In this study, we report the identification of two arsenic-binding proteins from Chinese hamster ovary (CHO) cells. The crude extract derived from CHO and SA7 (arsenic-resistant CHO cells) was applied to a phenylarsine oxide–agarose affinity column, and after extensive washing, the absorbed proteins were eluted with buffers containing 20mM 2-mercaptoethanol (2-ME) or dithiothreitol (DTT). Three differentially expressed proteins, galectin 1 (Gal-1; in the 2-ME-eluted fraction from CHO cells), glutathione S-transferase P-form (GST-P) and thioredoxin peroxidase II (TPX-II), respectively in the 2-ME- and DTT-eluted fractions from SA7 cells, were identified by partial amino acid sequence analysis after separation by SDS/PAGE. The GST-P protein has been previously shown to facilitate the excretion of sodium arsenite [As(III)] from SA7 cells. TPX II was detected predominately in SA7 cells [routinely cultured in As(III)-containing medium], but not in CHO or SA7N (a revertant of SA7 cells cultured in regular medium) cells. In contrast, Gal-1 was specifically identified in CHO and SA7N cells, but not in SA7 cells. The preferential expression of Gal-1 in CHO cells and TPX-II in SA7 cells was further illustrated by quantitative PCR analysis. The binding of Gal-1 and TPX-II with As(III) was further verified by both co-immunoprecipitation and co-elution of Gal-1 and TPX-II with As(III). It is suggested that Gal-1 and TPX-II are two proteins that serve as high-affinity binding sites for As(III) and thus both may be involved in the biological action of As(III).
Abbreviations used: CHO, Chinese hamster ovary; DMAIII, trivalent dimethylarsenic species; DTT, dithiothreitol; Gal-1, galectin-1; GST-P, glutathione S-transferase P-form; IPTG, isopropyl β-d-thiogalactoside; 2-ME, 2-mercaptoethanol; MMAIII, trivalent monomethylarsonous acid; Ni-NTA, Ni2+-nitrilotriacetate; PAO-agarose, phenylarsine oxide–agarose; RT-PCR, reverse transcriptase PCR; SRB, sulphorhodamine B; TPX-II, thioredoxin peroxidase II.
The nucleotide sequences reported here have been submitted to the GenBankTM/EMBL Nucleotide Sequence Databases under the accession number AF221841.