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Characterization of the role of protein–cysteine residues in the binding with sodium arsenite

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Abstract

To better characterize the interaction of protein–cysteines with sodium arsenite, arsenic-binding proteins were identified from the arsenic-resistant Chinese hamster ovary cell line SA7 using a p-aminophenylarsine oxide (PAO)-agarose matrix in combination with proteomic techniques. Twenty of the isolated arsenic-binding proteins were further peptide-mapped by MALDI-Q-TOF-MS. The binding capacity of PAO-agarose-retained proteins was then verified by re-applying Escherichia coli overexpressed recombinant proteins with various numbers of cysteine residues onto the PAO-agarose matrix. The results showed that recombinant heat shock protein 27 (HSP27, with one cysteine residue), reticulocalbin-3 (RCN3, with no cysteine residue), galectin-1 (GAL1, with six cysteine residues), but not peroxiredoxin 6 (Prdx6, with one cysteine residue but not retained by the PAO-agarose matrix), were bound to the PAO-agarose matrix. The six free cysteine residues in GAL1 were individually or double-mutated to alanine by means of site-directed mutagenesis and subjected to CD and ICP-MS analysis. The binding capacity of GAL1 for sodium arsenite was significantly attenuated in C16A, C88A and all double mutant clones. Taken together, our current data suggest that the cysteine residues in GAL1 may play a critical role in the binding of arsenic, but that in the case of RCN3 and Prdx6, this interaction may be mediated by other factors.

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Abbreviations

PAO:

p-Aminophenylarsine oxide

RCN3:

Reticulocalbin-3

GAL1:

Galectin-1

Prdx6:

Peroxiredoxin 6

CD:

Circular dichroism

ICP-MS:

Inductively coupled plasma mass spectrometry

2-DE:

Two-dimensional polyacrylamide gel electrophoresis

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Acknowledgments

We gratefully acknowledge the financial support provided by the National Science Council of Taiwan, Republic of China (NSC 97-2321-B-002-013-MY3). Proteomic mass spectrometry analyses were performed by the Core Facilities for Proteomics and Glycomics located at the Institute of Biological Chemistry, Academia Sinica, and were supported by a National Science Council grant (NSC 98-3112-P-001-023) and the Academia Sinica.

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Authors and Affiliations

  1. Department of Life Science, National Central University, No. 300 Jhongda Road, Taoyuan, 320, Taiwan

    Yu-Ying Chang & Yung-Hsi Kao

  2. Department of Agricultural Chemistry, National Taiwan University, Taipei, 106, Taiwan

    Chun-Hua Hsu

  3. Department of Biochemistry, Taipei Medical University, Taipei, 106, Taiwan

    Tai-Chih Kuo

  4. Department of Chemistry, National Central University, Taoyuan, 320, Taiwan

    Duen-Ren Hou

  5. Department of Entomology and Research Center for Plant Medicine, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, 106, Taiwan

    Rong-Nan Huang

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  1. Yu-Ying Chang
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Correspondence to Yung-Hsi Kao or Rong-Nan Huang.

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Chang, YY., Kuo, TC., Hsu, CH. et al. Characterization of the role of protein–cysteine residues in the binding with sodium arsenite. Arch Toxicol 86, 911–922 (2012). https://doi.org/10.1007/s00204-012-0828-0

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  • DOI: https://doi.org/10.1007/s00204-012-0828-0

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