Identification and cloning of a selenium dependent glutathione peroxidase from giant freshwater prawn, Macrobrachium rosenbergii.
A selenium dependent glutathione peroxidase (Se-GPx) cDNA was cloned from haemocyte by a reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA (RACE). The 913 bp cDNA contained an open reading frame (ORF) of 558 bp encoded a deduced amino acid sequence of 186 amino acids. The prawn Se-GPx sequence contains a selenocysteine (Sec) residue which is encoded by the unusual stop codon, (115)TGA(117). According to the molecular modeling analysis, the active site Sec residue, located in the loop between beta3 and alpha2 in a pocket on the protein surface, and hydrogen bonded to Gln(73) and Trp(141). A GPx signature motif 2, (63)LAFPCNQF(70) and active site motif, (151)WNFEKF(156), two arginine (R) residues, R(89) and R(167) contribute to the electrostatic architecture that directs the glutathione donor substrate, and two putative N-glycosylation site, (75)NNT(77) and (107)NGS(109) were observed in the prawn Se-GPx sequence. In addition, the eukaryotic selenocysteine insertion sequence element is conserved in the 3'-UTR. Comparison of amino acid sequences showed that prawn Se-GPx is more closely related to vertebrate GPx 1. The prawn Se-GPx was synthesized in haemocyte, hepatopancreas, muscle, stomach, gill, intestine, eyestalk, heart, epidermis, lymph organ, ventral nerve cord, testis and ovary. The increase of respiratory burst in haemocyte was observed in pathogen, Debaryomyces hansenii-injected prawn in order to kill the pathogen, and the up-regulation in SOD and GPx acitivity, and prawn Se-GPx mRNA transcription were involved with the protection against damage from oxidation.
Yeh
Shinn-Pyng
SP
Department of Aquaculture, National Pingtung University of Science and Technology, Pingtung 91201, Taiwan, ROC.
Liu
Kuan-Fu
KF
Chiu
Shieh-Tsung
ST
Jian
Shun-Ji
SJ
Cheng
Winton
W
Liu
Chun-Hung
CH
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D055165:Debaryomyces / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D005979:Glutathione Peroxidase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D033541:Palaemonidae / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology / Q000382:microbiology; D010802:Phylogeny; D017434:Protein Structure, Tertiary; D012643:Selenium / Q000378:metabolism*; D016415:Sequence Alignment
D018076:DNA, Complementary; D005979:Glutathione Peroxidase; D012643:Selenium