Characterization And Expression Regulation Of Cathepsin E-A-like Gene In Chicken (Gallus Gallus)

Aspartic proteinases are a group of proteolytic enzymes that characterised by two structurally similar aspartate residues localized in the active site,and are generally activated under acidic p H range to accomplish their catalytic function,which participate in many physiological processes.The cathepsin E-A-like is a new member of the aspartic protease family,also known as “similar to nothepsin” in chickens due to its amino acid sequence and protein structure are very similar to nothepsin in zebra fish.Though our laboratory and other previous studies have shown that the expression of cathepsin E-A-like is significantly up-regulation at the laying stage,the function role and regulation mechanism of the gene are not yet clear.In this study,the coding sequence of cathepsin E-A-like gene was cloned and analyzed by bioinformatics first.The expression levels of the gene in different tissues and different development stages were investigated by q PCR then.The expression regulation mechanism of the gene was further explored by in vitro and in vivoThe main results are as followings:1.According to the predicted Gallus gallus cathepsin E-A-like sequence published in NCBI,specific PCR primers were designed.The CDs of the gene was cloned by using template of liver c DNA which was extracted from 30 weeks old chickens.The result show that the full-length CDs of the cathepsin E-A-like(Genbank:KR559732)was 1215 bp,encodes a protein which is composed of 404 amino acids in2.The comparative sequence analysis of cathepsin E-A-like was couducted by using BLAST and Clustal X2 tools.The results revealed that cathepsin E-A-like shared 45% and 49% similarity to chicken cathepsin D and cathepsin E at the amino acid level,respectively.They all showed two highly conserved aspartic protease active sites.Analysis using SMART and Prosite tools revealed that cathepsin E-A-like has a typical structure of aspartic protease,including signal peptide,propeptide and function structure domain.methodschicken.3.Phylogenetic and Syntenic analysis indicated that cathepsin E-A-like gene and nothepsin gene are orthologous.4.The cathepsin E-A-like gene was found to be exclusively expressed in the liver of laying hens after the expression level of this gene in different tissues and different development stages were analyzed by q PCR.It is speculated that the expression of cathepsin E-A-like gene might be regulated by sex estrogen.5.To assess whether the expressions of cathepsin E-A-like was regulated by estrogen,10 weeks old pullets and embryonic primary hepatocytes were treated with different doses of 17β-estradiol.It turned out that the expression of cathepsin E-A-like significantly up-regulated after treatment with17β-estradiol.6.To further identify which ER subtype of estrogen mediates the expression of cathepsin E-A-like gene,embryonic primary hepatocytes were treated with17β-estradiol combined with different ER subtype antagonists,such as MPP、ICI182,780 and tamoxifen.The expression of cathepsin E-A-like was not altered compared with control group,but was significantly decreased(P< 0.05)when the hepatocytes were treated with 17β-estradiol combined with tamoxifen or ICI 182,780 compared with 17β-estradiol alone.Because MPP acts as a highly selective antagonist to ERα,whereas tamoxifen and ICI 182,780 antagonize nuclear ERs,our results indicate that the estrogens stimulated expression of chicken hepatic cathepsin E-A-like is mediated via ER-β.In conclusion,our results suggest that cathepsin E-A-like gene and nothepsin gene which was found in fish and other lower vertebrates are orthologous genes.Furthermore,cathepsin E-A-like is exclusively expressed in liver of the laying hens,and its expressions is regulated by sex estrogen predominantly mediated by ERβ.