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IDENTIFICATION OF A CALMODULIN‐BINDING SITE WITHIN THE DOMAIN I OF B ACILLUS THURINGIENSIS C ry3 A a TOXIN
Author(s) -
OchoaCampuzano Camila,
Sánchez Jorge,
GarcíaRobles Inmaculada,
Real M. Dolores,
Rausell Carolina,
Sánchez Jorge
Publication year - 2012
Publication title -
archives of insect biochemistry and physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.576
H-Index - 66
eISSN - 1520-6327
pISSN - 0739-4462
DOI - 10.1002/arch.21044
Subject(s) - toxin , proteolysis , calmodulin , biology , biochemistry , cleavage (geology) , trifluoperazine , midgut , microbiology and biotechnology , peptide , enzyme , paleontology , botany , fracture (geology) , larva
B acillus thuringiensis C ry3 A a toxin is a coleopteran specific toxin highly active against C olorado P otato B eetle ( CPB). We have recently shown that C ry3 A a toxin is proteolytically cleaved by CPB midgut membrane associated metalloproteases and that this cleavage is inhibited by ADAM metalloprotease inhibitors. In the present study, we investigated whether the C ry3 A a toxin is a calmodulin ( C a M ) binding protein, as it is the case of several different ADAM shedding substrates. In pull‐down assays using agarose beads conjugated with C a M , we demonstrated that C ry3 A a toxin specifically binds to C a M in a calcium‐independent manner. Furthermore, we used gel shift assays and 1 H NMR spectra to demonstrate that C a M binds to a 16‐amino acid synthetic peptide corresponding to residues N 256‐ V 271 within the domain I of C ry3 A a toxin. Finally, to investigate whether C a M has any effect on C ry3 A a toxin CPB midgut membrane associated proteolysis, cleavage assays were performed in the presence of the C a M ‐specific inhibitor trifluoperazine. We showed that trifluoperazine significantly increased C ry3 A a toxin proteolysis and also decreased C ry3 A a larval toxicity.