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. ß

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In many moths, mate-finding communication is mediated by the female sex pheromones. Since differentiation of sex pheromones is often associated with speciation, it is intriguing to know how the changes in female sex pheromone have been tracked by the pheromone recognition system of the males. A male-specific odorant receptor was found to have been conserved through the evolution of sex pheromone communication systems in the genus Ostrinia (Lepidoptera: Crambidae). In an effort to characterize pheromone receptors of O. scapulalis, which uses a mixture of (E)-11- and (Z)-11-tetradecenyl acetates as a sex pheromone, we cloned a gene (OscaOR1) encoding a male-specific odorant receptor. In addition, we cloned a gene of the Or83b family (OscaOR2). Functional assays using Xenopus oocytes co-expressing OscaOR1 and OscaOR2 have shown that OscaOR1 is, unexpectedly, a receptor of (E)-11-tetradecenol (E11-14:OH), a single pheromone component of a congener O. latipennis. Subsequent studies on O. latipennis showed that this species indeed has a gene orthologous to OscaOR1 (OlatOR1), a functional assay of which confirmed it to be a gene encoding the receptor of E11-14:OH. Furthermore, investigations of six other Ostrinia species have revealed that all of them have a gene orthologous to OscaOR1, although none of these species, except O. ovalipennis, a species most closely related to O. latipennis, uses E11-14:OH as the pheromone component. The present findings suggest that the male-specific receptor of E11-14:OH was acquired before the divergence of the genus Ostrinia, and functionally retained through the evolution of this genus.

. 519:215-220, 2002), by using monoclonal antibodies (MAbs) that block binding between the binding site and the receptor. First, we produced a series of MAbs against Cry1Aa and obtained two MAbs, MAbs 2C2 and 1B10, that were capable of blocking the binding between Cry1Aa and BmAPN1 (blocking MAbs). The epitope of the Fab fragments of MAb 2C2 overlapped the BmAPN1 binding site, whereas the epitope of the Fab fragments of MAb 1B10 did not overlap but was located close to the binding site. Using three approaches for epitope mapping, we identified two candidate epitopes for the blocking MAbs on Cry1Aa. We constructed two Cry1Aa toxin mutants by substituting a cysteine on the toxin surface at each of the two candidate epitopes, and the small blocking molecule N-(9-acridinyl)maleimide (NAM) was introduced at each cysteine substitution to determine the true epitope. The Cry1Aa mutant with NAM bound to Cys582 did not bind either of the two blocking MAbs, suggesting that the true epitope for each of the blocking MAbs was located at the site containing Val582, which also consisted of 508 STLRVN 513 and 582 VFTLSAHV 589 . These results indicated that the BmAPN1 binding site overlapped part of the region blocked by MAb 2C2 that was close to but excluded the actual epitope of MAb 2C2 on domain III of Cry1Aa toxin. We also discuss another area on Cry1Aa toxin as a new candidate site for BmAPN1 binding.Bacillus thuringiensis, a gram-positive bacterium, produces various insecticidal proteins called Cry toxins which kill only target insects. This bacterium is used as a microbial insecticide and for the genetic development of insect-resistant plants.Cry toxins are expressed in inclusion bodies as protoxins (70 to 140 kDa) during sporulation. When a protoxin is ingested by target insects, it is solubilized in the insect midgut and digested by proteolytic enzymes in the insect (2). After enzymatic activation, the toxic protease-resistant fragment, which is the 60-to 65-kDa activated toxin, binds to specific receptors located in the columnar cells of the midgut apical brush border membrane (28). The binding of the toxin to receptor molecules leads to a conformational change in the toxin. This allows the toxin to insert into the plasma membrane and subsequently generate pores or ion channels which lead to cellular swelling and lysis (27,35,65,68). Finally, the intoxicated insects die. The binding of the activated toxin to a specific gut receptor is considered to be one of the key factors for insect toxicity. The insecticidal specificity of Cry toxins seems to be largely dependent on this receptor recognition.The three-dimensional structures of Cry1Aa trypsin-activated toxin, Cry2A protoxin, Cry3A, and Cry3B have been analyzed by X-ray diffraction crystallography (14,25,43,51). These proteins are comprised of three domains. The Cry1Aa and Cry3A structures have many similar features. The N-terminal domain I is composed of a seven-alpha-helix bundle in which the alpha-5 helix is encircled by the other helices. Domain II consists of thre...

Aminopeptidase N (APN) and cadherin-like protein (BtR175) from Bombyx mori larvae were examined for their roles in Cry1Aa-and Cry1Ac-induced lysis of B. mori midgut epithelial cells (MECs). APNs and BtR175 were present in all areas of the midgut, were particularly abundant in the posterior region, and were found only on columnar cell microvilli and not on the lateral membrane that makes cell^cell contacts. This distribution was in accordance with the distribution of Cry1A-susceptible MECs in the midgut. The lytic activity of Cry1Aa and Cry1Ac on collagenase-dissociated MECs was linearly dependent on toxin concentration. Although pre-treatment of MECs with anti-BtR175 antibody was observed to partially inhibit the lytic activity exerted by 0.1^1 nM Cry1Aa toxin or 5 nM Cry1Ac toxin, no signi¢cant inhibition was observed when MECs were pre-treated with anti-APN antibody. These results suggest that BtR175 functions as a major receptor for Cry1A toxins in the midgut of B. mori larvae. ß