Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C.
Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.
International Center for Genetic Engineering and Biotechnology, New Delhi-110067, India.
Neema
N
Agrawal
Pawan
P
Malhotra
Raj K
RK
Bhatnagar
D000595:Amino Acid Sequence; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D001427:Bacterial Toxins; D018826:CD13 Antigens / Q000096:biosynthesis* / Q000145:classification / Q000235:genetics; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins; D008565:Membrane Proteins / Q000378:metabolism; D008871:Microvilli / Q000378:metabolism; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000378:metabolism; D018411:Spodoptera / Q000166:cytology / Q000201:enzymology*
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D008565:Membrane Proteins; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens