Insect Control: Biological and Synthetic Agents - Index of

260 7: Bacillus thuringiensis: Mechanisms and Use
In the case of Cry1Ac toxin, a sequential binding
mechanism to the APN receptor has been proposed
(Jenkins et al., 2000). The interaction of domain III
to an N-acetylgalactosamine moiety in the receptor
precedes the binding of loop regions of domain II
(Jenkins et al., 2000).
7.4.5. Receptor Binding Epitopes
In regard to the receptor binding epitopes, a region of
63 residues (Ile135–Pro198) involved in Cry1Aa binding
was identified by analysis of truncated derivatives
of B. mori APN. This site was specific for Cry1Aa
toxin since it was not involved in Cry1Ac binding
(Yaoi et al., 1999; Nakanishi et al., 2002). Nevertheless,
this binding region is present in other APN molecules
that do not bind Cry1Aa toxin when assayed by
toxin overlay assays (Nakanishi et al., 2002). This
result can be explained if the epitope mapped was
not accessible in native conditions. In fact it has been
shown that denaturation of M. sexta APN exposes
binding epitopes hidden under nondenaturating conditions
(Daniel et al., 2002). Therefore, the role of the
mapped binding epitope in B. mori APN in toxicity
remains to be analyzed.
Regarding cadherin-like receptors, a Cry1A binding
epitope was mapped in Bt-R1 and Bt-R175 receptor
molecules by the analysis of truncated
derivatives of these receptors in toxin overlay assays
(Nagamatsu et al., 1999; Dorsch et al., 2002). In
the case of Bt-R 1 and Bt-R 175, a toxin binding region
of 70 amino acid residues was mapped in the
cadherin repeat number 11 which is close to the
membrane spanning region (Nagamatsu et al., 1999;
Dorsch et al., 2002). The binding epitope was narrowed
to 12 amino acids ( 1331 IPLPASILTVTV 1342 )
by using synthetic peptides as competitors. Binding
of Cry1Ab toxin to the 70 residue toxin binding
peptide was inhibited by synthetic peptides
corresponding to loop a-8 and loop 2, suggesting
that these loop regions are involved in the interaction
with this receptor epitope (Gómez et al., 2003).
Using a library of single-chain antibodies displayed
in M13 phage, a second Cry1A toxin binding region
was mapped in the Bt-R1 receptor (Gómez et al.,
2001). An scFv antibody (scFv73) that inhibited
binding of Cry1A toxins to the cadherin-like receptor
Bt-R1, but not to APN, and reduced the toxicity
of Cry1Ab to M. sexta larvae was identified (Gómez
et al., 2001). Sequence analysis of CDR3 region of
the scFv73 molecule led to the identification of an
eight amino acid epitope of M. sexta cadherin-like
receptor, Bt-R1 ( 869 HITDTNNK 876 ) involved in
binding of Cry1A toxins. This amino acid region
maps in the cadherin repeat 7 (Gómez et al., 2001).
Using synthetic peptides of the exposed loop regions
of domain II of Cry1A toxins, loop 2 was identified
as the cognate binding epitope of the M. sexta
receptor Bt-R1 869 HITDTNNK 876 site (Gómez
et al., 2002a). This finding highlights the importance
of the 869 HITDTNNK 876 binding epitope
since extensive mutagenesis of loop 2 of Cry1A
toxins has shown that this loop region is important
for receptor interaction and toxicity (Rajamohan
et al, 1995, 1996b; Jenkins and Dean, 2000; Jenkins
et al., 2000). Nevertheless, binding to cadherin repeat
7 was only observed in small truncated derivatives of
Bt-R 1 (Gómez et al., 2003) in contrast with larger
truncated derivatives (Nagamatsu et al., 1999;
Dorsch et al., 2002). Analysis of the dissociation
constants of Cry1Ab binding to similar 70 amino
acid peptides containing both toxin binding regions
revealed that the toxin binds the epitope located
in cadherin repeat 7 with sixfold higher affinity than
cadherin repeat 11. Based on these results a sequential
binding mechanism was proposed where binding of
toxin to cadherin repeat 11 facilitates the binding
of toxin loop 2 to the epitope in cadherin repeat 7
(Gómez et al., 2003). Accumulating evidence indicate
that proteins can interact with amino acid sequences
displaying inverted hydropathic profiles (Blalock,
1995). The interactions of loop 2 with Bt-R1 865 NITI-
HITDTNN 875 region and of loops a-8 and 2 with
133 1IPLPASILTVTV 1342 region were shown to be
determined by hydropathic complementarity (Gómez
et al., 2002a, 2003).
As mentioned previously, it is generally accepted
that the toxic effect of Cry proteins is exerted by the
formation of a lytic pore. However, the fact that
Cry1A toxins interact with protein molecules
involved in cell–cell interactions (cadherin) within
susceptible hosts could be relevant for the intoxication
process as has been described for several other
pathogens (Dorsch et al., 2002). Targeting cell junction
molecules seems to be representative of those
bacteria that disrupt or evade epithelial barriers in
their hosts. In this regard, it is remarkable that
Cry1A toxins interact with at least two structural
regions that are not close together in the primary
sequence of Bt-R 1 (cadherin repeats 7 and 11).
Although we cannot exclude the possibility that
both sites could be located close together in the threedimensional
structure of Bt-R1, we speculate that
binding of Cry1A toxins could cause a conformational
change in cadherin molecule that could interact with
other cell-adhesion proteins, and consequently disrupt
the epithelial cell layer (Figure 7).
As mentioned previously, mutagenesis studies
have shown that besides domain II loop 2 and a-8,