Aminopeptidase-N from the Helicoverpa armigera (Hubner) Brush Border Membrane Vesicles as a Receptor of Bacillus thuringiensis Cry1Ac δ-Endotoxin

Abstract

Brush border membrane vesicles (BBMVs) were prepared from the 2nd instar larvae of Helicoverpa armigera. Binding of the activated Cry1Ac of Bacillus thuringiensis (Bt) toxin was shown by immunoblot. A 120-kDa protein was identified as a receptor for the Cry1Ac type delta-endotoxin. The aminopeptidase-N activity of BBMVs was measured as the hydrolysis of L-leucine p-nitroanilide. The specific activity was 35 units/mg protein. The BBMV preparation also showed low level of alkaline phosphatase activity. Zn++ chelating agents 2,2'-dipyridyl and 1,10-phenanthroline inhibited aminopeptidase activity at 10 mM concentration, indicating the presence of zinc-dependent aminopeptidase in the brush border of H. armigera. The aminopeptidase activity was increased with increasing concentration of delta-endotoxin. The purified 120-kDa binding protein was N-terminally sequenced. The first 10-amino-acid sequence showed 60-77% similarity with human cysteine-rich secretory protein-1 precursor, inhibin alpha chain precursor. Salmonella flagellar hook protein and yeast carboxypeptidase S.

Receptors of Garlic (Allium sativum) Lectins and Their Role in Insecticidal Action

“…Angelucci et al [2] have reported seven APN genes in H. armigera, four among them interact with Cry1Ac d-endotoxin. It is noteworthy that APNs have been comprehensively studied as receptors of d-endotoxins [2,44,49,87]. Fitches et al [30] observed the interaction of garlic bulb lectin with the membrane bound alanyl aminopeptidase N of Acyrthosiphon pisum.…”

Role of alkaline phosphatase in insecticidal action of Cry1Ac against Helicoverpa armigera larvae

“…Membrane-bound APN and ALP are important receptors for the d-endotoxins in several insects (Ingle et al 2001;Jurat-Fuentes and Adang 2004;Knight et al 1994;Krishnamoorthy et al 2007 (Griko et al 2008). Neonatal larvae of H. armigera are highly susceptible to Cry1Ac (personal observation).…”

“…Although, membrane-bound APN is a receptor for the d-endotoxins in Manduca Sexta (Knight et al 1994), Heliothis virescens (Luo et al1997), Helicoverpa armigera (Angelucci et al 2008;Ingle et al 2001) and Anopheles gambiae , its role as a functional receptor is debatable. Jenkins et al (1999) reported a mutation in domain III of Cry1Ac lowers its affinity towards APN without any significant impact on toxicity to the M. sexta larvae.…”

Cotton bollworm resistance to Bt transgenic cotton: A case analysis

“…A GPI-anchored APN in the midgut epithelium of H. armigera larvae was characterized as a Cry1A toxin receptor based on its APN-like activity and N-terminal sequence [49][50][51]. Recently, several in vivo studies confirmed the functional relevance of the midgut APN in the resistance of H. armigera to Cry1Ac toxin.…”