title: Characterization of the role of protein-cysteine residues in the binding with sodium arsenite.
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.
authors.affiliation: Department of Life Science, National Central University, No. 300 Jhongda Road, Taoyuan 320, Taiwan.
authors.forename: Yu-Ying
authors.initials: YY
authors.lastname: Chang
authors.forename: Tai-Chih
authors.initials: TC
authors.lastname: Kuo
authors.forename: Chun-Hua
authors.initials: CH
authors.lastname: Hsu
authors.forename: Duen-Ren
authors.initials: DR
authors.lastname: Hou
authors.forename: Yung-Hsi
authors.initials: YH
authors.lastname: Kao
authors.forename: Rong-Nan
authors.initials: RN
authors.lastname: Huang
mesh_terms: D000818:Animals; D001151:Arsenic / Q000378:metabolism / Q000633:toxicity*; D018053:Arsenites / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002352:Carrier Proteins / Q000378:metabolism*; D002460:Cell Line / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000378:metabolism*; D005260:Female; D006360:Heat-Shock Proteins / Q000187:drug effects / Q000378:metabolism; D010053:Ovary / Q000166:cytology / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D017670:Sodium Compounds / Q000378:metabolism*
chemical_list: D018053:Arsenites; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D006360:Heat-Shock Proteins; D017670:Sodium Compounds; C078865:reticulocalbin; C017947:sodium arsenite; D003545:Cysteine; D001151:Arsenic