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 3205bp in size and encodes a 1000-amino-acid protein, while H. armigera APN2 (haapn2) is 3116bp 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.
Abbreviations used: AP, alkaline phosphatase; APN, aminopeptidase N; BBMV, brush-border membrane vesicle; BCIP, 5-bromo-4-chloroindol-3-yl phosphate toluidine; Bt, Bacillus thuringiensis; Cry, crystalline inclusion protein; GPI, glycosyl-phosphatidylinositol; HaAPN, Helicoverpa armigera APN; ICP, insecticidal crystal protein; NBT, Nitroblue Tetrazolium; ORF, open reading frame; RACE, rapid amplification of cDNA ends; SPR, surface plasmon resonance.
Present address: Laboratory of Bacterial, Parasitic and Unconventional Agents, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20814, U.S.A.
Present address: Department of Microbiology, Faculty of Medicine, Kuwait University, Safat, 13110, Kuwait.