MOLECULAR-CLONING OF AN INSECT AMINOPEPTIDASE-N THAT SERVES AS A RECEPTOR FOR BACILLUS-THURINGIENSIS CRYIA(C) TOXIN

The Bacillus thuringiensis CryIA(c) insecticidal delta-endotoxin binds to a 120 kDa glycoprotein receptor in the larval midgut epithelia of the susceptible insect Manduca sexta. This glycoprotein has recently b een purified and identified as aminopeptidase N. We now report the clo ning of aminopeptidase N from a M. sexta midgut cDNA library. Two over lapping clones were isolated, and their combined 3095-nucleotide seque nce contains an open reading frame encoding a 990-residue prepro-prote in. The N-terminal amino acid sequence derived from the glycoprotein i s present in the open reading frame, immediately following a predicted cleavable signal peptide and a pro-peptide. There are four potential N-linked glycosylation sites. The C-terminal sequence contains a possi ble glycosylphosphatidylinositol (GPI) anchor signal peptide, which su ggests that, unlike most other characterized aminopeptidases, the lepi dopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sext a midgut brush border membranes by phosphatidylinositol-specific phosp holipase C. The deduced amino acid sequence shows significant similari ty to the zinc-dependent aminopeptidase gene family, particularly in t he region surrounding the consensus zinc-binding motif characteristic of these enzymes.