Molecular cloning of an insect aminopeptidase N that serves as a receptor for Bacillus thuringiensis CryIA(c) toxin

• Published in: The Journal of biological chemistry 1995-07, Vol.270 (30), p.17765-17770
• Main Authors: Knight, P.J.K. (University of Cambridge, Cambridge, UK.), Knowles, B.H, Ellar, D.J
• Format: Article
• Language: English
• Subjects: ADN; Amino Acid Sequence; AMINOPEPTIDASA; AMINOPEPTIDASE; Animals; BACILLUS THURINGIENSIS; Bacillus thuringiensis - metabolism; Bacillus thuringiensis Toxins; Bacterial Proteins - metabolism; Bacterial Toxins; Base Sequence; CD13 Antigens - genetics; CD13 Antigens - metabolism; CHIMIORECEPTEUR; CLONACION; CLONAGE; Cloning, Molecular; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; DNA, Complementary; ENDOTOXINAS; ENDOTOXINE; Endotoxins - metabolism; Hemolysin Proteins; Insect Proteins; Manduca - enzymology; Manduca sexta; Membrane Proteins - genetics; Membrane Proteins - metabolism; Molecular Sequence Data; Polymerase Chain Reaction; QUIMIORECEPTORES; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; SECUENCIA NUCLEOTIDICA; Sequence Homology, Amino Acid; SEQUENCE NUCLEOTIDIQUE; TOXINAS BACTERIANAS; TOXINE BACTERIENNE
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.30.17765

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 been purified and identified as aminopeptidase N. We now report the cloning of aminopeptidase N from a M. sexta midgut cDNA library. Two overlapping clones were isolated, and their combined 3095-nucleotide sequence contains an open reading frame encoding a 990-residue prepro-protein. The N-terminal amino acid sequence derived from the glycoprotein is 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 possible glycosylphosphatidylinositol (GPI) anchor signal peptide, which suggests that, unlike most other characterized aminopeptidases, the lepidopteran enzyme is anchored in the membrane by a GPI anchor. This was confirmed by partial release of aminopeptidase N activity from M. sexta midgut brush border membranes by phosphatidylinositol-specific phospholipase C. The deduced amino acid sequence shows significant similarity to the zinc-dependent aminopeptidase gene family, particularly in the region surrounding the consensus zinc-binding motif characteristic of these enzymes