identification of h2s3 and h2s produced by 3 mercaptopyruvate sulfurtransferase in the brain

www.nature.com/scientificreports OPEN Identification of H2S3 and H2S produced by 3-mercaptopyruvate sulfurtransferase in the brain received: 14 June 2015 accepted: 09 September 2015 Published: 06 October 2015 Yuka Kimura1, Yukiko Toyofuku1, Shin Koike1,2, Norihiro Shibuya1, Noriyuki Nagahara3, David Lefer4, Yuki Ogasawara2 & Hideo Kimura1 Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles We recently found H2Sn in the brain H2Sn induced some responses previously attributed to H2S but with much greater potency than H2S However, the number of sulfur atoms in H2Sn and its producing enzyme were unknown Here, we detected H2S3 and H2S, which were produced from 3-mercaptopyruvate (3 MP) by 3-mercaptopyruvate sulfurtransferase (3MST), in the brain High performance liquid chromatography with fluorescence detection (LC-FL) and tandem mass spectrometry (LC-MS/MS) analyses showed that H2S3 and H2S were produced from MP in the brain cells of wild-type mice but not 3MST knockout (3MST-KO) mice Purified recombinant 3MST and lysates of COS cells expressing 3MST produced H2S3 from MP, while those expressing defective 3MST mutants did not H2S3 was localized in the cytosol of cells H2S3 was also produced from H2S by 3MST and rhodanese H2S2 was identified as a minor H2Sn, and MP did not affect the H2S5 level The present study provides new insights into the physiology of H2S3 and H2S, as well as novel therapeutic targets for diseases in which these molecules are involved Hydrogen polysulfides (H2Sn) have a higher number of sulfane sulfur atoms than hydrogen sulfide (H2S), which has various physiological roles including neuromodulation, vascular tone regulation, and cytoprotection against ischemic insults1–7 Only the bound form of polysulfides, which bridges cysteine residues in proteins, has been observed in vitro8,9; 3-mercaptopyruvate (3 MP) is reported to be a substrate for its enzymatic production10 However, endogenous diffusible polysulfides (H2Sn) were not known to exist We recently found H2Sn in the brain and also showed that they activated transient receptor potential ankyrin (TRPA1) channels approximately 300 times more potently than did hydrogen sulfide (H2S)11–13 Anxiety-related behavior was observed in both 3-mercaptopyruvate sulfurtransferase knockout (3MST-KO)14 and TRPA1-KO mice15 as well as mice in which TRPA1 channels are pharmacologically inhibited15, suggesting the involvement of TRPA1 channels and 3MST in the induction of anxiety-like behavior H2Sn also facilitates the translocation of nuclear factor-like (Nrf2) to the nucleus by modifying its binding partner kelch-like ECH-associated protein (Keap1)16, regulates the activity of the tumor suppressor phosphatase and tensin homolog (PTEN)17, and reduces blood pressure by dilating vascular smooth muscle18 Despite these important physiological effects, its producing enzyme and the number of sulfur atoms in H2Sn were unknown Here, we identified H2S3 as an important H2Sn and 3MST as its producing enzyme H2S3 was also produced from H2S by 3MST and rhodanese Department of Molecular Pharmacology, National Institute of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawahigashi, Kodaira, Tokyo 187-8502, Japan 2Department of Analytical Biochemistry, Meiji Pharmaceutical University, 2-522-1 Noshio, Kiyose, Tokyo 204-8588, Japan 3Radioisotope Center, Nippon Medical School, 1-1-5 Sendagi, Bunkyo, Tokyo 113-8602, Japan 4Department of Pharmacology and Experimental Therapeutics and Cardiovascular Center of Excellence, LSU Health Science Center, New Orleans, LA 70112, USA Correspondence and requests for materials should be addressed to H.K (email: kimura@ncnp.go.jp) Scientific Reports | 5:14774 | DOI: 10.1038/srep14774 www.nature.com/scientificreports/ Results Production of H2S3 and H2S from MP by 3MST.  Because MP, a substrate of 3MST, produces polysulfides, which bridge cysteine residues in the proteins10,19, and cells expressing 3MST contain greater amounts of the form of polysulfides than controls6,19, we hypothesized that 3MST may also produce diffusible polysulfides (H2Sn) To address this problem, we examined H2Sn production using lysates of COS cells expressing 3MST as a source of the enzyme There are various methods to measure H2S including colorimetric methylene blue method, ion-selective or polarographic electrodes, gas chromatography, and monobromobimane assay20 The variations of endogenous levels of H2S in tissue and blood samples have been reported and are largely attributed to the contaminant of H2S released from acid-labile sulfur or bound sulfane sulfur mostly caused during the preparation of the samples rather than the methods21 The monobromobimane analysis with high performance liquid chromatography with fluorescence detection (LC-FL) was previously applied to the quantitative analysis of H2Sn13, and also to the analysis of H2S using both LC-FL and mass spectrometry22 In the present study LC-FL and LC-tandem mass spectrometry (LC-MS/MS) was used to analyze monobromobimane adducts of H2Sn and H2S H2S3 and H2S were produced from MP by 3MST in a concentration-dependent manner (Fig.  1a,b and Supplementary Fig 1) Rhodanese, which is homologous to 3MST and may produce the bound form of polysulfides10, generated neither H2S3 nor H2S from MP (Fig. 1a,b) Lysates of cells transfected with an empty vector did not produce either molecule (Fig. 1a,b) These observations suggest that 3MST produces H2S3 (as a major H2Sn) and H2S from MP H2S2 was detected as a minor product, and H2S5 increased approximately 40% from its basal level in the presence of MP (Fig. 2a,b) The relative levels for H2S2 and H2S5 are shown (Fig. 2a,b), as a H2S5 standard was not available and H2S2 was detected by LC-MS/MS but not clearly recognized by LC-FL, in which the H2S2 peak was buried within the H2S5 peak Production of H2S3 and H2S in whole cells.  