Cloning of catalase and expression patterns of catalase and selenium-dependent glutathione peroxidase from Exopalaemon carinicauda in response to low salinity stress

Catalase （CAT） and selenium-dependent glutathione peroxidase （Se-GPx） play a vital role in protecting organisms against various oxidative stresses by eliminating H202, The objective of this paper is to evaluate the roles of these antioxidant molecules in the ridgetail white prawn Exopalaemon carinicauda in response to low salinity stress. A complementary DNA （cDNA） containing the complete coding sequence of CAT was cloned from the hepatopancreas using reverse-transcription polymerase chain reaction （RT-PCR） and rapid amplification of cDNA ends. The full-length cDNA of CAT （2 649 bp） contains a 5＇-untranslated region （UTR） of 78 bp, a 3＇- UTR of 1 017 bp, with a poly （A） tail, and an open reading frame of 1 554 bp encoding a 517-amino-acid polypeptide with predicted molecular mass of 58.46 kDa and estimated isoelectric point of 6.64. This CAT sequence contained the proximal active site signature （60FDRERIPERWHAKGAG76）, proximal heme-ligand signature sequence （350RLFSYPDTH358） and three catalytic amino acid residues （His71, Asn144 and Tyr354）. Sequence comparison showed that the CAT deduced amino acid sequence of E. carinicauda shared 68%-92% of identities with those of other species. Quantitative real-time PCR analysis revealed that CAT mRNA was widely expressed in the hepatopancreas （highest）, hemocyte, eyestalk, heart, gill, muscle, ovary and stomach. Under low salinity stress, CAT and GPx mRNA expression levels both in the gill and hepatopancreas increased significantly at the first 48 h and 6 h respectively, indicating a tissue- and time-dependent antioxidant response in E. carinicauda. All these results indicate that E. carinicauda CAT is a member of the CAT family and might be involved in the acute response against low salinity stress.

Selenium Regulation of Selenium-dependent Glutathione Peroxidases in Animals and Transfected CHO Cells

Glutathione peroxidase (GPX1) was the first identified selenium-dependent enzyme, and this enzyme has been most useful as a biochemical indicator of selenium (Se) status and the parameter of choice for determining Se requirements. We have continued to study Se regulation of GPX1 to better understand the underlying mechanism and to gain insight into how cells themselves regulate nutrient status. In progressive Se deficiency in rats, GPX1 activity,protein and mRNA all decrease in a dramatic, coordinated and exponential fashion such that Se-deficient GPX1 mRNA levels are 6-15% of Sexadequate levels. mRNA levels for other Sedependent proteins are far less decreased in the same animals. The mRNA levels for a second Se-dependent peroxidase, phospholipid hydroperoxide glutathione peroxidase (GPX4 ), are little affected by Se deficiency, demonstrating that Se regulation of GPX1 is unique. Se regulation of GPX1 activity in growing male and female rats shows that the Se requirernent is 100 ng/g diet, based on liver GPX1 activity; use of GPX1 mRNA as the parameter indicates that the Se requirement is nearer to 50 ng Se/g diet in both male and female rats. This approach will readily detect an altered dietary Se requirement, as shown by the incremental increases in dietary Se requirement by 150, 100 or 50 ng Se/g diet in Seudeficient rat pups repleted with Se for 3, 7 or 14 d, respectively. Studies with CHO cells stably transfected with recombinant GPX1 also show that overexpression of GPX1 does not alter the minimum level of media Se necessary for Se-adequate levels of GPX1 activity or mRNA. We hypothesize that classical GPX1 has an integral biological role in the mechanism used by cells to regulate Se status,making GPX1 an especially useful and effective parameter for determining Se requirements in animals

A T-Cell Model for the Biological Role of Selenium-Dependent Glutathione Peroxidase

The cytosolic form of selenium-dependent glutathione peroxidase detoxifies both hydrogen and lipid peroxides and therefore represents a major component of the cellular anti-oxidant defenses. In order to study the biological role of this enzyme, we generated an expression construct in a retroviral vector, which when introduced into immortalized human T-cells, resulted in significant increases in the activity of this important enzyme. This effect is stable over extended maintenance in culture. The anti-oxidant defenses in these same cells are also shown to be attenuated hy chemically reducing cellular glutathione levels. Collectively, the abllity to both increase and decrease the anti-oxidant defenses in human T cells results in a useful model system for the study of oxidative stress and signaling in this cell type