Cloning and characterization of Manduca sexta and Plutella xylostella midgut aminopeptidase N enzymes related to Bacillus thuringiensis toxin-binding proteins.
We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1Ab5, 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-border membrane of Plutella xylostella, an insect species of which some populations acquired resistance against Cry1Ab5. Affinity chromatography on a Cry1Ab5 matrix was used to isolate a 120-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1Ab5 and the coleopteran-specific Cry3A delta-endotoxin. Internal amino 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 fragment was used to screen cDNA libraries of larval midguts from M. sexta and P. xylostella. From the M. sexta midgut cDNA library a 2973-bp nucleotide sequence was cloned. The ORF of the sequence encodes a 942-residue aminopeptidase N (M. sexta Apn2) containing two hydrophobic regions. The NH2-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdIns)-anchored proteins. Low-stringency 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. xylostella Apn1) displays 61% amino acid identity to M. sexta Apn2 and contains a COOH-terminal signal peptide for glycosyl-PtdIns anchor addition. Both M. sexta Apn2 and P. xylostella Apn1 contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P. xylostella apn1 gene with PtdIns-specific phospholipase C demonstrated that P. xylostella Apn1 is attached to the insect cell membrane by a glycosyl-PtdIns anchor.
Denolf
P
P
Plant Genetic Systems, Gent, Belgium. denolf@pgsgent.be
Hendrickx
K
K
Van Damme
J
J
Jansens
S
S
Peferoen
M
M
Degheele
D
D
Van Rie
J
J
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000818:Animals; D001413:Bacillus thuringiensis / Q000737:chemistry*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism* / Q000494:pharmacology; D001427:Bacterial Toxins; D001483:Base Sequence; D015152:Blotting, Northern; D018826:CD13 Antigens / Q000737:chemistry; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004731:Endotoxins / Q000378:metabolism* / Q000494:pharmacology; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry; D017261:Glycosylphosphatidylinositols / Q000737:chemistry; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007305:Insecticide Resistance; D007306:Insecticides / Q000378:metabolism / Q000494:pharmacology; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D009036:Moths / Q000201:enzymology* / Q000235:genetics; D011485:Protein Binding; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D018411:Spodoptera / Q000235:genetics / Q000378:metabolism
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006023:Glycoproteins; D017261:Glycosylphosphatidylinositols; D006460:Hemolysin Proteins; D019476:Insect Proteins; D007306:Insecticides; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000626:Aminopeptidases; D018826:CD13 Antigens