Binding of Bacillus thuringiensis delta-endotoxins Cry1Ac and Cry1Ba to a 120-kDa aminopeptidase-N of Epiphyas postvittana purified from both brush border membrane vesicles and baculovirus-infected Sf9 cells.
A 120-kDa protein was purified from brush border membrane vesicles of the tortricid moth Epiphyas postvittana (Walker) based both on its activity as an aminopeptidase and the ability to bind the Bacillus thuringiensis delta-endotoxin Cry1Ac. The purified enzyme had a pI of 5.6 and was a leucine aminopeptidase, with some isoleucine, phenylalanine and tryptophan aminopeptidase activity. Further characterisation showed that the protein was also able to bind Cry1Ba. During purification, the molecular weight of the protein decreased from 120 to 115 kDa due to the loss of a glycophosphatidinyl anchor. The protein was N-terminally sequenced and, using this information and conserved regions within other insect aminopeptidase-N (APN) sequences, redundant primers were designed to amplify the aminopeptidase coding sequence from E. postvittana midgut cDNA. The predicted protein sequence from the full-length cDNA was most closely related to the APN protein sequence from Heliothis virescens (61% identity) and shared other features of insect APNs including a Zn(2+) binding site motif and four conserved cysteines. The E. postvittana was expressed in Sf9 cells using baculovirus, yielding a protein of molecular weight 130 kDa, but with unchanged N-terminal sequence. Purified recombinant protein bound both Cry1Ac and Cry1Ba by ligand blot assays. However, despite the protein being expressed on the external surface of the Sf9 cells, it bound neither Cry1Ac nor Cry1Ba in vivo.
Simpson
R M
RM
Horticulture and Food Research Institute of New Zealand Limited, Private Bag 92-169, Auckland, New Zealand. rsimpson@hort.cri.nz
Newcomb
R D
RD
D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D016367:Baculoviridae; D001483:Base Sequence; D001665:Binding Sites / Q000502:physiology; D018826:CD13 Antigens / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D004731:Endotoxins / Q000378:metabolism*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008871:Microvilli / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths / Q000382:microbiology / Q000502:physiology* / Q000821:virology
D004731:Endotoxins; D019476:Insect Proteins; D007931:Leucyl Aminopeptidase; D018826:CD13 Antigens