کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
2815141 | 1159853 | 2016 | 9 صفحه PDF | دانلود رایگان |
• A putative silkworm Bombyx mori APH gene was cloned and sequenced in insects.
• Heterologous expression of BmAPH gene in E. coli showed that BmAPH has acylpeptide hydrolase activity toward the traditional substrate, Ac–Ala–pNA.
• The enzyme activity of BmAPH was inhibited by the OP compounds in vitro and in vivo, suggesting that it may be involved in OP insecticides resistance of silkworm.
• Immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes in head and midgut.
Acylpeptide hydrolase (APH) can catalyze the release of the N-terminal amino acid from acetylated peptides. There were many documented examples of this enzyme in various prokaryotic and eukaryotic organisms. However, knowledge about APH in insects still remains unknown. In this study, we cloned and sequenced a putative silkworm Bombyx mori APH (BmAPH) gene. The BmAPH gene encodes a protein of 710 amino acids with a predicted molecular mass of 78.5 kDa. The putative BmAPH and mammal APHs share about 36% amino acid sequence identity, yet key catalytic residues are conserved (Ser566, Asp654, and His686). Expression and purification of the recombinant BmAPH in Escherichia coli showed that it has acylpeptide hydrolase activity toward the traditional substrate, Ac–Ala–pNA. Furthermore, organophosphorus (OP) insecticides, chlorpyrifos, phoxim, and malathion, significantly inhibited the activity of the APH both in vitro and in vivo. In addition, BmAPH was expressed in all tested tissues and developmental stages of the silkworm. Finally, immunohistochemistry analysis showed that BmAPH protein was localized in the basement membranes. These results suggested that BmAPH may be involved in enhancing silkworm tolerance to the OP insecticides. In a word, our results provide evidence for understanding of the biological function of APH in insects.
Journal: Gene - Volume 580, Issue 1, 10 April 2016, Pages 8–16