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S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site

  • Molecular Toxicology
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Abstract

We previously developed a screening method to identify proteins that undergo aggregation through S-mercuration by methylmercury (MeHg) and found that rat arginase I is a target protein for MeHg (Kanda et al. in Arch Toxicol 82:803–808, 2008). In the present study, we characterized another S-mercurated protein from a rat hepatic preparation that has a subunit mass of 42 kDa, thereby facilitating its aggregation. Two-dimensional SDS–polyacrylamide gel electrophoresis and subsequent peptide mass fingerprinting using matrix-assisted laser desorption and ionization time-of-flight mass spectrometry revealed that the 42 kDa protein was NAD-dependent sorbitol dehydrogenase (SDH). With recombinant rat SDH, we found that MeHg is covalently bound to SDH through Cys44, Cys119, Cys129 and Cys164, resulting in the inhibition of its catalytic activity, release of zinc ions and facilitates protein aggregation. Mutation analysis indicated that Cys44, which ligates the active site zinc atom, and Cys129 play a crucial role in the MeHg-mediated aggregation of SDH. Pretreatment with the cofactor NAD, but not NADP or FAD, markedly prevented aggregation of SDH. Such a protective effect of NAD on the aggregation of SDH caused by MeHg is discussed.

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Notes

  1. The C164S mutant failed to refold from the insoluble inclusion bodies expressed in E. coli.

Abbreviations

MeHg:

Methylmercury

API:

Achromobacter protease I

SDH:

Sorbitol dehydrogenase

GSH:

Glutathione

MALDI-TOF/MS:

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

NEM:

N-ethylmaleimide

2-ME:

2-Mercaptoethanol

MnCl2 :

Manganese chloride

PVDF:

Polyvinylidene difluoride

BPM:

Biotin-PEAC5-maleimide

CBB:

Coomassie brilliant blue

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Acknowledgments

We thank Dr. Daigo Sumi (Faculty of Pharmaceutical Sciences, Tokushima Bunri University) and Dr. Akira Yasutake (National Institute for Minamata Disease) for helpful advice; Dr. Nobuhiro Shimojo (University of Tsukuba) for his encouragement; Drs. Tomomi Gotoh and Masataka Mori (Department of Molecular Genetics, Graduate School of Medical Sciences, Kumamoto University) for donating the antibody against arginase I. This work was supported by a grant-in-aid (23117703 to Y.K. for scientific research) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Akihiro Iwamatsu

    Present address: Laboratory for Vaccine Design, RIKEN Research Center for Allergy and Immunology, Yokohama, Kanagawa, 230-0045, Japan

Authors and Affiliations

  1. Doctoral Program in Biomedical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan

    Hironori Kanda, Takashi Toyama, Yasuhiro Shinkai, Makoto Kikushima & Yoshito Kumagai

  2. Central Laboratories for Key Technology, Kirin Brewery Co., Ltd., 1-13-5 Fukuura, Kanazawa-ku, Yokohama, Kanagawa, 236-0004, Japan

    Azusa Shinohara-Kanda & Akihiro Iwamatsu

  3. Department of Environmental Health, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Noda, Chiba, 278-8510, Japan

    Toshiyuki Kaji

  4. Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, 305-8575, Japan

    Yoshito Kumagai

  5. Protein Research Network, Inc., Yokohama, Kanagawa, 236-0004, Japan

    Akihiro Iwamatsu

Authors
  1. Hironori Kanda
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Correspondence to Yoshito Kumagai.

Additional information

Hironori Kanda and Takashi Toyama contributed equally to this work.

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Kanda, H., Toyama, T., Shinohara-Kanda, A. et al. S-Mercuration of rat sorbitol dehydrogenase by methylmercury causes its aggregation and the release of the zinc ion from the active site. Arch Toxicol 86, 1693–1702 (2012). https://doi.org/10.1007/s00204-012-0893-4

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