Identification of galectin I and thioredoxin peroxidase II as two arsenic-binding proteins in Chinese hamster ovary cells.
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 20 mM 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).
Chang
Kwang Ning
KN
Department of Life Science, National Central University, Chung-Li, Taoyuan, Taiwan 32054, Republic of China.
Lee
Te Chang
TC
Tam
Ming F
MF
Chen
Yi Chin
YC
Lee
Li Wen
LW
Lee
Shin Ying
SY
Lin
Pei Jung
PJ
Huang
Rong Nan
RN
D000595:Amino Acid Sequence; D000818:Animals; D001151:Arsenic / Q000378:metabolism*; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D037483:Galectin 1 / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D051379:Mice; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins; D010446:Peptide Fragments / Q000737:chemistry; D010544:Peroxidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D061105:Peroxiredoxin III; D054464:Peroxiredoxins; D051381:Rats; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D002352:Carrier Proteins; D017931:DNA Primers; D018076:DNA, Complementary; D037483:Galectin 1; D009363:Neoplasm Proteins; D010446:Peptide Fragments; C516116:Prdx3 protein, mouse; D010544:Peroxidases; C516115:PRDX3 protein, human; D061105:Peroxiredoxin III; D054464:Peroxiredoxins; D001151:Arsenic