Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella - Their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxin

Graduate School of Bio-Applications and Systems Engineering (BASE), Tokyo University of Agriculture and Technology, Edo, Tōkyō, Japan
FEBS Letters (Impact Factor: 3.17). 06/2002; 519(1-3):215-20. DOI: 10.1016/S0014-5793(02)02708-4
Source: PubMed


Novel aminopeptidase N (APN) isoform cDNAs, BmAPN3 and PxAPN3, from the midguts of Bombyx mori and Plutella xylostella, respectively, were cloned, and a total of eight APN isoforms cloned from B. mori and P. xylostella were classified into four classes. Bacillus thuringiensis Cry1Aa and Cry1Ab toxins were found to bind to specific APN isoforms from the midguts of B. mori and P. xylostella, and binding occurred with fragments that corresponded to the BmAPN1 Cry1Aa toxin-binding region of each APN isoform. The results suggest that APN isoforms have a common toxin-binding region, and that the apparent specificity of Cry1Aa toxin binding to each intact APN isoform seen in SDS-PAGE is determined by factors such as expression level in conjunction with differences in binding affinity.

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Available from: Katsuro Yaoi, Apr 09, 2014
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    • "This study confirms that the Cqm1 and Aam1 orthologs, from two closely related mosquito species, display dramatically different capacities to bind the Bin toxin likely associated to the nonconserved loop described in the text. A recent study demonstrated that aminopeptidase isoforms from the lepidopteran Ostrinia nubilalis have different binding capacities to the Cry1Ab and Cry1Fa toxins (Crava et al., 2013), while APNs isoforms from the different species, Bombyx mori and Plutella xylostella, seem to share a common binding region for the Cry1A toxins (Nakanishi et al., 2002), showing the differential roles assumed by these molecules. "
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    ABSTRACT: The Binary (Bin) toxin from the entomopathogenic bacterium Lysinibacillus sphaericus acts on larvae of the culicid Culex quinquefasciatus through its binding to Cqm1, a midgut-bound α-glucosidase. Specific binding by the BinB subunit to the Cqm1 receptor is essential for toxicity however the toxin is unable to bind to the Cqm1 ortholog from the refractory species Aedes aegypti (Aam1). Here, to investigate the molecular basis for the interaction between Cqm1 and BinB, recombinant Cqm1 and Aam1 were first expressed as soluble forms in Sf9 cells. The two proteins were found to display the same glycosilation patterns and BinB binding properties as the native α-glucosidases. Chimeric constructs were then generated through the exchange of reciprocal fragments between the corresponding Cqm1 and Aam1 cDNAs. Subsequent expression and binding experiments defined a Cqm1 segment encompassing residues S129 and A312 as critical for the interaction with BinB. Through site directed mutagenesis experiments, replacing specific sets of residues from Cqm1 with those of Aam1, the 158GG160 doublet was required for this interaction. Molecular modeling mapped these residues to an exposed loop within the Cqm1's structure, compatible with a target site for BinB and providing a possible explanation for its lack of binding to Aam1.
    Insect biochemistry and molecular biology 04/2014; 50(1). DOI:10.1016/j.ibmb.2014.04.004 · 3.45 Impact Factor
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    • "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. "
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    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.
    PLoS ONE 11/2013; 8(11):e79468. DOI:10.1371/journal.pone.0079468 · 3.23 Impact Factor
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    • "In addition, glycolipids were proposed to act as Cry toxin receptors in the nematode Caenorhabditis elegans (Griffitts et al., 2005). APN is the most extensively studied Cry toxin receptor with different isoforms from more than 20 lepidopteran species but the number of APN genes for each single species is uncertain (Angelucci et al., 2008; Herrero et al., 2005; Nakanishi et al., 2002; Pigott and Ellar, 2007; Simpson et al., 2008; Wang et al., 2005). So far, the phylogenetic analysis has shown that the APNs within a particular species can be clustered into eight different clades (Crava et al., 2010). "
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    ABSTRACT: Bacillus thuringiensis (Bt) crystal proteins (Cry) bind to aminopeptidase N (APN) receptors on insect midgut membrane leading to pore formation and subsequent death. However, evolution of insect resistance to Bt toxins threatens their long-term application. Therefore, search for new targets which could function as Cry toxin receptors is an immediate mandate. In the present study, two full-length APN cDNAs were cloned from Malpighian tubule and salivary gland tissues of the moth, Achaea janata. Both these APNs showed 99% and 32% sequence homology with fat body and midgut APNs respectively. Tissue distribution analysis revealed the presence of two different APN isoforms, one predominant in non-gut visceral tissues while the other was exclusively expressed in the midgut. Immunofluorescence and western blot analyses showed cross-reactivity in Malpighian tubule and salivary gland when probed with anti-fat body APN antiserum. These results clearly indicated the presence of non-gut (AjAPN1) and gut-specific (AjAPN4) isoforms in this moth. The expression of both the isoforms steadily increased during the larval development. Hormonal studies indicated regulation of the APN genes by the morphogenetic hormones, 20-hydroxyecdyone and juvenile hormone. Further, in vitro ligand-blotting studies demonstrated binding of Cry toxins to APNs in Malpighian tubule and salivary gland indicating their potential as alternate targets.
    Comparative biochemistry and physiology. Part B, Biochemistry & molecular biology 09/2013; 166(3-4). DOI:10.1016/j.cbpb.2013.09.005 · 1.55 Impact Factor
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