Characterization of a Novel Plasma Membrane Protein, Expressed in the Midgut Epithelia of Bombyx mori , That Binds to Cry1A Toxins
Open Access
- 1 August 2004
- journal article
- research article
- Published by American Society for Microbiology in Applied and Environmental Microbiology
- Vol. 70 (8) , 4604-4612
- https://doi.org/10.1128/aem.70.8.4604-4612.2004
Abstract
We describe the properties of a novel 252-kDa protein (P252) isolated from brush border membranes of Bombyx mori. P252 was found in a Triton X-100-soluble brush border membrane vesicle fraction, suggesting that it may be a component of the midgut epithelial cell membrane. P252 was purified to homogeneity, and the amino acid sequence of two internal peptides was determined, but neither of the peptides matched protein sequences in the available databases. The apparent molecular mass of the purified protein was estimated by denaturing gel electrophoresis to be 252 kDa, and it migrated as a single band on native gels. However, gel filtration chromatography indicated an apparent mass of 985 kDa, suggesting that P252 may exist as a homo-oligomer. The associations of P252 with Cry1Aa, Cry1Ab, and Cry1Ac were specific, and Kd constants were determined to be 28.9, 178.5, and 20.0 nM, respectively. A heterologous competition assay was also done. P252 did not exhibit Leu-pNA hydrolysis activity, and binding to the Cry1A toxins was not inhibited by GalNAc. Binding assays of P252 with various lectins indicated the presence of three antennal N-linked high-mannose-type as well as O-linked mucin-type sugar side chains. While the function of P252 is not yet clear, we propose that it may function with Cry1A toxins during the insecticidal response and/or Cry toxin resistance mechanism.Keywords
This publication has 46 references indexed in Scilit:
- Aminopeptidase N isoforms from the midgut of Bombyx mori and Plutella xylostella – their classification and the factors that determine their binding specificity to Bacillus thuringiensis Cry1A toxinFEBS Letters, 2002
- Cadherin‐like receptor binding facilitates proteolytic cleavage of helix α‐1 in domain I and oligomer pre‐pore formation of Bacillus thuringiensis Cry1Ab toxinFEBS Letters, 2002
- Identification of a Gene Associated with Bt Resistance in Heliothis virescensScience, 2001
- The Heliothis virescens 170kDa aminopeptidase functions as “Receptor A” by mediating specific Bacillus thuringiensis Cry1A δ-endotoxin binding and pore formationInsect Biochemistry and Molecular Biology, 1997
- Brush border membrane aminopeptidase-n in the midgut of the gypsy moth serves as the receptor for the CryIA(c) δ-endotoxin of Bacillus thuringiensisInsect Biochemistry and Molecular Biology, 1995
- Identification, Isolation, and Cloning of a Bacillus thuringiensis CryIAc Toxin-binding Protein from the Midgut of the Lepidopteran Insect Heliothis virescensPublished by Elsevier ,1995
- Dimeric Assembly of Enterocyte Brush Border EnzymesBiochemistry, 1994
- The receptor for Bacillus thuringiensis CrylA(c) delta‐endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase NMolecular Microbiology, 1994
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970