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Updated: Feb 22, 2026
Recombinant Protein Expression, Crystallization, and Biophysical Studies of a Bacillus-conserved Nucleotide Pyrophosphorylase, BcMazG
Published on: May 16, 2017
[Interaction of aminopeptidase (BmAPN5) and parasporal crystal (PC) toxin isolated from Bacillus bombysepticus]
Jianfeng Fu1, Ping Lin1, Tieshan Feng1
1State Key Laboratory of Silkworm Genome Biology, Southwest University, Chongqing 400715, China.
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.
Insights
Aminopeptidase N (APN) BmAPN5 from silkworms binds Bacillus bombysepticus (Bb) toxins. This interaction reveals BmAPN5 acts as a functional receptor, contributing to Bb pathogenicity and host damage.
Area of Science:
- Insect biochemistry
- Molecular biology
- Pathogen-host interactions
Background:
- Aminopeptidase N (APN) enzymes are zinc metalloproteinases involved in protein processing and pathogen toxin reception.
- In Bombyx mori (silkworms), 16 APN members exist, with BmAPN4 known to bind Bacillus bombysepticus (Bb) parasporal crystal (PC) toxin.
Purpose of the Study:
- To investigate if other APN family members in Bombyx mori interact with Bb PC toxin.
- To characterize the role of BmAPN5 in the pathogenicity of Bacillus bombysepticus.
Main Methods:
- Cloning and sequencing of the BmAPN5 gene from silkworm midgut.
- Purification of recombinant GST-BmAPN5 using a prokaryotic expression system.
- Experimental validation using Far-Western blotting, co-immunoprecipitation, ELISA, binding saturation assays, and cytotoxicity assays in Sf9 cells.
Main Results:
- BmAPN5 was cloned, encoding 953 amino acids with zinc peptidase_M1 and ERAP1_C domains.
- Trypsin-activated PC toxin binds to BmAPN5, confirmed by binding saturation assays.
- Sf9 cells expressing BmAPN5 showed significant swelling and lysis upon exposure to activated PC toxin, indicating BmAPN5's role as a functional receptor.
Conclusions:
- BmAPN5 functions as a receptor for trypsin-activated Bb PC toxin.
- BmAPN5 plays a role in the pathogenicity of Bacillus bombysepticus in Bombyx mori.
- These findings offer insights into the molecular mechanisms of Bb pathogenicity and host interactions.
