THE HELIOTHIS-VIRESCENS 170KDA AMINOPEPTIDASE FUNCTIONS AS RECEPTOR-ABY MEDIATING SPECIFIC BACILLUS-THURINGIENSIS CRY1A DELTA-ENDOTOXIN BINDING AND PORE FORMATION

The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry 1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens bru sh border membranes, Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-ac etylgalactosamine, The 140 kDa aminopeptidase has a cross-reacting det erminant typical of a cleaved glycosyl-phosphatidylinositol anchor. Af ter mild base treatment to de-acylate the glycosyl-phosphatidylinosito l linkage and incubation in phosphatidyl inositol phospholipase C, ant i-cross-reacting determinant antibody recognized the 170 kDa protein, Kinetic binding characteristics of Cry1A toxins to purified 170 kDa AP N were determined using surface plasmon resonance, Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN, Each Cr y1A toxin recognized two binding sites: a high affinity site with K-D ranging from 41 to 95 nM and a lower affinity site with K-D in the 325 to 623 nM range, N-acetylgalactosamine inhibited Cry1Ac but not Cry1A a and Cry1Ab binding to 170 kDa APN, When reconstituted into phospholi pid vesicles, the 170 kDa APN promoted toxin-induced Rb-86(+) release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry pro tein most toxic to H. virescens larvae, caused Rb-86(+) release at low er concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxi ns, The correlation between toxin-binding specificity and Rb-86(+) rel ease strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border me mbranes, (C) 1997 Elsevier Science Ltd, All rights reserved.