We examined whether whole cells were able to pro- duce H2S3 and H2S following exposure to MP Suspensions of brain cells prepared from wild-type and 3MST-KO mice were used (Fig. 1c–f) To provide MP intracellularly, whole cells prepared from wild-type mice were exposed to MP Wild-type cells produced significantly more H2S3 (0.23 ±  0.03 μ mol/g protein; ~65×  increase compared to controls) and H2S (0.02 ±  0.00 μ mol/g protein; ~5×  increase compared to controls) Conversely, H2S3 and H2S production did not significantly increase in cells obtained from 3MST-KO mice following MP exposure (Fig. 1e,f) These observations suggest that H2S3 is produced not only in in vitro enzymatic reactions, but also in whole cells Moreover, the concentration of H2S3 was approximately 10 times greater than that of H2S in whole cells The level of H2S2 was also increased in the presence of MP, while that of H2S5 was not significantly changed regardless of the presence or absence of MP (Fig. 2c,d) H2S3 was detected in cells prepared from wild-type mice even in the absence of MP exposure, suggesting the presence of endogenous H2S3 (3.4 ±  2.2 nmol/g protein in the brain) H2S was also detected at a level of 4.8 ±  1.6 nmol/g protein (Fig.  1e,f) The endogenous concentrations of H2S3 and H2S were lower in 3MST-KO mice, 1.9 ±  1.9 and 3.0 ±  0.4 nmol/g protein, respectively (Fig. 1e,f), but these values did significantly differ from those found in wild-type mice H2S3 and H2S production depends on 3MST catalytic site.  We examined the affinity of 3MST for MP using purified recombinant 3MST produced by bacteria (Fig.  3a,b) 3MST produced H2S3 from MP in a concentration-dependent manner up to 1.5 mM The Km value of 3MST for MP was 4.5 ±  1.5 mM To confirm the production of H2S3 and H2S by 3MST, the production of both molecules by defective 3MST mutants was compared with that of wild-type 3MST using lysates of COS cells expressing the mutant or wild-type 3MST as a source of the enzymes23,24 Introduction of the mutant, in which the active site cysteine 247 was replaced with serine (C274S), resulted in greatly decreased production of H2S3 and H2S to 8.5% and 1.2%, respectively, of that produced in the presence of wild-type 3MST (Fig. 3c,d) The R187G mutant produced H2S3 and H2S at 55.9% and 12.2%, respectively, of that produced by wild-type 3MST; R196G produced H2S3 and H2S at 94.1% and 105.5%, respectively, of that produced by the wild-type (Fig.  3c,d) These observations suggest that the production of H2S3 and H2S greatly depends on the 3MST catalytic site, C247 H2S3 is localized in the cytosol.  We examined the cellular localization of H2S3 produced by 3MST by using COS cells expressing 3MST and loaded with SSP4, a polysulfide-sensitive fluorescence probe25 H2S3 produced de novo from MP incorporated into cells was localized in the cytosol (Fig.  4a) H2S3 was also localized in the cytosol in primary neuronal cultures (Fig. 4b) H2S3 production from H2S by 3MST.  Because H2Sn can also be generated from H2S26, it is possible that 3MST is involved in its generation We examined this problem using lysates of COS cells expressing 3MST Although H2S was oxidized to generate H2S3, even in the absence of 3MST, 3MST significantly accelerated the production of H2S3 from Na2S, a sodium salt of H2S; control lysates did not generate H2S3 (Fig. 5a) Scientific Reports | 5:14774 | DOI: 10.1038/srep14774 www.nature.com/scientificreports/ a 0.5 0.6 H S production (µmole/min/g protein) H S production (µmole/min/g protein) b 0.5 0.4 0.4 0.3 ## 0.2 0.3 ## 0.2 ## 0.1 0.1 10 30 3MP dose (µM) 100 c 10 30 3MP dose (µM) 100 d 3MST ← 578 bp GAPDH ← 346 bp (+/+) (+/+) (+/-) (-/-) (-/-) Male 3MST GAPDH (+/+) (-/-) Female f 0.3 ## 0.25 ## 0.2 0.15 0.05 0.1 control 3MP H S conc (µmole/g protein) H S conc (µmole/g protein) e 0.03 0.025 0.02 0.015 0.01 0.005 control 3MP Figure 1.  H2S3 and H2S are produced by 3MST from MP (a,b) The production of H2S3 (a) and H2S (b) from MP with lysates of COS cells expressing 3MST (●), rhodanese (☐), or empty vector (○) as a source of the enzymes incubated for 15 min (c,d) Determination of the 3MST genotype by polymerase chain reaction (PCR) (c) Western blot analysis (d) of 3MST in the brains of wild-type (+ /+ ) and 3MST-KO (− /− ) mice (+ /− ): heterozygote GAPDH was used as a control (e,f) Concentrations of H2S3 (e) and H2S (f) in whole cells prepared from wild-type (open bar) and 3MST-KO-mice (filled bar) Suspensions of brain cells were exposed to 500 μ M MP (distilled water for a control) for 15 min (the intracellular MP concentration reached 0.40 ±  0.09 μ mol/g protein), and the concentrations of H2S3 (e) and H2S (f) were measured as monobromobimane adducts from cell lysates ** and ##p