Alanine substitution mutations in the Cry1Ac domain III region, from amino acid residues 503 to 525, were constructed to study the functional role of domain III in the toxicity and receptor binding of the protein to Lymantria dispar, Manduca sexta, and Heliothis virescens. Five sets of alanine block mutants were generated at the residues 503SS504,506NNI508, 509QNR511,522ST523, and 524ST525. Single alanine substitutions were made at the residues509Q, 510N, 511R, and513Y. All mutant proteins produced stable toxic fragments as judged by trypsin digestion, midgut enzyme digestion, and circular dichroism spectrum analysis. The mutations,503SS504-AA,506NNI508-AAA,522ST523-AA,524ST525-AA, and 510N-A affected neither the protein’s toxicity nor its binding to brush border membrane vesicles (BBMV) prepared from these insects. Toward L. dispar and M. sexta, the509QNR511-AAA, 509Q-A,511R-A, and 513Y-A mutant toxins showed 4- to 10-fold reductions in binding affinities to BBMV, with 2- to 3-fold reductions in toxicity. Toward H. virescens, the509QNR511-AAA, 509Q-A,511R-A, and 513Y-mutant toxins showed 8- to 22-fold reductions in binding affinities, but only509QNR511-AAA and 511R-A mutant toxins reduced toxicity by approximately three to four times. In the present study, greater loss in binding affinity relative to toxicity has been observed. These data suggest that the residues509Q, 511R, and 513Y in domain III might be only involved in initial binding to the receptor and that the initial binding step becomes rate limiting only when it is reduced more than fivefold.

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) brush border membrane vesicle (BBMV). CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or CryIC, CryIIA, and CryIIIA toxins showed weak binding to APN. An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN. Inhibition of short circuit current for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specific phospholipase C, which is known to release APN from the midgut membrane. In contrast, addition of phosphatidylinositol-specific phospholipase C had only a marginal effect on the inhibition of short circuit current for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV. A ligand blotting experiment demonstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIAa and CryIAb recognized a 210-kDa molecule in L. dispar BBMV. In contrast, CryIAa and CryIAb bound to both the 120-and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide. The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean agglutinin, indicating that N-acetylgalactosamine is a component of this glycoprotein.

The delta-endotoxin family of toxic proteins represents the major component of the insecticidal capability of the bacterium Bacillus thuringiensis. Domain I of the toxins, which is largely alpha-helical, has been proposed to unfold at protein entry into the membrane of a target insect, following models known as the penknife and umbrella models. We extended the analysis of a previous work in which four disulfide bridges were constructed in domain I of the Cry1Aa delta-endotoxin that putatively prevented unfolding during membrane partitioning. Using bioassays and voltage clamping of whole insect midgut instead of artificial lipid bilayers, it was found that, while toxicity and inhibition of the short-circuit current were reduced, only one of the disulfide bridges eliminated the activity of the toxins in the insect midgut membrane, and in that case, the loss of toxicity was due to the single amino acid substitution, R99C. It is proposed that at least alpha helices 4, 5, 6, and 7 and domain II partition in the midgut membranes of target insects, in support of an insertion model in which the whole protein translocates into the midgut membrane.

Background: Bacillus thuringiensis Cry1Aa insecticidal protein is the most active known B. thuringiensis toxin against the forest insect pest Lymantria dispar (gypsy moth), unfortunately it is also highly toxic against the non-target insect Bombyx mori (silk worm).