CLONING AND CHARACTERIZATION OF MANDUCA-SEXTA AND PLUTELLA-XYLOSTELLAMIDGUT AMINOPEPTIDASE-N ENZYMES RELATED TO BACILLUS-THURINGIENSIS TOXIN-BINDING PROTEINS

We report the purification, cloning and characterization of an aminope ptidase N from the midgut epithelium of Manduca sexta that binds Cry1A b5, an insecticidal crystal protein [ICP] from Bacillus thuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning of an aminopeptidase N from the midgut brush-bord er membrane of Plutella xylostella, an insect species of which some po pulations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from t he larval midgut of the lepidopteran M. sexta. On ligand blots the pur ified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal a mino acid sequences from the 120-kDa protein were used for the design of degenerate oligonucleotides. From a nested PCR with M. sexta midgut cDNA as template, a DNA fragment was obtained which shows similarity to prokaryotic and eukaryotic aminopeptidase N genes, This PCR fragmen t was used to screen cDNA libraries of larval midguts from M. sexta an d P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucle otide sequence was cloned. The ORF of the sequence encodes a 942-resid ue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions . The NH2-terminal hydrophobic region corresponds to a secretory signa l sequence and the COOH-terminal hydrophobic region is typical of glyc osylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stri ngency hybridization of the P. xylostella midgut cDNA library with M. sexta apn2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N (P. xy lostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and c ontains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addi tion. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys resid ues, which are highly conserved among eukaryotic aminopeptidase N mole cules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene w ith PtdIns-specific phospholipase C demonstrated that P. xylostella Ap n1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor .