Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata

Dutta-Gupta A (2013) Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata. PLoS ONE 8(11): e79468. doi:10.1371/journal.pone.0079468 Functional Interpretation of a Non-Gut Hemocoelic Tissue Aminopeptidase N (APN) in a Lepidopteran Insect Pest Achaea janata Thuirei Jacob Ningshen 0 Polamarasetty Aparoy 0 Venkat Rao Ventaku 0 Aparna Dutta-Gupta 0 Omprakash Mittapalli, The Ohio State University/OARDC, United States of America 0 1 Department of Animal Sciences, School of Life Sciences, University of Hyderabad , Hyderabad, Andhra Pradesh , India , 2 Centre for Computational Biology and Bioinformatics, School of Life Sciences, Central University of Himachal Pradesh , Dharamshala, Himachal Pradesh , India 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. - Funding: This work was supported by University Grants Commission, India (Grant No. 36-305/2008/SR). The funder had no role in study design, conduct of experiments, results analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. 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]. However, they are mainly studied for their role as receptors in Cry toxin-induced pathogenesis in insects [6,7]. The Cry proteins produced by a gram positive bacterium Bacillus thuringiensis are in the form of protoxins which upon ingestion by larvae of susceptible insects, are cleaved by the midgut proteinases to form active toxins. The activated toxins then bind to specific midgut receptors resulting in oligomerization and insertion of toxins into the membranes to generate pores leading to cell lysis and finally, the death of the insect [5,8]. Though cadherin-like proteins [9], GPI-anchored alkaline phosphatases (ALPs) [10], glycolipids [11] and glyconjugates [5] are reported receptors for Cry toxins, the GPI-anchored APNs [12,13] by far are the most widely studied and well characterized Cry toxin receptors. Apart from midgut, APN expression in fat body [14,15], Malpighian tubule [4,16,17,18], salivary gland [18] of lepidopteran insects has now been reported. Pore forming ability of Cry toxins on in vitro cultured fat body cells indicated the possibility of Cry toxins binding to fat body membrane proteins and causing toxic effects to the cells [19]. Transgenic expression of Manduca sexta midgut APN in Drosophila melanogaster induced sensitivity to the lepidopteranspecific insecticidal Cry1Ac which otherwise is not toxic [20]. Further, Sivakumar et al also 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. In cases where the experimental determination of protein threedimensional (3D) structure is not possible, homology modeling is the most widely used approach. To date, there are no reports on crystal structure of insect APNs. However, molecular models of midgut-specific APNs from M. sexta [22] and Spodoptera litura [23] have been generated using homology modeling strategy. RNAimediated knockdown of gene expression in lepidopteran insects either by feeding or intra-hemocoelic injection has been commonly used to identify potential target genes for pest control [13,21,24,25]. Gene silencing studies revealed the functional role of midgut APNs as a Cry toxin receptor in S. litura [13] and H. armigera [21]. In A. janata, AjAPN1 is the APN isoform which is predominantly expressed in fat body, Malpighian tubule and salivary gland [18]. However till date, proper functional characterization of non-gut hemocoelic tissue APNs in insects has been lacking. In the present study, we employed homology modeling and RNAi strategies to decipher the functional role of AjAPN1 expression in non-gut hemocoelic tissues of A. janata larvae. We demonstrated specific in vitro interaction of Cry1Aa toxin with the 113 kDa AjAPN1 membrane protein of larval fat body, Malpighian tubule and salivary gland. High similarity of 3D molecular structure of AjAPN1 of A. janata with that of Bombyx mori midgut APN (Genbank AAC33301), especially in the Cry1Aa toxin binding region as well as in vitro binding of Cry1Aa toxin to it further supported its potential role in Cry toxin interaction and toxicity. RNAi-mediated silencing not only down-regulated AjAPN1 expression in fat body and Malpighian tubule but also induced adverse physiological effects, which suggest that it plays important physiological role during growth, development as well as me (...truncated)