Chemical modification of Bacillus thuringiensis activated  -endotoxin and its effect on toxicity and binding to Manduca sexta midgut membranes

Abstract

The effect of chemical modification of aromatic and basic residues of the Bacillus thuringiensis insecticidal CrylA(c) toxin on toxicity and receptor binding was studied. Modification of four or more of the 43 arginine residues resulted in abolition of toxicity towards Manduca sexfa and Pieris brassicae in vivo and a Choristoneura fumiferana cell line in witro. Modification of seven or more of the 27 tyrosine residues resulted in reduction of toxicity. Upon modification of most of the 10 tryptophan residues, toxicity was reduced. Modification of histidine residues had no effect on toxicity. A quantitative binding assay was developed and optimized to compare the binding of derivatives with native toxin t o M. sexta brush border membrane vesicles. The reduction or abolition of toxicity observed upon selective modification of tyrosine or arginine residues was reflected in the binding abilities of the derivatives. However a non-toxic derivative, modified at tryptophan residues, retained its ability to bind vesicles. These results suggest that tyrosine and arginine residues are involved in the binding interaction of toxin with receptor but tryptophan residues are involved in some subsequent step.

Mutagenesis of three surface-exposed loops of a Bacillus thuringiensis insecticidal toxin reveals residues important for toxicity, receptor recognition and possibly membrane insertion

“…However when R369 was replaced by isoleucine both toxicity and binding were dramatically lower. The importance of arginine residues is again mirrored in the results obtained by Cummings & Ellar (1994), where chemical modification of four or more arginines in the CryIAc toxin was enough to abolish toxicity and dramatically reduce binding to M. sexta BBMV. The results for loop 2 match the findings of Lu e t al.…”

“…Replacement of Y367 with serine produced a marked drop in toxicity and some reduction in binding (compare YRKP and SKRP). The role of tyrosine in binding and toxicity has been highlighted in a previous study, where chemical modification of seven or more of the 27 tyrosines in the CryIAc toxin completely abolished toxicity and decreased binding to M. sexta BBMV (Cummings & Ellar, 1994). Replacement of either R368 or R369 with the slightly shorter lysine side-chain was without effect provided Y367 was unchanged.…”

“…The possibility of regions outside domain I1 being involved in receptor binding was raised by a mutagenesis study in which residues S503 and S504 in a domain I11 loop of CryIAc were reported to be involved in toxicity and binding to M. sexta and H. virescens (Aronson et al, 1995). Chemical modification of CryIAc residues showed that modifying 4 of the 43 arginine residues or 7 of the 27 tyrosines resulted in greatly reduced toxicity and binding to M. s o d a BBMV (Cummings & Ellar, 1994). Finally, mutational analysis of predicted residues in loops 1 and 2 of CryIC toxin showed their importance in specificity determination for Aedes aegypti and a Spodoptera frugiPerda (SB) cell line (Smith & Ellar, 1994).…”

The α-Helix 4 Residue, Asn135, Is Involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis Toxins

“…Evidence suggests that ␣-helix 4 is orientated to face the lumen of the pore and, as such, may be involved in channel function (30). Conversely, ␣-helix 5 appears to be in contact with the lipid milieu of the membrane and plays a vital role in the oligomerization of the hairpin (2,10,15). Oligomerization is believed to occur following insertion of the ␣-helix 4/␣-helix 5 hairpin (13,30) and to proceed to a final complex of four monomers (23,30,42).…”

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“…Other regions were also found to be involved in the phenomenon 29 . Chemical modifications of 4 Arg or 7 Tyr residues significantly reduced toxicity and binding 30 . The loops (β2-β3 and β4-β5) probably interact with the receptor through both hydrophobic and electrostatic interactions.…”