Insect Control: Biological and Synthetic Agents - Index of

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unpublished data).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