Activation process of the mosquitocidal delta-endotoxin Cry39A produced by Bacillus thuringiensis subsp. aizawai BUN1-14 and binding property to Anopheles stephensi BBMV.
Most delta-endotoxins produced by Bacillus thuringiensis require proteolytic processing in order to become active. The in vitro and in vivo activation processes of Cry39A, a delta-endotoxin that is highly toxic to Anopheles stephensi, were investigated. Cry39A with a molecular mass of 72 kDa was processed in vitro into a 60 kDa fragment by trypsin and gut extract from A. stephensi larvae. N-terminal amino acid sequencing of the 60 kDa fragment revealed that trypsin and the protease(s) in the gut extract cleaved Cry39A between Arg(61) and Gly(62). In contrast, 40 and 25 kDa polypeptides were generated in vivo by intramolecular cleavage of the 60 kDa fragment in A. stephensi larvae. Further, a co-precipitation assay was used to investigate the binding property of the activated Cry39A to A. stephensi BBMV. Cry39A bound to A. stephensi BBMV specifically and did not compete with the Cry4Aa toxin. This indicated that the binding molecule(s) for Cry39A might differ from those for Cry4A. In addition, Cry39A preferentially bound to the Triton X-100-insoluble membrane fraction.
Ito
Takeshi
T
Department of Applied Bioscience, Graduate School of Agriculture, Hokkaido University, N9 W9, Sapporo 060-8589, Japan. ito-t@abs.agr.hokudai.ac.jp <ito-t@abs.agr.hokudai.ac.jp>
Bando
Hisanori
H
Asano
Shin-Ichiro
S
D000818:Animals; D000852:Anopheles / Q000187:drug effects* / Q000254:growth & development; D001413:Bacillus thuringiensis / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D004731:Endotoxins / Q000096:biosynthesis* / Q000235:genetics / Q000633:toxicity; D066298:In Vitro Techniques; D008871:Microvilli / Q000378:metabolism; D010572:Pest Control, Biological; D011485:Protein Binding; D022161:Transport Vesicles / Q000378:metabolism*
D004731:Endotoxins