Cadherin‐like receptor binding facilitates proteolytic cleavage of helix α‐1 in domain I and oligomer pre‐pore formation of Bacillus thuringiensis Cry1Ab toxin

Abstract

Cry toxins form lytic pores in the insect midgut cells. The role of receptor interaction in the process of protoxin activation was analyzed. Incubation of Cry1Ab protoxin with a single chain antibody that mimics the cadherin-like receptor and treatment with Manduca sexta midgut juice or trypsin, resulted in toxin preparations with high pore-forming activity in vitro. This activity correlates with the formation of a 250 kDa oligomer that lacks the helix K K-1 of domain I. The oligomer, in contrast with the 60 kDa monomer, was capable of membrane insertion as judged by 8-anilino-1-naphthalenesulfonate binding. Cry1Ab protoxin was also activated to a 250 kDa oligomer by incubation with brush border membrane vesicles, presumably by the action of a membrane-associated protease. Finally, a model where receptor binding allows the efficient cleavage of K K-1 and formation of a pre-pore oligomeric structure that is efficient in pore formation, is presented. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Permeability Changes of Manduca sexta Midgut Brush Border Membranes Induced by Oligomeric Structures of Different Cry Toxins

“…In the case of the Cry1A toxins, it was reported that they interact sequentially with two protein receptors located in the apical membrane of Manduca sexta larvae midgut cells [3,4]. The interaction of the monomeric Cry1A with the cadherin receptor induces toxin oligomerization [4,5]. The oligomerization of Cry1A toxin increases its affinity to the second receptor, aminopeptidase-N. Aminopeptidase-N localizes the toxin in membrane microdomains, where it is inserted and induces the formation of ionic pores [4,6].…”

“…These ionic pores shunt the potential difference gradient and disrupt the K + and H + gradients, affecting nutritional uptake with eventual lysis of the midgut cells [2]. It was proposed that the oligomeric structure of Cry1A toxin is an intermediary in the process of membrane insertion; it is defined as pre-pore since it is formed outside of the membrane after interacting with cadherin and the cleavage of helix a-1 [5]. The pre-pore is an insertion-competent structure that produces stable channels in black lipid bilayers with high open probability in contrast to the monomeric structure [7].…”

Cytotoxicity of Bacillus thuringiensis Cry1Ab toxin depends on specific binding of the toxin to the cadherin receptor BT-R1 expressed in insect cells

“…37 The toxin oligomers, usually in dimeric and tetrameric form, are SDS-resistant and could be detected by SDS-PAGE Western blotting. 21,26,38 We were interested in determining whether Cry1Ab interacts with the cytoplasmic membrane of living cells in a similar way, and, if so, whether these interactions contribute to cytotoxicity. Therefore, we analyzed toxin-membrane interactions in S5 and H5 cells by examining protein samples prepared from membrane and cytoplasmic Antibody against Cry1Ab was used to detect the toxin in protein fractions corresponding to membrane pellet (P) and supernatant containing soluble proteins (S).…”

“…[22][23][24][25] In the pore-forming model of Cry toxin action, the toxin monomers are considered precursors to oligomeric assembly. 21,25 The formation of toxin oligomer also has been postulated to be the result of the interaction of monomers that were bound to the cadherin receptor. 48 Our results clearly show differential association profiles for the oligomeric and monomeric forms of Cry1Ab toxin in cell membrane, the consequence of two distinct modes of interaction with the insect cells.…”

“…18 Like many other bacterial toxins, Cry toxins incorporate into lipid bilayer rafts as well as brush border membrane vesicles (BBMV). 19 Lipid bilayerand BBMV-associated Cry toxin molecules have been reported to appear in oligomeric (dimeric and tetrameric) and monomeric forms, 20,21 and these associations have been correlated with changes in membrane permeability. 22 The oligomeric forms of Cry toxin were presumed to be pores that cause cell death by osmotic lysis.…”