Abstract
Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa) was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w). If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice) equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.
Highlights
Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive sporulating bacterium that produces several entomopathogenic proteins
Whereas the concentration of the smaller bands decreased with the incubation time, the 66 kDa band seemed to become more intense as the incubation proceeded
The accumulation with time of the 66 kDa band became even more evident, and at the same time the smaller bands eventually disappeared (Figure 1b,c). This phenomenon was observed when trypsin digestion was performed at a lower temperature (4 ◦ C), as well as when the Tris buffer was substituted by carbonate buffer
Summary
Bacillus thuringiensis (Bt) is a ubiquitous Gram-positive sporulating bacterium that produces several entomopathogenic proteins. Other proteins associated with insecticidal activity, including the Vip proteins, are secreted into the medium during the vegetative growth phase [1]. Vip proteins (such as Vip3A proteins) show high insecticidal activity against a wide range of insect species The proposed mode of action of Vip3A proteins shares some similarities with that of the Cry proteins, in that both undergo activation (proteolytic processing) in the insect midgut, bind to receptors on the surface of the midgut cells, and, make pores that lead to cell lysis, septicemia, and eventually death of the insect [3,4,5,6,7].
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