Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.
Molecular cloning, expression and characterization of acylpeptide hydrolase in the silkworm, Bombyx mori.
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DOI：
10.1016/j.gene.2015.12.069
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发表时间：
2016-04
期刊：
影响因子：
3.5
通讯作者：
Ping Fu;Wei Sun;Ze Zhang
中科院分区：
文献类型：
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作者：
Ping Fu;Wei Sun;Ze Zhang
酰基肽水解酶（APH）可以催化乙酰化肽N端氨基酸的释放。在各种原核生物和真核生物中有许多这种酶的记录实例。然而，关于APH在昆虫中的知识仍然是未知的。本研究克隆了家蚕APH基因（BmAPH），并进行了序列测定。BmAPH基因编码710个氨基酸的蛋白质，预测分子量为78.5 kDa。推定的BmAPH和哺乳动物APH共享约36%的氨基酸序列同一性，但关键的催化残基是保守的（Ser 566，Asp 654和His 686）。重组BmAPH在大肠杆菌中的表达和纯化表明，该重组蛋白对传统底物Ac-Ala-pNA具有酰基肽水解酶活性。有机磷杀虫剂毒死蜱、辛硫磷和马拉硫磷对APH活性有明显的抑制作用。此外，BmAPH在家蚕各组织和发育阶段均有表达。免疫组化结果显示BmAPH蛋白定位于基底膜。这些结果表明，BmAPH可能参与提高家蚕对OP杀虫剂的耐受性。总之，本研究结果为了解APH在昆虫体内的生物学功能提供了依据。
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 silkwormBombyx moriAPH (BmAPH) gene. TheBmAPHgene 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 inEscherichia colishowed 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 bothin vitroandin vivo. In addition,BmAPHwas 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.