1 research outputs found
Oligomerization of Cry9Aa in solution without receptor binding, is not related with insecticidal activity
Background: Bacillus thuringiensis Cry toxins bind with different
insect midgut proteins leading to toxin oligomerization, membrane
insertion and pore formation. However, different Cry toxins had been
shown to readily form high molecular weight oligomers or aggregates in
solution in the absence of receptor interaction. The role of Cry
oligomers formed in solution remains uncertain. The Cry9A proteins show
high toxicity against different Lepidoptera, and no-cross resistance
with Cry1A. Results: Cry9Aa655 protein formed oligomers easily in
solution mediated by disulfide bonds, according to SDS-PAGE analysis
under non-reducing and reducing conditions. However, oligomerization is
not observed if Cry9Aa655 is activated with trypsin, suggesting that
cysteine residues, C14 and C16, located in the N-terminal end that is
processed during activation participate in this oligomerization. To
determine the role of these residues on oligomerization and in toxicity
single and double alanine substitution were constructed. In contrast to
single C14A and C16A mutants, the double C14A\u2013C16A mutant did not
form oligomers in solution. Toxicity assays against Plutella xylostella
showed that the C14A\u2013C16A mutant had a similar insecticidal
activity as the Cry9Aa655 protein indicating the oligomers of Cry9Aa
formed in solution in the absence of receptor binding are not related
with toxicity. Conclusions: The aggregation of Cry9Aa655 polypeptides
was mediated by disulfide bonds. Cry9Aa655 C14 and C16C are involved in
oligomerization in solution. These aggregate forms are not related to
the mode of action of Cry9Aa leading to toxicity