our measurements× − of MST± and TST activities. of TST in KO mice was significantly greater than in wild-type mice. This result is consistent with the results of our measurements of MST and TST activities.

Figure 3. Real-time quantitative PCR analysis for TST mRNA. When GAPDH was used as an internal control gene, mRNA expression of TST in KO mice was approximately 3.3 times greater than expression Figure 3. Real-time quantitative 3PCR analysis for TST mRNA. When GAPDH was used as an internal in wild-type mice (p = 4.43 10− , n = 3). On the other hand, when β-actin was used as the internal control gene, mRNA expression× of TST in KO mice was3 approximately 3.3 times greater than control gene, the ratio was approximately 9.1 (p = 6.69 10− , n = 3). expression in wild-type mice (p = 4.43 × 10−3, n = 3). On× the other hand, when β-actin was used as the 3.3. Westerninternal Blot control Analysis gene, ofthe Mitochondrial ratio was approximately Fractions for 9.1 TST (p = and6.69MST × 10−3, n = 3). Previous western blot analysis without blocking for TST revealed that the band representing TST molecules3.3. Western was Blot more Analysis intense of thanMitochondrial the band Fractions representing for TST MST, and and MST their molecular masses were 33,180 and 32,808,Previous respectively western [blot2,3]. analysis Anti-MST without polyclonal blocking antibody for TST cross-reacted revealed withthat TSTthe [band3]. In representing the present study,TST molecules 30 mg samples was more of mitochondrial intense than fractionthe band proteins representing from wild-type MST, and and their KO molecular mice were masses loaded were in gels.33,180 The and band 32,808, representing respectively MST [2,3]. molecules Anti-MST was polyclonal not detected antibody in KO cross-reacted mice (Figure with4). On TST the [3]. other In the hand,present the study, band representing30 mg samples TST of moleculesmitochondrial of KO fraction mice was proteins more from prominent wild-type than and that KO of wild-typemice were Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 9 miceloaded (Figure in gels.4), which The band is consistent representing with ourMST measurement molecules was of thenot enzyme detected activities. in KO mice (Figure 4). On the other hand, the band representing TST molecules of KO mice was more prominent than that of wild-type mice (Figure 4), which is consistent with our measurement of the enzyme activities.

FigureFigure 4. 4.Western Western blot blot analysis analysis of mitochondrialof mitochondrial fractions fracti forons TST for andTST MST.and MST. The band The representingband representing TST moleculesTST molecules from KO from mice KO is moremice prominentis more prominent than that than of wild-type that of micewild-type in the mice mitochondrial in the mitochondrial fractions. fractions.

4. Discussion TST and MST activity assays and western blot analysis of mitochondrial fractionation revealed that TST was overexpressed in MST-KO mice. Moreover, real-time quantitative PCR analysis supported this result. TST overexpression is, therefore, most likely compensating for the effect of the MST defect. TST and MST genes are aligned on both sides (NC_005106.4) (Figures 1 and 5) and transcribed bidirectionally. When we first tried MST-KO mice production, we failed to obtain heterozygous KO mice [15]. The gene targeting was extended to 2714 bp from the initiation codon ATG of the promoter region, which in the germ line may cause an embryonic lethal phenotype. Therefore, a common regulatory region of transcription may be located in this area. In fact, the transcription of β-globin genes in erythroid cells is enhanced by the locus control region (LCR), which is localized in non-coding region at a long distance from the gene [32].

Figure 5. Proposed overexpression mechanism of TST. Decrease in cytosolic H2Sn may directly or indirectly affect the promoter region or the cis-regulatory element between TST and MST structural genes. Then it may facilitate transcription of TST gene, resulting in overexpression of TST. Trx:

thioredoxin; TS: thiosulfate; H2Sn: polysulfides. ?: unspecified factors.

