ABSTRACT
Bacillus thuringiensis Cry toxins are insecticidal proteins used widely for pest control. They are lethal to a restricted range of insects via specific interactions with insect receptors such as the ABC transporter subfamily members C2 (ABCC2) and C3 (ABCC3). However, it is still unclear how these different receptors contribute to insect susceptibility to Cry1A toxins. Here, we investigated the differences between the silkworm (Bombyx mori) ABCC2 (BmABCC2_S) and ABCC3 (BmABCC3) receptors in mediating Cry toxicity. Compared with BmABCC2_S, BmABCC3 exhibited 80- and 267-fold lower binding affinities to Cry1Aa and Cry1Ab, respectively, and these decreased affinities correlated well with the lower receptor activities of BmABCC3 for these Cry1A toxins. To identify the amino acid residues responsible for these differences, we constructed BmABCC3 variants containing a partial amino acid replacement with extracellular loops (ECLs) from BmABCC2_S. Replacing three amino acids from ECL 1 or 3 increased BmABCC3 activity toward Cry1Aa and enabled its activity toward Cry1Ab. Meanwhile, BmABCC2_S and BmABCC3 exhibited no receptor activities for Cry1Ca, Cry1Da, and Cry3Bb, correlating with markedly lower binding affinities for these Cry toxins. ABCC2 from a Cry1Ab-resistant B. mori strain (BmABCC2_R), which has a tyrosine insertion in ECL 2, displayed 93-fold lower binding affinity to Cry1Ab compared with BmABCC2_S but maintained high binding affinity to Cry1Aa. These results indicate that the Cry toxin-binding affinities of ABCC transporters are largely linked to the level of Cry susceptibility of ABCC-expressing cells and that the ABCC ECL structures determine the specificities to Cry toxins.
Subject(s)
ATP-Binding Cassette Transporters/chemistry , ATP-Binding Cassette Transporters/metabolism , Bacterial Proteins/metabolism , Bombyx/metabolism , Endotoxins/metabolism , Hemolysin Proteins/metabolism , Insect Proteins/chemistry , Insect Proteins/metabolism , Insecticides/metabolism , Animals , Bacillus thuringiensis Toxins , Bacterial Proteins/chemistry , Bombyx/growth & development , Endotoxins/chemistry , HEK293 Cells , Hemolysin Proteins/chemistry , Humans , Insecticides/chemistry , Multidrug Resistance-Associated Protein 2 , Protein Conformation , Substrate SpecificityABSTRACT
Human intestinal absorption is estimated using a human colon carcinoma cell line (Caco-2) cells from human colorectal adenocarcinoma, intestinal perfusion, or a mammalian model. These current evaluation systems are limited in their ability to estimate human intestinal absorption. In addition, in vivo evaluation systems using laboratory animals such as mice and rats entail animal ethics problems, and it is difficult to screen compounds on a large scale at the drug discovery stage. Thus, we propose the use of Bombyx mori larvae for evaluation of intestinal absorption of compounds as an alternative system in this study. First, to compare the characteristics among Caco-2 cells, human intestine, and B. mori larval midgut, we analyzed their RNA-seq data, and we found 26 drug transporters common to humans and B. mori. Next, we quantitatively developed an oral administration technique in B. mori and established a method using silkworm B. mori larvae that can easily estimate the intestinal permeability of compounds. Consequently, we could determine the dose and technique for oral administration in B. mori larvae. We also developed a B. mori model to evaluate the intestinal permeability of orally administered. Our constructed evaluation system will be useful for evaluating intestinal permeability in medical drug development.
