Insect Control: Biological and Synthetic Agents - Index of
Insect Control: Biological and Synthetic Agents - Index of
Insect Control: Biological and Synthetic Agents - Index of
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Figure 7 Structural regions on M<strong>and</strong>uca sexta cadherin receptor<br />
(Bt-R1) involved in Cry1A binding. Ectodomains (EC) 7 <strong>and</strong><br />
11 contain Cry1A binding epitopes.<br />
loop 3 <strong>of</strong> Cry1A toxins is important for receptor<br />
interaction <strong>and</strong> toxicity (Rajamohan et al., 1996a;<br />
Lee et al., 2001). The Bt-R1 <strong>and</strong> the APN epitope<br />
involved in binding loop 3 regions still remains to<br />
be identified.<br />
7.4.6. Cry Toxin–Receptor Binding Function<br />
in Toxicity<br />
It is proposed that, following binding, at least part <strong>of</strong><br />
the toxin inserts into the membrane resulting in pore<br />
formation (Schnepf et al., 1998). However, it is still<br />
largely unknown what is the role <strong>of</strong> receptor binding<br />
in promoting the insertion <strong>and</strong> oligomerization <strong>of</strong><br />
the toxin. In the case <strong>of</strong> Cry1A toxins the differences<br />
<strong>of</strong> binding affinities between APN <strong>and</strong> Bt-R 1<br />
(100 nM versus 1 nM Kd respectively) suggest that<br />
binding to Bt-R1 is the first event in the interaction <strong>of</strong><br />
Cry1A toxins with microvilli membranes.<br />
In other noninsecticidal pore forming toxins,<br />
receptor binding facilitates a complete proteolytic<br />
activation <strong>of</strong> the toxin resulting in the formation <strong>of</strong><br />
functional oligomers that are membrane insertion<br />
competent (Abrami <strong>and</strong> Van der Goot, 1999). In<br />
the case <strong>of</strong> Cry1A toxins, Gómez et al. (2002b,<br />
2003) provided evidence that interaction <strong>of</strong> Cry<br />
toxin with its cadherin-like receptor is a necessary<br />
step for a complete proteolytic <strong>of</strong> the toxin. Incubation<br />
<strong>of</strong> Cry1Ab protoxin with the single-chain<br />
antibody scFv73 that mimics the cadherin-like<br />
receptor or with the toxin-binding peptides <strong>of</strong><br />
Bt-R 1, <strong>and</strong> treatment with M. sexta midgut juice,<br />
resulted in toxin preparations with high pore formation<br />
activity in vitro. In contrast toxin preparations<br />
7: Bacillus thuringiensis: Mechanisms <strong>and</strong> Use 261<br />
activated with same midgut juice proteases in the<br />
absence <strong>of</strong> the antibody showed very low pore formation<br />
activity (Gómez et al., 2002b, 2003). The<br />
high pore formation activity correlated with the<br />
formation <strong>of</strong> a 250 kDa oligomer composed <strong>of</strong> four<br />
Cry toxins that lacked the a-1 helix <strong>of</strong> domain I. The<br />
oligomer, in contrast to the 60 kDa monomer, has<br />
higher hydrophobicity as judged by 8-anilino-1naphthalenesulfonate<br />
binding (Gómez et al., 2002b).<br />
The oligomer was membrane insertion competent in<br />
contrast with the monomer, as judged by measuring<br />
toxin membrane insertion using the intrinsic fluorescence<br />
<strong>of</strong> tryptophan residues (Raussel <strong>and</strong> Bravo,<br />
unpublished data). The 250 kDa oligomer could also<br />
be obtained by incubation <strong>of</strong> the Cry1Ab protoxin<br />
with brush border membrane vesicles isolated in the<br />
absence <strong>of</strong> protease inhibitors, presumably by the<br />
action <strong>of</strong> a membrane associated protease (Gómez<br />
et al., 2002b). Therefore toxin binding to cadherin<br />
facilitates the complete proteolytic activation <strong>of</strong> the<br />
toxin <strong>and</strong> the formation <strong>of</strong> a prepore structure that<br />
consists <strong>of</strong> four monomers. Characterization <strong>of</strong><br />
the membrane insertion capabilities <strong>of</strong> the prepore,<br />
the kinetic properties <strong>of</strong> the ionic pore formed by this<br />
structure, <strong>and</strong> solving its three-dimensional structure<br />
could be important steps towards underst<strong>and</strong>ing<br />
the role <strong>of</strong> this insertion-intermediate structure in<br />
the mode <strong>of</strong> action <strong>of</strong> Cry toxins.<br />
As mentioned previously APN receptors are also<br />
key molecules involved in the toxicity <strong>of</strong> Cry1<br />
toxins. Cry1A APN receptors are anchored to the<br />
membrane by a GPI anchor. An indication <strong>of</strong> its<br />
possible role came from the characterization <strong>of</strong><br />
membrane microdomains (lipid rafts) from microvilli<br />
membranes <strong>of</strong> M. sexta <strong>and</strong> H. virescens<br />
(Zhuang et al., 2002). Like their mammalian counterparts,<br />
H. virescens <strong>and</strong> M. sexta lipid rafts are<br />
enriched in cholesterol, sphingolipids, <strong>and</strong> glycosylphosphatidylinositol<br />
anchored proteins (Zhuang<br />
et al., 2002). Lipid rafts have been implicated in<br />
membrane <strong>and</strong> protein sorting <strong>and</strong> in signal transduction<br />
(Simons et al., 2000). They have also been<br />
described as portals for different viruses, bacteria<br />
<strong>and</strong> toxins. The interaction <strong>of</strong> different bacterial<br />
toxins with their receptors located in lipid rafts is<br />
a crucial step in the oligomerization <strong>and</strong> insertion<br />
<strong>of</strong> toxins into the membrane (Cabiaux et al., 1997;<br />
Abrami <strong>and</strong> van der Goot, 1999; Rosenberg et al.,<br />
2000). Several Cry1A receptors, including the GPI<br />
anchored proteins, 120 <strong>and</strong> 170 kDa APNs from<br />
H. virescens, <strong>and</strong> the 120 kDa APN from M. sexta<br />
were preferentially partitioned into lipid rafts.<br />
After toxin exposure, Cry1A toxins were associated<br />
with lipid rafts <strong>and</strong> the integrity <strong>of</strong> these