Abstract
CryIA(c) delta-endotoxin, a member of the CryI family of Bacillus thuringiensis insecticidal proteins, specifically recognizes and binds with high affinity to target proteins in the midgut of susceptible insects. Protein blots of Manduca sexta brush-border membranes probed with 125I-CryIA(c) identify a major binding protein of 120 kDa and a minor binding protein of 65 kDa. Monoclonal antibodies were raised against the 120-kDa toxin binding protein. Using isoelectric focusing and monoclonal antibodies (2B3, 8G1, and 12B8) 120- and 65-kDa brush-border proteins were isolated. Labeled CryIA(c) and monoclonal antibodies probed to blots of the affinity-selected proteins recognized the 120- and 65-kDa proteins. When reconstituted into phospholipid vesicles, antibody-selected proteins increased toxin binding (35%) and enhanced toxin-induced 86Rb+ release up to 1000-fold. The 120-kDa protein was identified as aminopeptidase N (EC 3.4.11.2). A CryIA(c)-sensitive phosphatase was also present in the 120/65-kDa protein mixture. These findings provide the first identification of B. thuringiensis toxin binding proteins, although confirmation is needed in vivo.
Highlights
From the $Department of Entomology, University of Georgia, Athens, Georgia 30602-2603a n d 5Ecogen Inc., Langhorne, Pennsylvania 19047
Monoclonal an- concentration required for pore formation
Tibodies were raised against the 120-kDa toxin binding protein
Summary
With 5 pl of toxin in 66 mM KOH and incubated for 4 min. Release of RfiRb' was initiatebdy diluting thevesicle/toxin mixture 10-fold into 25 mM Tris-HCI, pH 8.5,lOOmM choline chloride. These are thebackground nonsaturable interaction of toxin with phospholipid (data not shown) and the saturable binding mediatebdy the additionof the toxin binding proteins These experiments indicated CthraytIA(c)recognized mAb-selected proteins both on protein blots and when inserted into membranes. The vesicles used in the assay showed no spontaneous efflux of 86Rb+over the duration of the experiments.As shown in Fig. 4A when external of the vesicle preparation was incubated with'''I-cryIA(c) in the pres- CryIA(c) approaches micromolar concentrationsitinduces ence of increasing concentrationsof unlabeled CryIA(c). Vesicles Toxin was resistant to purified peptidase activity as detercontaining 120- and 65-kDa proteinrequired 1000-fold less mined with '251-CryIA(c)(data not shown). The data shown inFig. 4 indicate a positive Manduca 125/65-kDa protein mixture had phosphatase activcorrelation between the amount of toxin binding protein (i.e. 120/65-kDa protein) and the extenotf"Rb' efflux. CryIA(c) did not change the activity of bovine alkaline phosphatase (Table 11)
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