MST functions as an antioxidant [12–17], a producer of hydrogen sulfide and polysulfides [18– 22], and as a possible producer of sulfur oxide [32]. After sulfuration of the catalytic site cysteine (Cys-S− or Cys S-S−), reduction by thioredoxin produced hydrogen sulfide and polysulfide [20]. On the other hand, after oxidation of the sulfurated catalytic site cysteine to Cys-thiosulfenate (Cys-Sγ-SO−), Cys-thiosulfinate (Cys-Sγ-SO2−), and Cys-thiosulfonate (Cys-Sγ-SO3−), reduction of these molecules by thioredoxin was proposed to produce sulfur dioxides [33]. Hydrogen sulfide activates N-methyl-D-aspartic acid receptors via reduction of cysteine disulfide bonds in a ligand binding domain [23]; hydrogen sulfide directly acts on a functional protein in this case. Hydrogen sulfide also facilitates transcriptional factor, nuclear factor (NF)-κB via sulfuration [34]. Polysulfides Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 9

Figure 4. Western blot analysis of mitochondrial fractions for TST and MST. The band representing AntioxidantsTST molecules2019, 8, 116 from KO mice is more prominent than that of wild-type mice in the mitochondrial 6 of 9 fractions.

4. Discussion TST and MST activity assays and westernwestern blotblot analysisanalysis ofof mitochondrialmitochondrial fractionationfractionation revealed that TSTTST was was overexpressed overexpressed in MST-KO in MST-KO mice. mice. Moreover, Moreover, real-time real-time quantitative quantitative PCR analysis PCR supported analysis thissupported result. this TST result. overexpression TST overexpression is, therefore, is, therefor most likelye, most compensating likely compensating for the for eff ectthe ofeffect the of MST the defect.MST defect. TST and TST MST and genes MST aregenes aligned are onaligned both sideson both (NC_005106.4) sides (NC_005106.4) (Figures1 and(Figures5) and 1 transcribedand 5) and bidirectionally.transcribed bidirectionally. When we first When tried MST-KOwe first tri miceed production,MST-KO mice we failedproduction, to obtain we heterozygous failed to obtain KO miceheterozygous [15]. The KO gene mice targeting [15]. The was gene extended targeting to 2714 wa bps extended from the initiationto 2714 bp codon from ATG the initiation of the promoter codon region,ATG of whichthe promoter in the germ region, line which may causein the an germ embryonic line may lethal cause phenotype. an embryonic Therefore, lethal phenotype. a common β regulatoryTherefore, regiona common of transcription regulatory region may be of located transcri inption this area.may Inbe fact, located the transcriptionin this area. ofIn fact,-globin the genestranscription in erythroid of β-globin cells isenhanced genes in byerythroid the locus cells control is enhanc regioned (LCR), by the which locus is localizedcontrol region in non-coding (LCR), regionwhich is at localized a long distance in non-coding from the region gene [ 32at ].a long distance from the gene [32].

Figure 5. Proposed overexpression mechanism of TST. Decrease in cytosolic H2Sn may directly or Figure 5. Proposed overexpression mechanism of TST. Decrease in cytosolic H2Sn may directly or indirectly affect the promoter region or the cis-regulatory element between TST and MST structural indirectly affect the promoter region or the cis-regulatory element between TST and MST structural genes. Then it may facilitate transcription of TST gene, resulting in overexpression of TST. Trx: genes. Then it may facilitate transcription of TST gene, resulting in overexpression of TST. Trx: thioredoxin; TS: thiosulfate; H2Sn: polysulfides. ?: unspecified factors. thioredoxin; TS: thiosulfate; H2Sn: polysulfides. ?: unspecified factors. MST functions as an antioxidant [12–17], a producer of hydrogen sulfide and polysulfides [18–22], and asMST a possiblefunctions producer as an antioxidant of sulfur [12–17], oxide [a32 producer]. After of sulfuration hydrogen ofsulfide the catalyticand polysulfides site cysteine [18– 22], and as a possible producer of sulfur oxide [32]. After sulfuration of the catalytic site cysteine (Cys-S− or Cys S-S−), reduction by thioredoxin produced hydrogen sulfide and polysulfide [20]. − − On(Cys-S the or other Cyshand, S-S ), reduction after oxidation by thioredoxin of the sulfurated produced catalytic hydrogen site sulfide cysteine and topolysulfide Cys-thiosulfenate [20]. On the other hand, after oxidation of the sulfurated catalytic site cysteine to Cys-thiosulfenate (Cys-Sγ-SO−), Cys-thiosulfinate (Cys-Sγ-SO2−), and Cys-thiosulfonate (Cys-Sγ-SO3−), reduction of − − − these(Cys-S moleculesγ-SO ), Cys-thiosulfinate by thioredoxin was(Cys-S proposedγ-SO2 ), toand produce Cys-thiosulfonate sulfur dioxides (Cys-S [33γ].-SO Hydrogen3 ), reduction sulfide of activatesthese molecules N-methyl-D-aspartic by thioredoxin acid was receptors proposed via toreduction produce sulfur of cysteine dioxides disulfide [33]. bondsHydrogen in a sulfide ligand bindingactivates domain N-methyl-D-aspartic [23]; hydrogen acid sulfide receptors directly via acts reduction on a functional of cysteine protein disulfide in this bonds case. in Hydrogen a ligand sulfidebinding also domain facilitates [23]; transcriptionalhydrogen sulfide factor, directly nuclear acts factor on a functional (NF)-κB via protein sulfuration in this [34 case.]. Polysulfides Hydrogen facilitatesulfide also the facilitates nuclear translocation transcriptional of transcriptional factor, nuclear factor factor NF-E2 (NF)- relatedκB via sulfuration factor 2 (Nrf2) [34]. via Polysulfides sulfuration of Kelch-like erythroid-derived central nervous system homology factor associated protein 1 (Keap 1) [35]; polysulfides act on a transcriptional protein in this case. On the other hand, decrease in hydrogen sulfide, polysulfides, and sulfur oxide or increase in thiosulfate could directly or indirectly regulate a functional protein; TST, a family enzyme of MST could be up-regulated (Figure4), however, the transcription mechanism, cis-element of TST gene has not been clarified. Further, TST and MST are family enzymes and both enzymes catalyze the same substrates, although Km and Kcat are different [1,2]. Thiosulfate, a substrate for both TST and MST [1,2], was accumulated in MST-KO mice (unpublished data), which may induce overexpression of TST (Figure5). Antioxidants 2019, 8, 116 7 of 9

