Cloning and complete sequence characterization of two gypsy moth aminopeptidase-N cDNAs, including the receptor for Bacillus thuringiensis Cry1Ac toxin

• Published in: Insect biochemistry and molecular biology 1999-06, Vol.29 (6), p.527-535
• Main Authors: Garner, Karen J, Hiremath, Shiv, Lehtoma, Kirsten, Valaitis, Algimantas P
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acid sequences; Aminopeptidase-N; aminopeptidases; Aminopeptidases - genetics; Animals; apn1; APN1 gene; APN2 gene; Bacillus thuringiensis; Bacillus thuringiensis receptor; Bacterial Proteins; Baculovirus; Base Sequence; binding sites; cDNA sequence; Cell Line; cloning; Cloning, Molecular; complementary DNA; Cry1Ac protein; crystal proteins; d-endotoxin; DNA Probes; DNA, Complementary; endotoxins; Expression; genbank/af126442; genbank/af126443; Gene Expression; Genetic Vectors; Gypsy moth; Insect Proteins; lambda-apn2; Lymantria dispar; messenger RNA; midgut; Molecular Sequence Data; Moths - enzymology; Moths - genetics; Nucleopolyhedrovirus; nucleotide sequences; receptors; Receptors, Cell Surface - genetics; Reverse Transcriptase Polymerase Chain Reaction; sequence alignment; Sequence Homology, Amino Acid; signal peptide; Spodoptera - cytology; toxin receptors
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/S0965-1748(99)00027-2

The complete cDNAs corresponding to two distinct gypsy moth ( Lymantria dispar) larval gut aminopeptidases, APN1 and λAPN2, were cloned and sequenced. The 3.4 kilobasepair cDNA of APN1 which encodes a 1017 amino acid prepro-protein corresponds to the previously-identified gypsy moth APN (APN-1) that specifically binds the Cry1Ac δ-endotoxin of Bacillus thuringiensis. Analysis of the primary structure of APN1 revealed a cluster of five potential N-linked glycosylation sites near the N-terminus and a C-terminal sequence characteristic of a putative glycosylphosphatidyl-inositol (GPI) anchor signal sequence. The cDNA of APN1 encodes the N-terminal peptide sequence and nine internal sequences obtained from the purified brush border membrane vesicle Cry1Ac receptor by protein sequencing. The λAPN2 cDNA encodes a shorter protein with 51% similarity to APN1 that also appears to have a GPI anchor signal sequence. Expression of the APN1 cDNA in a baculovirus vector was confirmed by immunoblotting.