Molecular cloning and expression of chicken neuropathy target esterase activity domain.
Neuropathy target esterase (NTE) is recognized as the initial target during the process of organophosphate-induced delayed neuropathy (OPIDN). Adult hens are usually used as the animal model for experimental studies of OPIDN. However, the molecular cloning and characteristics of chicken NTE is unknown. On the basis of the predicted chicken NTE gene middle sequence and its 3'-end cDNA sequence, we cloned the gene sequence of chicken NTE activity domain (cNEST). The cloned cNEST gene is 1740 base pairs and encodes 579 amino acids, showing high identity with human and mouse NEST at amino acid level. The serine hydrolase signature motif GXSXG and the patatin domain were found in the cNEST sequence. Over-expression of cNEST tagged with enhanced green fluorescence protein (EGFP) in monkey kidney COS7 cells increased NTE activity significantly. The increased extent is similar to that in over-expression of hNEST cells. Moreover, over-expression of cNEST led to an accumulation of partial cNEST on the cytoplasmic surface of the endoplasmic reticulum. Partial cNEST located in the cytoplasm by comparing the distribution of cNEST and hNEST. After inhibition with different concentrations of mipafox for 60min, the calculated I(50) value was 4.95microM for COS7 cells over-expressing cNEST. These results firstly confirmed that the protein sequence, enzymatic activity, and cellular location of cNEST are very similar to that of hNEST at molecular level. The inhibition curve of mipafox on NTE activity of cNEST in mammalian cells was also reported here.
Chang
Ping-An
PA
Key Laboratory of Molecular Biology, College of Bio-information, Chongqing University of Posts and Telecommunications, Chongqing 400065, PR China.
Long
Ding-Xin
DX
Wu
Yi-Jun
YJ
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D002265:Carboxylic Ester Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D002645:Chickens / Q000235:genetics*; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005260:Female; D006801:Humans; D007531:Isoflurophate / Q000031:analogs & derivatives / Q000494:pharmacology; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary / Q000235:genetics; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D014162:Transfection
D018076:DNA, Complementary; D007531:Isoflurophate; C005238:mipafox; D002265:Carboxylic Ester Hydrolases; C021859:neurotoxic esterase