家蚕氨肽酶 (BmAPN5) 与黑胸败血芽孢杆菌伴孢晶体 (PC) 毒素相互作用

Interaction of aminopeptidase (BmAPN5) and parasporal crystal (PC) toxin isolated from Bacillus bombysepticus

摘要:

氨肽酶N (APN) 属于锌金属肽酶M1 (Peptidase_M1) 家族的成员，不仅参与蛋白水解过程，而且也作为毒素受体参与病原微生物的致病过程。家蚕氨肽酶家族含有16个成员，其中BmAPN4结合黑胸败血芽孢杆菌产生的伴孢晶体 (PC) 毒素，为研究该基因家族其他成员是否与PC毒素结合，参与其致病过程。本文克隆家蚕中肠特异表达的氨肽酶家族成员BmAPN5基因，全长3 313 bp，编码953个氨基酸，含有1个锌金属肽酶M1和ERAP1_C结构域。构建原核表达载体，表达和纯化获得可溶性GST-BmAPN5重组蛋白。Far-Western blotting、免疫共沉淀和ELISA等实验结果表明BmAPN5和活化的PC毒素相互结合。通过构建BmAPN5细胞转染载体，转染Sf9细胞系，与PC毒素共孵育，导致细胞形态改变和裂解死亡；同时，乳酸脱氢酶含量测定结果 (LDH) 表明BmAPN5参与PC毒素致病过程，导致细胞裂解死亡，使细胞培养基中的乳酸脱氢酶升高。上述结果表明BmAPN5作为一种功能性受体，PC毒素与其相互作用，参与了病原物的致病过程，为进一步揭示病原微生物黑胸败血芽孢杆菌与宿主相互作用的致病机制研究奠定了基础。

Abstract:

Aminopeptidase N (APN) belonging to zinc-dependent metalloproteinase, not only catalyzes protein proteolytic process, but also is involved in the pathogenic process as the receptor of pathogenic toxin. In Bombyx mori, APN gene family consists of 16 members, of which BmAPN4 binds trypsin-activated parasporal crystal (PC) toxin isolated from Bacillus bombysepticus (Bb). In order to verify whether or not other APNs interact with PC toxin during the pathogenesis of Bb, we cloned BmAPN5, a member of aminopeptidase family, from the silkworm midgut. The full length of BmAPN5 is 3313 bp, encoding 953 amino acids, containing a zinc peptidase_M1 and ERAP1_C domains. A recombinant GST-BmAPN5 was purified by a prokaryotic expression system. Far-Western blotting, co-immunoprecipitation and ELISA. Binding saturation assays demonstrated that PC after activated by trypsin could be bound by BmAPN5. Additionally, cytotoxic activity of trypsin-activated PC in Sf9 cells transfected with BmAPN5 showed that cells exhibited dramatic cytological changes, including swelling and lysis, revealing BmAPN5 serves as a functional receptor that participates in Bb and PC pathogenicity. These provide some clues for further exploring the pathogenesis relationships of Bb and host.