. 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...

To identify and gain a better understanding of the cadherin‐like receptor‐binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin‐like protein‐binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175 kDa cadherin‐like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, aminopeptidase N1, from Bo. mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops 2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N‐(9‐acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175‐binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175‐binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo. mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.