Bacillus thuringiensis Cry toxins bound specifically to various proteins via domain III, which had a galactose-binding domain-like fold.

Cry toxins have been reported to bind not only to receptors on insect cells but also to several unrelated proteins. In this study, we investigated the binding properties of Bacillus thuringiensis Cry toxins, focusing on domain III, a Cry toxin region with a structure that of the galactose-binding domain-like. Cry1Aa, Cry1Ac, and Cry8Ca specifically bound to several proteins unrelated to insect midgut cells. Cry1Aa binding to Cry toxin-binding proteins was inhibited by a monoclonal antibody, 2C2, indicating that Cry1Aa binds to these Cry toxin-binding proteins through domain III. Cry1Aa binding to Bombyx mori aminopeptidase N and other Cry toxin-binding proteins was inhibited by carbonic anhydrase, a Cry toxin-binding protein. The binding regions of carbonic anhydrase and Bombyx mori aminopeptidase N were narrowed to regions of less than 20 amino acids that did not have any similarity, suggesting that Cry toxin domain III has a binding pocket for multiple proteins.

Novel RNA sequences associated with late male killing in Homona magnanima.

Maternally inherited female-biased sex ratios have been documented in many invertebrate species. One cause of such biased sex ratios is male killing, i.e. only males die. In most species, male killing occurs during embryonic stages (early male killing) and is associated with cytoplasmic bacteria, including Wolbachia, Spiroplasma, Rickettsia, Flavobacteria and gamma proteobacteria. However, the oriental tea tortrix, Homona magnanima, is one of the few species in which male death occurs in the larval or pupal stage, and is thus an example of late male killing. We partially purified the agent causing late male killing in H. magnanima and showed that it consists of two RNA sequences. This represents an entirely novel agent causing late male killing.

A novel 96-kDa aminopeptidase localized on epithelial cell membranes of Bombyx mori midgut, which binds to Cry1Ac toxin of Bacillus thuringiensis.

Proteins in the brush border membrane (BBM) of the midgut binding to the insecticidal Cry1Ac toxin from Bacillus thuringiensis were investigated to examine the lower sensitivity of Bombyx mori to Cry1Ac, and new aminopeptidase N that bound to Cry1Ac was discovered. DEAE chromatography of Triton X-100-soluble BBM proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4-kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to approximately 190-kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of the amino acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. K(D) of the APN96-Cry1Ac interaction was determined to be 1.83 +/- 0.95 microM. The lectin binding assay suggested that APN96 had an N-linked bi-antennal oligosaccharide or an O-linked mucin type one. The role of APN96 was discussed in relation to the insensitivity of B. mori to Cry1Ac.

Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.

We analyzed the binding site on Cry1Aa toxin for the Cry1Aa receptor in Bombyx mori, 115-kDa aminopeptidase N type 1 (BmAPN1) (K. Nakanishi, K. Yaoi, Y. Nagino, H. Hara, M. Kitami, S. Atsumi, N. Miura, and R. Sato, FEBS Lett. 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 508STLRVN513 and 582VFTLSAHV589. 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.

A cadherin-like protein functions as a receptor for Bacillus thuringiensis Cry1Aa and Cry1Ac toxins on midgut epithelial cells of Bombyx mori larvae.

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 significant 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.

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.

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.