Diversity of aminopeptidases, derived from four lepidopteran gene duplications, and polycalins expressed in the midgut of Helicoverpa armigera: Identification of proteins binding the δ-endotoxin, Cry1Ac of Bacillus thuringiensis

Abstract

Helicoverpa armigera midgut proteins that bind the Bacillus thuringiensis (Bt) δ-endotoxin Cry1Ac were purified by affinity chromatography. SDS-PAGE showed that several proteins were eluted with N-acetylgalactosamine and no further proteins were detected after elution with urea. Tandem mass spectral data for tryptic peptides initially indicated that the proteins resembled aminopeptidases (APNs) from other lepidopterans and cDNA sequences for seven APNs were isolated from H. armigera through a combination of cloning with primers derived from predicted peptide sequences and established EST libraries. Phylogenetic analysis showed lepidopteran APN genes in nine clades of which five were part of a lepidopteran-specific radiation. The Cry1Ac-binding proteins were then identified with four of the seven HaAPN genes. Three of those four APNs are likely orthologs of APNs characterised as Cry1Ac-binding proteins in other lepidopterans. The fourth Cry1Ac-binding APN has orthologs not previously identified as Cry1Ac-binding partners. The HaAPN genes were expressed predominantly in the midgut through larval development. Each showed consistent expression along the length of the midgut but five of the genes were expressed at levels about two orders of magnitude greater than the remaining two. The remaining mass spectral data identified sequences encoding polycalin proteins with multiple lipocalin-like domains. A polycalin has only been previously reported in another lepidopteran, Bombyx mori, but polycalins in both species are now linked with binding of Bt Cry toxins. This is the first report of hybrid, lipocalin-like domains in shorter polycalin sequences that are not present in the longest sequence. We propose that these hybrid domains are generated by alternative splicing of the mRNA.

Differential alteration of two aminopeptidases N associated with resistance to Bacillus thuringiensis toxin Cry1Ac in cabbage looper

“…2). More recently, APN6 has been identified from lepidopteran larvae, and affinity purification of H. armigera midgut proteins using Cry1Ac as a ligand indicated that APN6 was not among the Cry1Ac-binding proteins (22). Similarly, results of the present study indicate that APN6 in the midgut BBMVs from the Cry1Ac-resistant T. ni is significantly more abundant than that from the Cry1Ac-susceptible strain (Fig.…”

Extensive synteny conservation of holocentric chromosomes in Lepidoptera despite high rates of local genome rearrangements

“…Several gene clusters were covered by our analysis. The aminopeptidase N (APN) of lepidopteran midgut has received wide attention because of its role in the mode of action of Cry toxins of Bacillus thurigiensis (25), and multiple APN cDNAs from a variety of species are available (26). We show here that APN genes are organized in a single, large cluster of nine genes on chromosome 9 of B. mori.…”

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

“…Several forms of APNs have been isolated and characterized. Presence of N-or O-linked glycosylation has also been reported [2,12,49]. N-terminal signal peptide directs APNs to the outer surface of the cytoplasmic membrane where they bind to the membrane by GPI anchor [19,50,53,82].…”

“…N-terminal signal peptide directs APNs to the outer surface of the cytoplasmic membrane where they bind to the membrane by GPI anchor [19,50,53,82]. 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].…”

“…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.…”