title: Recombinantly expressed isoenzymic aminopeptidases from Helicoverpa armigera (American cotton bollworm) midgut display differential interaction with closely related Bacillus thuringiensis insecticidal proteins.
abstract: Several investigators have independently identified membrane-associated aminopeptidases in the midgut of insect larvae as the initial interacting ligand to the insecticidal crystal proteins of Bacillus thuringiensis. Though several isoenzymes of aminopeptidases have been identified from the midgut of an insect and their corresponding cDNA cloned, only one of the isoform has been expressed heterologously and studied for its binding to Cry toxins. Here we report the cloning and expression of two aminopeptidases N from Helicoverpa armigera (American cotton bollworm) (HaAPNs). The full-length cDNA of H. armigera APN1 (haapn1) is 3205 bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116 bp in size and corresponds to a 1012-amino-acid protein. Structurally these proteins show sequence similarity to other insect aminopeptidases and possess characteristic aminopeptidase motifs. Both the genes have been expressed in Trichoplusia ni (cabbage looper) cells using a baculovirus expression vector. The expressed aminopeptidases are membrane-associated, catalytically active and glycosylated. Ligand-blot analysis of both these aminopeptidases with bioactive Cry1Aa, Cry1Ab and Cry1Ac proteins displayed differential interaction. All the three toxins bound to HaAPN1, whereas only Cry1Ac interacted with HaAPN2. This is the first report demonstrating differential Cry-toxin-binding abilities of two different aminopeptidases from a susceptible insect.
authors.affiliation: Insect Resistance Group, International Centre for Genetic Engineering and Biotechnology (ICGEB), PO Box 10504, Aruna Asaf Ali Marg, New Delhi 110 067, India.
authors.forename: R
authors.initials: R
authors.lastname: Rajagopal
authors.forename: Neema
authors.initials: N
authors.lastname: Agrawal
authors.forename: Angamuthu
authors.initials: A
authors.lastname: Selvapandiyan
authors.forename: S
authors.initials: S
authors.lastname: Sivakumar
authors.forename: Suhail
authors.initials: S
authors.lastname: Ahmad
authors.forename: Raj K
authors.initials: RK
authors.lastname: Bhatnagar
mesh_terms: D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins; D003001:Cloning, Molecular; D004064:Digestive System / Q000201:enzymology; D004731:Endotoxins / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D006460:Hemolysin Proteins; D007527:Isoenzymes / Q000737:chemistry / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D007915:Lepidoptera / Q000033:anatomy & histology / Q000201:enzymology* / Q000235:genetics; D008024:Ligands; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D007527:Isoenzymes; D008024:Ligands; D011994:Recombinant Proteins; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000626:Aminopeptidases