Cloning and Characterization of Manduca Sexta and Plutella Xylostella Midgut Aminopeptidase N Enzymes Related to Bacillus Thuringiensis Toxin‐Binding Proteins

Abstract

We report the purification, cloning and characterization of an aminopeptidase N from the midgut epithelium of Manduca sexta that binds Cry1 Ab5, an insecticidal crystal protein [ICP] from Bacillus fhuringiensis. Sequence information derived from this M. sexta aminopeptidase N was used for the cloning o f an aminopeptidase N from the midgut brush-border membrane of Plutrlla xylostella, an insect species of which some populations acquired resistance against Cry1 Ab5. Affinity chromatography on a CrylAb5 matrix was used to isolate a 220-kDa glycoprotein from the larval midgut of the lepidopteran M. sexta. On ligand blots the purified 120-kDa protein discriminates between the lepidopteran-specific Cry1 Ab5 and the coleopteran-specific Cry3A d-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. sextu 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 NH,-terminal hydrophobic region corresponds to a secretory signal sequence and the COOH-terminal hydrophobic region is typical of glycosylphosphatidylinositol (glycosyl-PtdTns)-anchored proteins. Low-stringency hybridization of the tl xylostella midgut cDNA library with M. sexta u p 2 probes enabled the isolation of a 3118-bp sequence with an ORF encoding a 946-residue preproprotein. This aminopeptidase N ( P xylostella Apnl) 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. .xy'lostella Apnl contain four Cys residues, which are highly conserved among eukaryotic aminopeptidase N molecules. Treatment of Sf9 cells expressing the P xylostellu apnl gene with PtdIns-specific phospholipase C demonstrated that tl xylostella Apnl is attached to the insect cell tnembrane by a glycosyl-Ptdlns anchor.

Bacillus thuringiensis Cry1Ac toxin interaction with Manduca sexta aminopeptidase N in a model membrane environment

“…APN was also identified as a receptor for Cry1Aa in Bombyx mori (silkworm) [14], and for Cry1C and Cry1Ab in Manduca sexta (tobacco hornworm) [15,16]. The APN proteins in M. sexta are distinct, forming a family of related putative toxin receptors.…”

Knockdown of Aminopeptidase-N from Helicoverpa armigera Larvae and in Transfected Sf21 Cells by RNA Interference Reveals Its Functional Interaction with Bacillus thuringiensis Insecticidal Protein Cry1Ac

“…Two membrane proteins, cadherin and aminopeptidase-N (APN), have been implicated as possible receptors to insecticidal proteins. The putative receptor molecules have been cloned, heterologously expressed, and have been shown to bind Cry1A proteins by ligand blot analysis (4)(5)(6)(7)(8)(9)(10)(11). Although functional proof for cadherin as a Cry1A protein receptor has been demonstrated by cytolysis of insect cells expressing lepidopteran cadherin genes on exposure to Cry1Ab/Cry1Aa (10,12,13), a similar characterization of APN-Cry1A interaction has not been investigated yet.…”

Identification of Two Isoforms of Aminopeptidase N in Aedes aegypti Larval Midgut

“…This is again not surprising considering the fact that various insects also have multiple isoforms of APN that are expressed in the midgut tissue. Specifically, four, three, and two APN isoforms have been identified and cloned from B. mori, Plutella xylostella (Chang et al, 1999;Nakanishi et al, 2002;AJ222699), and M. sexta (Knight et al, 1995;Denolf et al, 1997) larval midgut, respectively. All of these isoforms, in general, have been found to be able to bind to Bt Cry toxins.…”