Contents - Faperta

266 Biotechnological Approaches for Pest Management and Ecological Sustainability
Wells, 1995; Novillo, Castanera, and Ortego, 1997). The Cry1A protoxins are digested to a
65 kDa protein starting at the C-terminus, and proceeds towards the 55 to 65 kDa toxic core
(Chestukhina et al., 1982; Choma et al., 1990). The carboxy-terminal end of the protoxin is
clipped off in 10 kDa sections (Choma, Surewicz, and Kaplan, 1991). The mature Cry1A
toxin is cleaved at R28 at the amino-terminal end (Nagamatsu et al., 1984), while Cry1Ac is
cleaved at K623 on the carboxy-terminal end (Bietlot et al., 1989). Activated toxin binds to
specifi c receptors on the apical brush border membrane vesicles (BBMV) of the midgut
(Hofmann and Luthy, 1986; Hofmann et al., 1988). Irreversible binding is associated with
membrane insertion (Van Rie et al., 1989; Ihara et al., 1993; Rajamohan et al., 1995) and
requires the insertion of domain I (Flores et al., 1997). Cry1Aa and Cry1Ab bind to purifi ed
M. sexta aminopeptidase N (APN) (Masson et al., 1995). Cry1Ac also binds irreversibly to
purifi ed L. dispar APN (Vadlamudi, Ji, and Bulla, 1993; Valaitis et al., 1997). Cry1Ab receptor
is believed to be a cadherin-like 210 kDa membrane protein (Francis and Bulla, 1997;
Keeton and Bulla, 1997), while the Cry1Ac and Cry1C receptors have been identifi ed as
APN proteins with molecular weights of 120 and 106 kDa, respectively (Knight, Crickmore,
and Ellar, 1994; Sangadala et al., 1994; Luo, Lu, and Adang, 1996).
Incorporation of purifi ed 120 kDa APN into planar lipid bilayers catalyzes channel formation
by Cry1Aa, Cry1Ac, and Cry1C (Schwartz et al., 1997). There is some evidence that
domain II from either Cry1Ab or Cry1Ac promotes binding to the larger protein, while
domain III of Cry1Ac promotes binding to APN (de Maagd et al., 1996a, 1996b). Alkaline
phosphatase has also been proposed to be a Cry1Ac receptor (Sangadala et al., 1994). In
H. virescens, three aminopeptidases bind to Cry1Ac. The 170 kDa APN binds to Cry1Aa,
Cry1Ab, and Cry1Ac, but not Cry1C or Cry1E. N-Acetylgalactosamine inhibits the binding
of Cry1Ac, but not of Cry1Aa or Cry1Ab. In gypsy moth, L. dispar, the Cry1Ac receptor
seems to be APN, while Cry1Aa and Cry1Ab bind to a 210 kDa BBMV protein (Valaitis
et al., 1995, 1997). In P. xylostella (Luo, Tabashnik, and Adang, 1997) and B. mori (Yaoi et al.,
1997), the APN appears to function as a Cry1Ac binding protein. A gene encoding a
Cry1Ab-binding APN has been cloned in M. sexta and its homolog in P. xylostella (Denolf
et al., 1997). Insertion into the apical membrane of the columnar epithelial cells follows
the initial receptor-mediated binding, rendering the toxin insensitive to proteases and
monoclonal antibodies (Wolfersberger, Hofmann, and Luthy, 1986), inducing ion channels
or nonspecifi c pores in the target membrane.
Several studies have demonstrated the association of insect specifi city of a toxin with its
affi nity for specifi c receptors on BBMV (Hofmann and Luthy, 1986; Hofmann et al., 1988;
Van Rie et al., 1989). Insect specifi city of Cry1Aa and Cry1Ac is localized in the central
domain of the toxin in B. mori and T. ni, and the central and C-terminal domain for H. virescens
(Ge, Shivarova, and Dean, 1989; Ge et al., 1991; Bosch and Honee, 1993). Specifi city
and binding domains were colinear for Cry1Aa against B. mori (Lee et al., 1992). However,
Wolfersberger (1990) observed that Cry1Ab was more active than Cry1Ac against gypsy
moth, L. dispar, larvae, despite exhibiting a relatively weaker binding affi nity. A number of
other examples indicating the lack of correlation between receptor binding affi nity and
insecticidal activity have also been reported (Van Rie et al., 1989; Garczynski, Crim, and
Adang, 1991; Sanchis and Ellar, 1993). Liang, Patel, and Dean (1995) observed that affi nity
of Cry1Ab was not directly related to toxin activity in gypsy moth, but observed a direct
correlation between the irreversible binding rate and toxicity. Minor changes in binding
usually do not have a major effect on toxicity. Binding affi nity as measured by competition
binding or irreversible binding may effect toxicity. The same mutation in a toxin can have
different effects in different insects. Different toxins may have the same amino acid
sequence in the loops of domain II (e.g., Cry1Ab and Cry1Ac), and yet bind to different