Cry毒素の受容体結合領域と受容体利用能力に関する研究

概要

Cry toxins, insecticidal proteins produced by the soil bacterium Bacillus thuringiensis, have specific toxicity only against the narrow group of insects. Since it is not toxic to humans and other mammals, the Cry toxin has been used as gene sources for pest-resistant genetically modified crops and has continued to support agriculture. This insecticidal specificity is thought to depend on the specific binding between Cry toxins and insect’s receptors. Elucidating the mechanism of action of Cry toxins, particularly the mechanism that generates specificity, is one of the additional evidences that ensures the safety of Cry toxins. This requires more detailed information on Cry toxin-receptor binding. After binding to the receptor, Cry toxin undergo structural changes and insert into the cell membrane, then show toxicity to target cells by opening small toxin pore in the cell membrane. There are several reports of membrane proteins that bind to Cry toxins, but only molecules that can bind to Cry toxins and promote the formation of toxin-pore can be defined as Cry toxin receptors. Cadherin-like protein and ATP binding cassette transporter (ABC transporter) seems to be important receptors, because insects gain the high resistance to Cry1 toxins when the genes were broken. Furthermore, recent studies showed that Cry toxins extensively use insect ABC transporter family molecules as main receptors in intoxication.

In this doctoral thesis, I tried to clarify where and how Cry toxin bind to the receptor, focusing on analysis of receptor binding region on Cry toxin. The main subject of analysis was an ABC transporter that had little knowledge of its binding relationship with Cry toxins because it was difficult to handle due to its complex structure. In Chapters 1 and 2, I analyzed the regions on Cry1Aa that affect the binding and cytotoxic activity against Bombyx mori ABC transporter C2 (BmABCC2). In briefly, after investigating which domain of Cry1Aa is important for binding to BmABCC2, I made various mutants of Cry1Aa and carefully analyzed the effects on binding affinity and cytotoxic activity against BmABCC2. In addition, the binding region on Cry1Aa for B. mori cadherin-like protein (BtR175), which were already known, was analyzed again by the same method as in BmABCC2 and carefully considered whether the regions were overlapped with BmABCC2. Furthermore, in Chapter 2, I examined how the receptor-binding region is conserved among different Cry toxins. Based on the results, I inferred what kind of molecular evolution of Cry toxins changed the usage of receptors. Finally, the analysis in Chapter 3 began with an attempt to find the receptor for Cry1Ca from ABC transporter family molecule expressed on Sf9 cells, which are ovary-derived cultured cells of Spodoptera frugiperda and are sensitive to Cry1Ca. Furthermore, based on the RNA-seq data derived from the midgut of the B. mori, I analyzed the type and expression level of the ABC transporters expressed in the midgut of the B. mori and searched carefully whether they had the function of Cry toxin receptor. The results suggested that ABC transporters available by a certain Cry toxin are located within a close range of the phylogenetic tree. This probably means that the closeness of Cry toxin is likely to correlate to some extent with the closeness of ABC transporter.

In summary, in this doctoral thesis, I tried to infer some of the aspects of Cry toxins that have become specific to a wide range of insect species by changing their amino acid sequence and structure little by little and expanding the usage of receptors.