Identification, Isolation and Cloning of cDNA Encoding Aminopeptidase from the Midgut of the Egyetian Cotton Leaf Worm That Serves as a Receptor for CRY Toxin

Abstract

Bacillus thuringiensis (Bt) is an aerobic, spore-forming bacterium that produces crystalline inclusions during the sporulation phase of growth that are composed of proteins known as δ-endotoxins (Bravo et al., 2005). The δ-endotoxins comprise two multigenic families, cry and cyt. Cry proteins are specifically toxic to different insect orders, Lepidoptera, Coleoptera, Hymenoptera, Diptera or nematodes (Go´mez et al., 2007). The crystal inclusions ingested by susceptible larvae, dissolve in the alkaline environment of the gut, and the solubilized inactive protoxins are cleaved by midgut proteases yielding active monomeric 60–70 kDa protease-resistant toxin (Pardo-Lo´ pez et al., 2009; Bravo et al., 2005), then the Cry toxic fragment binds to specific receptors located in the microvilli of the apical membrane of midgut epithelial cells that leads to membrane insertion, pore formation, cell lysis and the eventual death of the host (Bravo et al., 2004 & 2005).
For Cry1A toxins (lepidopteran specific toxins), at least four different binding-proteins have been described as receptors for the Cry toxins; a cadherin-like protein (CADR), a glycosylphos-phatidyl–inositol (GPI) - anchored amin-opeptidase-N (APN), a GPI-anchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugate (Vadlamudi et al., 1995; Knight et al., 1994; Jurat Fuentes et al.,
2004; Valaitis et al., 2001).
APN is the most extensively studied Cry receptor identified and isolated from several lepidopteran insects. APN serves as a binding molecule for Cry1C in Manduca sexta (Luo et al., 1996) and Spodoptera litura (Agrawal et al., 2002), for Cry1Aa in Bombyx mori (Nakanishi et al., 2002; Jenkins and Dean 2001; Yaoi et al., 1999) and for Cry1Ac in M. sexta (Garczynski et al., 1995; Knight et al., 1994 & 1995; Sangadala et al., 1994), Lymantria dispar (Lee et al., 1996; Valaitis et al., 1995) and Heliothis virescens (Gill et al., 1995; Luo et al., 1997; Oltean et al., 1999)
The characterization of receptors in the economically important pests by studying their binding to different Cry toxins will help in evaluating the molecular mechanism of action of Cry toxin and will lead to a better understanding of the mechanism of development of resistance to these proteins in insects.
The Egyptian cotton leaf worm (Spodoptera littoralis Boisduval) is an important pest in Mediterranean and Asian countries; it populates fields in relatively close locations (Hamal et al., 1991) and is considered a totally polyphagous pest (Lutfallah et al., 1993) affecting various economically important crops in Egypt such as cotton, maize, rice, soybeans and vegetables (Salama and Matter, 1991).
To identify the precise toxin binding targets in insects, midgut cell membranes have been electrophoretically separated and incubated with Bt cry toxins in Western blot analysis. We have identified the proteins involved in CryIAc toxicity to Spodoptera littoralis. A 109-kDa B. thuringiensis toxin-binding protein from the midgut brush-border membrane that binds the Cry1Ac toxin was determined. A cDNA clone encoding this protein was cloned, sequenced and characterized. The analysis of the predicted amino acids sequence reveals that the protein belongs to the aminopeptidase N family of proteins.