Research On Proteolysis And Oligomerization Mechanism Of Bacillus Thuringiensis Toxin Cry2Ab

Cry toxin,an insecticidal crystal protein derived from Bacillus thuringiensis(Bt),is widely applied as a bio-insecticides to control agricultural pests all over the world.The mechanism action of Cry toxin included proteolysis of Cry protoxin,interaction with insect midgut receptors,oligomerization and pore-formation.Nowdays most studies were devoted into "interaction of Cry toxin with midgut receptors" since it was an essential step for the insecticidal activities of Cry toxin,however,researches on proteolysis,oligomerization and pore-formation mechanism of Cry toxin were few reported.In present study,we systematically evaluated the proteolytic activation of Bt toxin Cry2Ab.The oligomerization action of Cry2Ab were further analysed and the key residues responsible for Cry2Ab oligomerization were identified.Our studies highlighted the importance of proteolysis and oligomerization in the insecticidal mode of Cry2Ab that could point out new insight for the mechanism action of Cry2Ab.The main results were as follows:Proteolytic processing of Cry2Ab protoxin by Plutella xylostella midgut juice(PxMJ)was first assessed using N-terminal sequencing.Both trypsin and chymotrypsin were identified involved in the proteolysis of Cry2Ab,and cleaved Cry2Ab at Arg139 and Leu144 respectively.Three Cry2Ab mutants(R139A,L144A and R139A-L144A)were constructed by replacing residues Arg139,Leu144 and Arg139-Leu144 with alanine.Proteolysis assays demonstrated that wild-type Cry2Ab,R139A and L144A but not R139A-L144A could be cleaved into 50 kDa activated-toxin by PxMJ.Bioassays showed that wild-type Cry2Ab,R139A and L144A were high toxic against P.xylostella larvae,while mutant R139A-L144A was almost non-insecticidal.Those results suggested that proteolysis by PxMJ was associated with Cry2Ab's insecticidal activity against P.xylostella.It also revealed that either trypsin or chymotrypsin was enough for awakening Cry2Ab protoxin.This characteristic was regarded as a belt-and-braces approach and might contribute to control of resistances development in target insects.Proteolytic activation of Cry2Ab cleaved and removed the first three a-helices,and uncovered an active region(helices ?4-?5)in Domain I.Either the masking or the delection of helices ?4-?5 mediated the pesticidal activity of Cry2Ab.The exposure of helices a4-a5 did not facilitate the binding of Cry2Ab toP.xylostella midgut receptors,but did induce Cry2Ab monomer to aggregate and assemble a 250 kDa pre-pore oligomer.Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site-directed on the helices a4-a5,and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P.xylostella.Taken together,those results suggested that oligomer formation of Cry2Ab was tightly linked with its toxicity against P.xylostella and highlighted the importance of helices a4-a5 in the mode of action of Cry2Ab.In conclusion,our data systematically assessed the role of proteolysis and oligomerization in insecticidal mechanism of Cry2Ab and demonstrated that both proteolysis and oligomerization were required for the pesticidal activity of Cry2Ab.Our study could lead to more detailed studies on the mode of action of Cry2Ab and provide new direction for the modification of Cry toxins.