Location of the Bombyx mori 175kDa cadherin-like protein-binding site on Bacillus thuringiensis Cry1Aa toxin.
To identify and gain a better understanding of the cadherin-like receptor-binding site on Bacillus thuringiensis Cry toxins, it is advantageous to use Cry1Aa toxin, because its 3D structure is known. Therefore, Cry1Aa toxin was used to examine the locations of cadherin-like protein-binding sites. Initial experiments examining the binding compatibility for Cry1Aa toxin of partial fragments of recombinant proteins of a 175kDa cadherin-like protein from Bombyx mori (BtR175) and another putative receptor for Cry1Aa toxin, amino peptidaseN1, from Bo.mori (BmAPN1), suggested that their binding sites are close to each other. Of the seven mAbs against Cry1Aa toxin, two mAbs were selected that block the binding site for BtR175 on Cry1Aa toxin: 2A11 and 2F9. Immunoblotting and alignment analyses of four Cry toxins revealed amino acids that included the epitope of mAb 2A11, and suggested that the area on Cry1Aa toxin blocked by the binding of mAb 2A11 is located in the region consisting of loops2 and 3. Two Cry1Aa toxin mutants were constructed by substituting a Cys on the area blocked by the binding of mAb 2A11, and the small blocking molecule, N-(9-acridinyl)maleimide, was introduced at each Cys substitution to determine the BtR175-binding site. Substitution of Tyr445 for Cys had a crippling effect on binding of Cry1Aa toxin to BtR175, suggesting that Tyr445 may be in or close to the BtR175-binding site. Monoclonal antibodies that blocked the binding site for BtR175 on Cry1Aa toxin inhibited the toxicity of Cry1Aa toxin against Bo.mori, indicating that binding of Cry1Aa toxin to BtR175 is essential for the action of Cry1Aa toxin on the insect.
Atsumi
Shogo
S
Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Japan.
Inoue
Yukino
Y
Ishizaka
Takahisa
T
Mizuno
Eri
E
Yoshizawa
Yasutaka
Y
Kitami
Madoka
M
Sato
Ryoichi
R
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D001665:Binding Sites; D012831:Bombyx; D015820:Cadherins / Q000378:metabolism*; D004731:Endotoxins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D000939:Epitopes; D006460:Hemolysin Proteins / Q000737:chemistry* / Q000235:genetics / Q000633:toxicity; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016415:Sequence Alignment
D000911:Antibodies, Monoclonal; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D015820:Cadherins; D004731:Endotoxins; D000939:Epitopes; D006460:Hemolysin Proteins; D011993:Recombinant Fusion Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis