Insect Control: Biological and Synthetic Agents - Index of

and Cry1Ac proteins bind to a 120 kDa aminopeptidase-N
(APN) (Knight et al., 1994; Garczynski
and Adang, 1995; Denolf et al., 1997) and to a
210 kDa cadherin-like protein (Bt-R1) (Belfiore
et al., 1994; Vadlamudi et al., 1995). Cadherins
represent a large family of glycoproteins that are
classically responsible for intercellular contacts.
These proteins are transmembrane proteins with a
cytoplasmic domain and an extracellular ectodomain
with several cadherin repeats (12 in the case
of Bt-R1). The ectodomain contain calcium binding
sites, integrin interaction sequences, and cadherin
binding sequences. In Bombyx mori, Cry1Aa binds
to a 175 kDa cadherin-like protein (Bt-R 175)
(Nagamatsu et al., 1998, 1999) and to a 120 kDa
APN (Yaoi et al., 1997). In H. virescens Cry1Ac
binds to two proteins of 120 kDa and 170 kDa
both identified as APN (Gill et al., 1995; Oltean
et al., 1999). Also, a cadherin-like protein is
involved in the mode of action of Cry1Ac toxin in
H. virescens (Gahan et al., 2001). In P. xylostella and
Lymantria dispar APNs were identified as Cry1Ac
receptors (Valaitis et al., 1995; Lee et al., 1996;
Denolf et al., 1997; Luo et al., 1997). In L. dispar,
besides APN and cadherin-like receptors, a high
molecular weight anionic protein (Bt-R270) that
binds Cry1A toxins with high affinity was identified
(Valaitis et al., 2001). For Cry1C toxin an APN
receptor molecule was identified in Spodoptera
litura (Agrawal et al., 2002). Sequence analysis of
various APN from lepidopteran insects suggests that
APN’s group into at least four classes (Oltean et al.,
1999). Plutella xylostella and B. mori produce the
four classes of APN and it is anticipated that several
other lepidopteran insects may also produce the four
isoforms (Nakanishi et al., 2002). Cry1A binding to
the different B. mori APN isoforms revealed that this
toxin only binds the 115 kDa isoform in ligand blot
binding analysis (Nakanishi et al., 2002).
Surface plasmon resonance experiments showed
that the binding affinity of Cry1A toxins to the
M. sexta APN is in the range of 100 nM (Jenkins
and Dean, 2000), while that of cadherin-like receptors
(Bt-R1) is in the range of 1 nM (Vadlamudi et al.,
1995). This difference in the binding affinities between
APN and Bt-R 1 suggest that binding to Bt-R 1
might be the first event on the interaction of Cry1A
toxins with microvilli membranes and, therefore,
the primary determinant of insect specificity.
Different experimental evidence supports the involvement
of both Cry1A toxins receptors (APN
and cadherin-like) in toxicity. Expression of the
M. sexta and B. mori cadherin-like proteins, Bt-R 1
and Bt-R 175 respectively, on the surface of different
cell lines render these cells sensitive to Cry1A toxins,
7: Bacillus thuringiensis: Mechanisms and Use 257
although the toxicity levels were low (Nagamatsu
et al., 1999; Dorsch et al., 2002; Tsuda et al., 2003).
Cry1Aa toxin was shown to lyse isolated midgut
epithelial cells; this toxic effect was inhibited if the
cells were preincubated with anti-Bt-R 175 antisera in
contrast with the treatment with anti-APN antisera,
suggesting that cadherin-like protein Bt-R175 is a
functional receptor of Cry1Aa toxin (Hara et al.,
2003). Also, a single-chain antibody (scFv73) that
inhibits binding of Cry1A toxins to cadherin-like
receptor, but not to APN, reduced the toxicity of
Cry1Ab to M. sexta larvae (Gómez et al., 2001).
Moreover, disruption of a cadherin gene by a retrotransposon-mediated
insertion and its linkage
to high resistance to Cry1Ac toxin in H. virescens
YHD2 (a laboratory selected line) larvae supports
a role for cadherin as a functional receptor (Gahan
et al., 2001). Overall, these results suggest that
binding to the cadherin-like receptor is an important
step in the mode of action Cry1A toxins (Figure 6).
Regarding the APN receptor, several reports
argue against the involvement of this protein in
toxin activity. Mutants of Cry1Ac toxin affected
on APN binding retained similar toxicity levels to
M. sexta as the wild-type toxin (Burton et al., 1999).
However, there are several reports describing point
mutations of Cry toxins located in domain II that
affected both APN binding and toxicity (Jenkins and
Dean, 2000). Expression of APN in heterologous
systems did not result in Cry1A sensitivity (Denolf
et al., 1997) and, as mentioned above, an APN
antibody did not protect B. mori midgut cells from
Cry1Aa toxic effect in contrast to a cadherin antibody
(Hara et al., 2003). However, two recent
reports clearly demonstrate the importance of this
molecule on the mode of action of Cry1 toxins.
Inhibition of APN production in S. litura larvae
by dsRNA interference showed that insects with
low APN levels became resistant to Cry1C toxin
(Rajagopal et al., 2002). Also, heterologous expression
of M. sexta APN in midguts and mesodermal
tissues of transgenic Drosophila melanogaster
caused sensitivity to Cry1Ac toxin (Gill and Ellar,
2002). Additionally, previous reports demonstrated
that incorporation of M. sexta APN into black lipid
bilayers lowers the concentration of toxin needed
for pore formation activity of Cry1Aa toxin
(Schwartz et al., 1997b). Finally, all Cry1A APN
receptors are anchored to the membrane by a glycosyl
phosphatidylinositol (GPI) (Knight et al., 1994;
Garczynski and Adang, 1995; Denolf et al., 1997;
Oltean et al., 1999; Agrawal et al., 2002; Nakanishi
et al., 2002). And treatment of Trichoplusia ni brush
border membrane vesicles with PI-PLC, which
cleaves GPI-anchored proteins from the membrane,