5. Conclusions The ratio of TST activity in MST-KO to wild-type mice was approximately 2.5. Real-time quantitative polymerase chain reaction analysis revealed the ratio was approximately 3.3. These facts led us to conclude that TST was overexpressed in the KO mice. Double (MST and TST)-KO mice, therefore, should be produced to investigate the pathophysiology of defects in H2S and H2Sn in mitochondria.

Author Contributions: N.N. supervised the work, analyzed the results, and drafted the manuscript. All authors analyzed the samples and contributed to data interpretation. All authors approved the submitted version and agreed to be personally accountable for the authors’ own contributions. Funding: This research received no external funding. Acknowledgments: We wish to thank Masatoshi Nagano, Yasunori Mikagahara, and Hidenori Suzuki, Department of Pharmacology, Nippon Medical School for providing KO mice. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Nagahara, N.; Okazaki, T.; Nishino, T. Cytosolic mercaptopyruvate sulfurtransferase is evolutionarily related to mitochondrial rhodanese. Striking similarity in amino acid sequence and the increase in the mercaptopyruvate sulfurtransferase activity of rhodanese by site-directed mutagenesis. J. Biol. Chem. 1995, 270, 16230–16345. [PubMed] 2. Nagahara, N.; Nishino, T. Role of amino acid residues in the active site of rat liver mercaptopyruvate sulfurtransferase. cDNA cloning, overexpression, and site-directed mutagenesis. J. Biol. Chem. 1996, 271, 27395–27401. [CrossRef][PubMed] 3. Nagahara, N.; Ito, T.; Kitamura, H.; Nishino, T. Tissue and subcellular distribution of mercaptopyruvate sulfurtransferase in the rat: Confocal laser fluorescence and immunoelectron microscopic studies combined with biochemical analysis. Histochem. Cell Biol. 1998, 110, 243–250. [CrossRef][PubMed] 4. Ludwig, S.; Chanutin, A. Distribution of enzymes in the liver of control and x-irradiated rats. Arch. Biochem. 1950, 441–445. 5. Nagahara, N.; Sreeja, V.G.; Li, Q.; Shimizu, T.; Tsuchiya, T.; Fujii-Kuriyama, Y. A point mutation in a silencer module reduces the promoter activity for the human mercaptopyruvate sulfurtransferase. Biochim. Biophys. Acta 2004, 1680, 176–184. [CrossRef][PubMed] 6. Amberger, J.S.; Bocchini, C.A.; Schiettecatte, F.; Scott, A.F.; Hamosh, A. OMIM.org: Online Mendelian Inheritance in Man, an online catalog of human genes and genetic disorders. Nucleic Acids Res. 2015, 43, D789–D798. [CrossRef] 7. Crawhall, J.C. Beta-mercaptolactate-cysteine disulfiduria. In Metabolic Basis of Inherited Disease; Stanbury, J.B., Wyngaarden, J.B., Fredrickson, D.S., Eds.; McGraw-Hill: New York, NY, USA, 1978; pp. 504–513. 8. Crawhall, J.C.; Parker, R.; Sneddon, W.; Young, E.P.; Ampola, M.G.; Efron, M.L.; Bixby, E.M. Beta mercaptolactate-cysteine disulfide: Analog of cystine in the urine of a mentally retarded patient. Science 1968, 160, 419–420. [CrossRef][PubMed] 9. Law, E.A.; Fowler, B. Beta-mercaptolactate cysteine disulphiduria in a mentally retarded Scottish male. J. Ment. Def. Res. 1976, 20, 99–104. [CrossRef] 10. Niederwieser, A.; Giliberti, P.; Baerlocher, K. Beta-mercaptolactate-cysteine disulfiduria in two normal sisters. Isolation and characterization of beta-mercaptolactate cysteine disulfide. Clin. Chim. Acta 1973, 43, 405–416. [CrossRef] 11. Hannestad, U.; Mårtensson, J.; Sjödahl, R.; Sörbo, B. 3-Mercaptolactate cysteine disulfiduria: Biochemical studies on affected and unaffected members of a family. Biochem. Med. 1981, 26, 106–114. [CrossRef] 12. Nagahara, N. Regulation of mercaptopyruvate sulfurtransferase activity via intrasubunit and intersubunit redox-sensing switches. Antioxid. Redox Signal. 2013, 19, 1792–1802. [CrossRef][PubMed]

