Location of theBombyx moriAminopeptidase N Type 1 Binding Site onBacillus thuringiensisCry1Aa Toxin

Abstract

. 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...

Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

“…A third region that has been proposed to act as an APN binding site was first characterized for Cry1Aa (6). This region was identified at the interface between domain II and III and was shown to interact with the 64-amino-acid toxin-binding fragment of class 1 B. mori APN (193).…”

“…The smallest fragment that retained toxin binding affinity corresponded to APN amino acids residues Ile135 to Pro198. More recently, Cry1Aa binding to the same APN fragment was demonstrated under nondenaturing conditions by enzymelinked immunosorbent assay (6).…”

A Spodoptera exigua Cadherin Serves as a Putative Receptor for Bacillus thuringiensis Cry1Ca Toxin and Shows Differential Enhancement of Cry1Ca and Cry1Ac Toxicity

“…Cry1Ac and Cry1Ca were activated toxins and were obtained from Envirologix Inc. Their purities and molec-ular weights were confirmed by SDS-PAGE, and their quantities were determined by Bradford's method. Activated Cry1Ac and Cry1Ca were biotinylated with the EZ-Link sulfo-N-hydroxysuccinimide (NHS) liquid chromatography (LC) biotinylation kit (Pierce, FL, USA), similar to the method described by Atsumi et al (46).…”

Specific Epitopes of Domains II and III of Bacillus thuringiensis Cry1Ab Toxin Involved in the Sequential Interaction with Cadherin and Aminopeptidase-N Receptors in Manduca sexta

“…Regarding interaction of Cry1A toxins with APN, Cry1Ac toxin binds to APN receptor by means of domain III that specifically recognizes N-acetylgalactosamine (GalNAc) moieties in contrast to Cry1Aa and Cry1Ab toxins that show no GalNAc binding capacities (18). Based on the use of monoclonal antibodies that competed binding of Cry1Aa with Bombix mori APN, the Cry1Aa-APN interacting epitopes were recently mapped in domain III ␤16 ( 508 STLRVN 513 ) and ␤22 ( 582 VFTLSAHV 589 ) residues, which are exposed and in close proximity in the three-dimensional structure (24,25).…”