The Heliothis virescens 170kDa aminopeptidase functions as "receptor A" by mediating specific Bacillus thuringiensis Cry1A δ-endotoxin binding and pore formation

被引:126
作者
Luo, K
Sangadala, S
Masson, L
Mazza, A
Brousseau, R
Adang, MJ [1 ]
机构
[1] Univ Georgia, Dept Biochem & Mol Biol, Athens, GA 30602 USA
[2] Univ Georgia, Dept Entomol, Athens, GA 30602 USA
[3] Natl Res Council Canada, Biotechnol Res Inst, Quebec City, PQ H4P 2R2, Canada
关键词
Bacillus thuringiensis; Cry1A; delta-endotoxins; Heliothis virescens; aminopeptidase N; receptor; GPI anchor; surface plasmon resonance; pore formation;
D O I
10.1016/S0965-1748(97)00052-0
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The relationship between Bacillus thuringiensis Cry1Aa, Cry1Ab and Cry1Ac delta-endotoxin binding and pore formation was investigated using a purified 170 kDa aminopeptidase N (APN) from Heliothis virescens brush border membranes, Aminopeptidases with molecular sizes of 110, 140 and 170 kDa were eluted from a Cry1Ac toxin affinity column using N-acetylgalactosamine, The 140 kDa aminopeptidase has a cross-reacting determinant typical of a cleaved glycosyl-phosphatidylinositol anchor. After mild base treatment to de-acylate the glycosyl-phosphatidylinositol linkage and incubation in phosphatidyl inositol phospholipase C, anti-cross-reacting determinant antibody recognized the 170 kDa protein, Kinetic binding characteristics of Cry1A toxins to purified 170 kDa APN were determined using surface plasmon resonance, Cry1Aa, Cry1Ab and Cry1Ac, but not Cry1C and Cry1E toxins recognized 170 kDa APN, Each Cry1A 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 Cry1Aa and Cry1Ab binding to 170 kDa APN, When reconstituted into phospholipid vesicles, the 170 kDa APN promoted toxin-induced Rb-86(+) release for Cry1A toxins, but not Cry1C toxin. Furthermore Cry1Ac, the Cry protein most toxic to H. virescens larvae, caused Rb-86(+) release at lower concentrations, and to a greater extent than Cry1Aa and Cry1Ab toxins, The correlation between toxin-binding specificity and Rb-86(+) release strongly suggests that the purified 170 kDa APN is the functional receptor A in the H. virescens midgut epithelial cell brush border membranes, (C) 1997 Elsevier Science Ltd, All rights reserved.
引用
收藏
页码:735 / 743
页数:9
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