13. Nagahara, N. Multiple role of 3-mercaptopyruvate sulfurtransferase: Antioxidative function, H2S and polysulfide production and possible SOx production. Br. J. Pharmacol. 2018, 175, 577–589. [CrossRef] Antioxidants 2019, 8, 116 8 of 9

14. Nagahara, N.; Katayama, A. Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis. J. Biol. Chem. 2005, 280, 34569–34576. [CrossRef][PubMed] 15. Nagahara, N.; Nagano, M.; Ito, T.; Shimamura, K.; Akimoto, T.; Suzuki, H. Antioxidant enzyme, 3-mercaptopyruvate sulfurtransferase-knockout mice exhibit increased anxiety-like behaviors: A model for human mercaptolactate-cysteine disulfiduria. Sci. Rep. 2013, 3, 1986. [CrossRef][PubMed] 16. Nagahara, N.; Nagano, M.; Ito, T.; Suzuki, H. Chapter Thirteen—Redox Regulation of Mammalian 3-Mercaptopyruvate Sulfurtransferase. In Method Enzymol; Cadenas, E., Packer, L., Eds.; Academic Press: New York, NY, USA, 2015; Volume 554, pp. 229–254. 17. Nagahara, N.; Yoshii, T.; Abe, Y.; Matsumura, T. Thioredoxin-dependent enzymatic activation of mercaptopyruvate sulfurtransferase: An intersubunit disulfide bond serves as a redox switch for activation. J. Biol. Chem. 2007, 282, 1561–1569. [CrossRef] 18. Mikami, Y.; Shibuya, N.; Kimura, Y.; Nagahara, N.; Ogasawara, Y.; Kimura, H. Thioredoxin and dihydrolipoic acid are required for 3-mercaptopyruvate sulfurtransferase to produce hydrogen sulfide. Biochem. J. 2011, 439, 479–485. [CrossRef] 19. Mikami, Y.; Shibuya, N.; Kimura, Y.; Nagahara, N.; Yamada, M.; Kimura, H. Hydrogen sulfide protects the retina from light-induced degeneration by the modulation of Ca2+ influx. J. Biol. Chem. 2011, 286, 39379–39386. [CrossRef]

