| 1.
図書 |
図書
|
edited by Hideo Utsumi, Katsuhiko Nakamuro
|
||||||
| 2.
図書 |
図書
|
edited by M. Okamoto, Y. Ishimura, H. Nawata
|
||||||
| 3.
学位論文(リポジトリ) |
学位
|
Shitomi, Yasuyuki
概要:
Bombyx mori, hybrid Shunrei x Shogetsu, is susceptible to Cry1Aa and insensitive to Cry1Ac. Toxicity of Cry toxin can be
…
correlated with the presence of a specific receptor in midgut epithelial cell membrane in insect. In surface plasmon resonance (SPR) and ligand blot analysis, however, many kinds of brush border membrane vesicle (BBMV) proteins were shown to bind to Cry1Ac with almost equal intensity in both resistance and susceptible insects. This suggests that majority of the bindings between Cry1Ac and BBM proteins of B. mori may be pseudo-binding. Therefore, we have hypothesized that these pseudo-bindings to Cry1Ac should not lead to insect death. Cry1A must pass through the peritrophic membrane (PM) before getting to BBM, thus PM plays a vital role for the action of Cry1A toxin as well as BBM. To elucidate the Cry1Ac insusceptibility of B. mori, we searched Cry1Ac binding proteins which are involved in the pseudo-binding in PM and BBM of B. mori. Here, we present the evidences showing some PM and BBM proteins can act as pseudo-receptor for Cry1Ac in B. mori. First of all, we evaluated permeability of Cry1Aa or Cry1Ac through the PM. An apparatus for the estimation of passing through PM was constructed, then BSA (66kDa), Carbonic anhydrase (29 kDa) and Cry1Aa were passed through the PM at 0.37 μg/mm2PM/hr. These passages seemed to be a diffusion because the rate was almost same, even though molecular size was different from each other. In contrast, Cry1Ac didn't pass through the PM for the first 2 hrs. However, during 3rdhr, Cry1Ac passed through the PM at 0.34 μg/mm2PM/hr, which may be due to the destruction of the PM or the saturation of Cry1Ac binding site on the PM. The Cry1Ac trapping during the first 2 hrs can be closely correlated with the interaction of toxins to some PM proteins. We performed ligand blot analysis of Cry1A toxins with PM proteins. As expected, Cry1Ac binding proteins were detected in detergent soluble fractions. Particularly, 125 (P125), 100 (P100), 95 (P95) and 55 (P55) kDa proteins, from class 2 which were obtained with 1% Triton X-100 and 165 (PI65) kDa proteins of class 3 which was extracted with 2% SDS and 2% mercaptoethanol, were shown to bind to Cry1Ac only but not Cry1Aa. These results suggested the prevention of Cry1Ac from passing through the PM happens through the trapping of PM proteins. Cry1Ac recognizes N-acetylgalactosamine (GalNAc) occurring as sugar side chain of proteins localizing in BBM. Cry1Ac trapping by the whole class 2 and 3 PM proteins had 70% inhibition in the presence of GalNAc, but Cry1Aa trapping was not inhibited.<br />In ligand blot, the bindings between Cry1Ac with P125, P95, P55 and P165 were almost completely inhibited by GalNAc. We estimated the GalNAc effect on the Cry1Ac passage through the PM and co-inoculation of Cry1Ac and GalNAc was shown to facilitate the Cry1Ac passage through the PM with the rate of 0.45 μg/mm2PM/Hr. GalNAc residue of PM protein seemed to be involved in Cry1Ac trapping. Secondly, we also detected many kinds of Cry1Ac binding proteins from B. mori whole BBM protein, Triton X-100-soluble and -insoluble BBMV proteins. Although Cry1Ac has no toxicity to B. mori, many kinds of Cry1Ac binding proteins, such as 252, 115, 105, 100, 96 and 75 kDa, were detected. DEAE chromatography of Triton X-100-soluble BBMV proteins from the midgut revealed 96-kDa aminopeptidase that bound to Cry1Ac. The enzyme was purified to homogeneity and estimated to be a 96.4 kDa molecule on a silver-stained SDS-PAGE gel. However, the native protein was eluted as a single peak corresponding to ～190 kDa on gel filtration and gave a single band on native PAGE. The enzyme was determined to be an aminopeptidase N (APN96) from its substrate specificity. Antiserum to class 3 B. mori APN (BmAPN3) recognized APN96, but peptide mass fingerprinting revealed that 54% of amino the acids of matched peptides were identical to those of BmAPN3, suggesting that APN96 was a novel isoform of the APN3 family. On ligand blots, APN96 bound to Cry1Ac but not Cry1Aa or Cry1Ab, and the interaction was inhibited by GalNAc. The KD value of the APN96-Cry1Ac interaction was determined to be 1.83± 0.95μM. The lectin binding assay suggested that APN96 had an N-linked oligosaccharide or an O-linked mucin type one. The role of APN96 and other Cry1Ac binding proteins were discussed in relation to the insensitivity of B. mori to Cry1Ac. We believe that majority of these Cry1Ac binding proteins from both PM and BBM may act as pseudo-receptors to quench the toxicity of Cry1Ac, and propose here the model of mechanism for detoxification of Cry1Ac in B. mori. Activated Cry1Ac is initially trapped by PM proteins. In case, some Cry1Ac molecules pass through the PM and bind to BBM proteins, such as APN96, P252, 100, 105 and 75 kDa proteins. The bindings with these proteins reduce further the toxicity of Cry1Ac. It was reported that both the cadherin like protein and APN were shown to be related with Cry1Ab toxicity in Manduca sexta. We guess, however, there is no such involvement of these proteins in CrylAc binding in B. mori. CrylAc must bind to PM and/or BBMV proteins and stay there for a while without modification, thus the Cry1Ac can not form pores on BBMV.<br />新大院博（学）甲第167号
続きを見る
|
||||||
| 4.
図書 |
図書
|
福井希一, 福住俊一, ルーク・上田サーソン編
|
