Aminopeptidase N from Bombyx Mori as a Candidate for the Receptor of Bacillus Thuringiensis Cry1Aa Toxin

Abstract

CrylAa toxin-binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin-susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that CrylAa toxin bound to a 120-kDa protein. A part of the 120-kDa protein was solubilized from the membrane vesicles with phosphatidylinositol-specific phospholipase C, resulting in a 110-kDa protein which therefore may be linked to a glycosyl-phosphatidylinositol anchor. The 120-kDa and 110-kDa Cryl Aa toxin-binding proteins were solubilized with detergent or pohosphatidylinositol-specific phospholipase C, respectively, and purified using anion-exchange chromatography. Scatchard plot analysis for the specific binding of purified 110-kDa protein to Cryl Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N-terminal and internal amino acid sequences of the 120-kDa and 110-kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative CrylAc toxin receptor, reported in Munducu sexta and Heliothis virescens. On this basis, the 120-kDa CrylAa toxin-binding protein from B. mori was identified as a member of the aminopeptidase family.Keywords: Bacillus thuringiensis; S-endotoxin ; Bombyx mori; receptor; aminopeptidase N.Bacillus thuringiensis, a gram-positive bacterium, produces various types of insecticidal proteinaceous crystal inclusions during spomlation [l-31. When this bacterium is ingested by susceptible insects, these crystal inclusions, composed of protoxins [4], are solubilized in the alkaline environment of the insect midgut and processed proteolytically to yield smaller active toxins [3, 51. The toxin is believed to bind specifically to receptor molecules on the midgut epithelial cells of host insects [7-101, disrupting the ion permeability of midgut cell membranes [ll]. This results in a net influx of ions and an accompanying influx of water, so that the cells swell and lyse [6, 121. The formation of cation-selective [13-151 or small non-specific [16] pores in the membrane has been proposed as a possible mechanism of the toxin action [17].Each toxin shows a specific insecticidal spectrum in vivo. In some cases, this activity spectrum correlates with the presence of specific toxin receptors in the brush border membrane vesicles (BBMV) [7][8][9][10] 181, while in other cases the binding of the toxin to the BBMV was unrelated to in vivo toxic activity. In the latter cases, toxin bound to BBMV, but very low-level in vivo toxicity resulted [18, 191. Although the binding of toxin to specific receptors on the midgut epithelial cells may be one of the requirements for the insecticidal activity in vivo, other factors, such as the ability of the toxin to interact with the epithelium membrane lipid layer, might also be involved. However, it is not clear what the normal function of the receptor molecules might be or how these molecules are involved in toxin action. Insects are able to acquire resistance to ...

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

“…APN was also identified as a receptor for Cry1Aa in Bombyx mori (silkworm) [14], and for Cry1C and Cry1Ab in Manduca sexta (tobacco hornworm) [15,16]. The APN proteins in M. sexta are distinct, forming a family of related putative toxin receptors.…”

Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac

“…Two membrane proteins, cadherin and aminopeptidase-N (APN), have been implicated as possible receptors to insecticidal proteins. The putative receptor molecules have been cloned, heterologously expressed, and have been shown to bind Cry1A proteins by ligand blot analysis (4)(5)(6)(7)(8)(9)(10)(11). Although functional proof for cadherin as a Cry1A protein receptor has been demonstrated by cytolysis of insect cells expressing lepidopteran cadherin genes on exposure to Cry1Ab/Cry1Aa (10,12,13), a similar characterization of APN-Cry1A interaction has not been investigated yet.…”

Mapping the Epitope in Cadherin-like Receptors Involved inBacillus thuringiensis Cry1A Toxin Interaction Using Phage Display

“…In Manduca sexta, Cry1Aa, Cry1Ab, and Cry1Ac proteins bind to a 120-kDa aminopeptidase N (APN) (11)(12)(13) and to a 210-kDa cadherin-like protein (Bt-R 1 ) (14,15). In Bombyx mori, Cry1Aa binds to a 175-kDa cadherin-like protein (Bt-R 175 ) (16,17) and to a 120-kDa APN (18). In Heliothis virescens, Cry1Ac binds to two proteins of 120 and 170 kDa, both identified as APN (20,21).…”