title: Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis.
abstract: Vip3 proteins are secretable proteins fromBacillus thuringiensiswhose 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 withAgrotis ipsilonmidgut 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.
authors.affiliation: ERI BIOTECMED and Department of Genetics, Universitat de València, Dr. Moliner, 50, BURJASSOT, 46100 Valencia, Spain. yolanda.bel@uv.es.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Bel
authors.affiliation: ERI BIOTECMED and Department of Genetics, Universitat de València, Dr. Moliner, 50, BURJASSOT, 46100 Valencia, Spain. nuria.banyuls@uv.es.
authors.forename: Núria
authors.initials: N
authors.lastname: Banyuls
authors.affiliation: ERI BIOTECMED and Department of Genetics, Universitat de València, Dr. Moliner, 50, BURJASSOT, 46100 Valencia, Spain. chakrounmaissa7@gmail.com.
authors.forename: Maissa
authors.initials: M
authors.lastname: Chakroun
authors.affiliation: ERI BIOTECMED and Department of Genetics, Universitat de València, Dr. Moliner, 50, BURJASSOT, 46100 Valencia, Spain. baltasar.escriche@uv.es.
authors.forename: Baltasar
authors.initials: B
authors.lastname: Escriche
authors.affiliation: ERI BIOTECMED and Department of Genetics, Universitat de València, Dr. Moliner, 50, BURJASSOT, 46100 Valencia, Spain. juan.ferre@uv.es.
authors.forename: Juan
authors.initials: J
authors.lastname: Ferré
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism / Q000633:toxicity; D007419:Intestinal Secretions / Q000378:metabolism; D007814:Larva / Q000187:drug effects; D007915:Lepidoptera / Q000187:drug effects; D010455:Peptides / Q000378:metabolism; D017433:Protein Structure, Secondary; D059748:Proteolysis; D014357:Trypsin / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D010455:Peptides; C447139:Vip3A protein, Bacillus thuringiensis; D014357:Trypsin
keywords: Bacillus thuringiensis; SDS-PAGE artefact; Vip proteins; bacterial secreted proteins; protease stability; proteolytic activation; toxin activation; trypsin inhibitors