Identification of novel Cry1Ac binding proteins in midgut membranes from Heliothis virescens using proteomic analyses.
Proteins such as aminopeptidases and alkaline phosphatases, both glycosyl-phosphatidyl-inositol (GPI) anchored proteins, were previously identified as Cry1Ac binding proteins in the Heliothis virescens midgut. To identify additional toxin binding proteins, brush border membrane vesicles from H. virescens larvae were treated with phosphatidyl inositol phospholipase C, and released proteins were resolved by two-dimensional electrophoresis. Protein spots selected by their ability to bind Cry1Ac were identified by MALDI-TOF mass spectrometry coupled to peptide mass fingerprinting (PMF) and database searching. As in previous studies, H. virescens alkaline phosphatase was identified as a Cry1Ac binding protein. V-ATP synthase subunit A and actin were identified as novel Cry1Ac binding proteins in H. virescens. Additional toxin-binding proteins were predicted based on MS/MS fragmentation and de novo sequencing, providing amino acid sequences that were used in database searches to identify a phosphatase and a putative protein of the cadherin superfamily as additional Cry1Ac binding proteins.
Krishnamoorthy
M
M
Departments of Entomology, University of Georgia, Athens, GA 30602-2603, USA.
Jurat-Fuentes
J L
JL
McNall
R J
RJ
Andacht
T
T
Adang
Michael J
MJ
D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004731:Endotoxins / Q000378:metabolism*; D041981:Gastrointestinal Tract / Q000737:chemistry / Q000166:cytology; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006460:Hemolysin Proteins / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry*; D013058:Mass Spectrometry; D008871:Microvilli / Q000737:chemistry*; D009036:Moths / Q000737:chemistry*; D010449:Peptide Mapping; D040901:Proteomics; D011956:Receptors, Cell Surface / Q000737:chemistry*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; C095416:Cry toxin receptors; D004731:Endotoxins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D019476:Insect Proteins; D011956:Receptors, Cell Surface; C030956:insecticidal crystal protein, Bacillus Thuringiensis