Abstract
Insect midgut membrane-anchored aminopeptidases N (APNs) are Zn++ dependent metalloproteases. Their primary role in dietary protein digestion and also as receptors in Cry toxin-induced pathogenesis is well documented. APN expression in few non-gut hemocoelic tissues of lepidopteran insects has also been reported but their functions are widely unknown. In the present study, we observed specific in vitro interaction of Cry1Aa toxin with a 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland of Achaea janata. Analyses of 3D molecular structure of AjAPN1, the predominantly expressed APN isoform in these non-gut hemocoelic tissues of A. janata showed high structural similarity to the Cry1Aa toxin binding midgut APN of Bombyx mori, especially in the toxin binding region. Structural similarity was further substantiated by in vitro binding of Cry1Aa toxin. RNA interference (RNAi) resulted in significant down-regulation of AjAPN1 transcript and protein expression in fat body and Malpighian tubule but not in salivary gland. Consequently, reduced AjAPN1 expression resulted in larval mortality, larval growth arrest, development of lethal larval-pupal intermediates, development of smaller pupae and emergence of viable defective adults. In vitro Cry1Aa toxin binding analysis of non-gut hemocoelic tissues of AjAPN1 knockdown larvae showed reduced interaction of Cry1Aa toxin with the 113 kDa AjAPN1 protein, correlating well with the significant silencing of AjAPN1 expression. Thus, our observations suggest AjAPN1 expression in non-gut hemocoelic tissues to play important physiological role(s) during post-embryonic development of A. janata. Though specific interaction of Cry1Aa toxin with AjAPN1 of non-gut hemocoelic tissues of A. janata was demonstrated, evidences to prove its functional role as a Cry1Aa toxin receptor will require more in-depth investigation.
Highlights
Insect midgut aminopeptidases N (APNs) are Zn++ dependent gluzincin family M1 metalloproteases [1] attached to brush border membrane of the epithelial cells through a glycosylphosphatidylinositol (GPI) anchor [2,3]
In vitro Cry1Aa Toxin Binding Analysis The observations from ligand binding studies presented in figure 1 clearly showed strong binding of Cry1Aa toxin predominantly to a 113 kDa membrane protein of fat body, Malpighian tubule and salivary gland
Immunoprecipitation of Cry1Aa Toxin Interacting Protein The specificity of interaction between the 113 kDa membrane protein (Malpighian tubule and salivary gland) and Cry1Aa toxin was confirmed by co-immunoprecipitation
Summary
Insect midgut aminopeptidases N (APNs) are Zn++ dependent gluzincin family M1 metalloproteases [1] attached to brush border membrane of the epithelial cells through a glycosylphosphatidylinositol (GPI) anchor [2,3]. In midgut of lepidopteran insect larvae, APNs are primarily involved in dietary protein digestion whereby they cleave a single amino acid residue from the Nterminus of oligopeptides, preferentially the neutral amino acids [4,5] They are mainly studied for their role as receptors in Cry toxin-induced pathogenesis in insects [6,7]. Sivakumar et al demonstrated that Sf21 insect cells expressing Helicoverpa armigera midgut APN which allowed high sensitivity to Cry1Ac, upon down-regulation by RNA interference (RNAi) resulted in reduced sensitivity [21]. These studies suggest the possibility of Cry toxins causing insecticidal effects on cells where APNs are expressed
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