20. Nagahara, N.; Koike, S.; Nirasawa, T.; Kimura, H.; Ogasawara, Y. H2S and polysulfides production from sulfurated catalytic cysteine of reaction intermediates of 3-mercaptopyruvate sulfurtransferase. Biochem. Biophys. Res. Commun. 2018, 496, 648–653. [CrossRef][PubMed] 21. Shibuya, N.; Mikami, Y.; Kimura, Y.; Nagahara, N.; Kimura, H. Vascular endothelium expresses 3-mercaptopyruvate sulfurtransferase and produces hydrogen sulfide. J. Biochem. 2009, 146, 623–626. [CrossRef][PubMed] 22. Shibuya, N.; Tanaka, M.; Yoshida, M.; Ogasawara, Y.; Togawa, T.; Ishii, K.; Kimura, H. 3-Mercaptopyruvate sulfurtransferase produces hydrogen sulfide and bound sulfane sulfur in the brain. Antioxid. Redox Signal. 2009, 11, 703–714. [CrossRef] 23. Kimura, Y.; Toyofuku, Y.; Koike, S.; Shibuya, N.; Nagahara, N.; Lefer, D.; Ogasawara, Y.; Kimura, H.

Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain. Sci. Rep. 2015, 5, 14774. [CrossRef][PubMed] 24. Brancaleone, V.; Vellecco, V.; Matassa, D.S.; di Villa Bianca, R.; Sorrentino, R.; Ianaro, A.; Bucci, M.;

Esposito, F.; Cirino, G. Crucial role of androgen receptor in vascular H2S biosynthesis induced by testosterone. Br. J. Pharmacol. 2015, 172, 1505–1515. [CrossRef][PubMed] 25. Di Villa Bianca, R.D.E.; Mitidieri, E.; Donnarumma, E.; Tramontano, T.; Brancaleone, V.; Cirino, G.; Bucci, M.; Sorrentino, R. Hydrogen sulfide is involved in dexamethasone-induced hypertension in rat. Nitric Oxide 2015, 46, 80–86. [CrossRef][PubMed] 26. Kimura, H. Physiological roles of hydrogen sulfide and polysulfides. Nihon Yakurigaku Zasshi 2016, 147, 23–29. [CrossRef][PubMed] 27. Suwanai, Y.; Nagahara, N. Pharmacological usage of an —A selective inhibitor of 3-mercaptopyruvate sulfurtransferase to control generation of polysulfide. In Nanoscale Fabrication, Optimization, Scale-up and Biological Aspects of Pharmaceutical Nanotechnology; Grumezescu, A.M., Ed.; Elsevier: Cambridge, MA, USA, 2018; Volume 15, pp. 579–617. 28. Morrison, T.B.; Weis, J.J.; Wittwer, C.T. Quantification of low-copy transcripts by continuous SYBR green I monitoring during amplification. Biotechniques 1998, 24, 954–958. 29. Vachek, H.; Wood, J.L. Purification and properties of mercaptopyruvate sulfur of Escherichia coli. Biochim. Biophys. Acta 1972, 258, 133–146. [CrossRef] 30. Sörbo, B.H. Crystalline rhodanese. I. Purification and physicochemical examination. Acta Chem. Scand. 1953, 7, 1129–1136. [CrossRef] 31. GenBank Sequence Database; National Center for Biotechnology Information: Bethesda, MD, USA, 2019. 32. Li, Q.; Peterson, K.R.; Fang, X. Locus control regions. Blood 2002, 100, 3077–3086. [CrossRef][PubMed] 33. Nagahara, N.; Nirasawa, T.; Yoshii, T.; Niimura, Y. Is novel signal transducer sulfur oxide involved in the redox cycle of persulfide at the catalytic site cysteine in a stable reaction intermediate of mercaptopyruvate sulfurtransferase? Antioxid. Redox Signal. 2012, 16, 747–753. [CrossRef] Antioxidants 2019, 8, 116 9 of 9

34. Sen, N.; Paul, B.D.; Gadalla, M.M.; Mustafa, A.K.; Sen, T.; Xu, R.; Kim, S.; Snydewr, S.H. Hydrogen sulfide-linked sulfhydration of NF-κB mediates its antiapoptotic actions. Mol. Cell 2012, 45, 13–24. [CrossRef] 35. Koike, S.; Ogasawara, Y.; Shibuya, N.; Kimura, H.; Ishii, K. Polysulfide exerts a protective effect against cytotoxicity caused by t-buthylhydroperoxide through Nrf2 signaling in neuroblastoma cells. FEBS Lett. 2013, 587, 3548–3555. [CrossRef][PubMed]

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).