Interaction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal crystal protein Cry1C

Abstract

Insecticidal toxins produced by Bacillus thuringiensis interact with specific receptors located in the midguts of susceptible larvae, and the interaction is followed by a series of biochemical events that lead to the death of the insect. In order to elucidate the mechanism of action of B. thuringiensis toxins, receptor protein-encoding genes from many insect species have been cloned and characterized. In this paper we report the cloning, expression, and characterization of Cry toxin-interacting aminopeptidase N (APN) isolated from the midgut of a polyphagous pest, Spodoptera litura. The S. litura APN cDNA was expressed in the Sf21 insect cell line by using a baculovirus expression system. Immunofluorescence staining of the cells revealed that the expressed APN was located at the surface of Sf21 cells. Treatment of Sf21 cells expressing S. litura APN with phosphatidylinositol-specific phospholipase C demonstrated that the APN was anchored in the membrane by a glycosylphosphatidylinositol moiety. Interaction of the expressed receptor with different Cry toxins was examined by immunofluorescence toxin binding studies and ligand blot and immunoprecipitation analyses. By these experiments we showed that the bioactive toxin, Cry1C, binds to the recombinant APN, while the nonbioactive toxin, Cry1Ac, showed no interaction.

FIG. 1.

(a) Deduced amino acid sequence of APN from S. litura. The 2,856-bp cDNA clone encodes a 952-amino-acid polypeptide. The putative NH₂-terminal cleavable peptide and the GPI signal peptide at the COOH terminus are double underlined. The zinc metallopeptidase signature is indicated with a dotted line. The putative N-glycosylation sites are underlined. The four Cys residues that are conserved among eukaryotic aminopeptidases are in boldface. (b) Schematic representation and putative signature domains of S. litura APN.

FIG. 2.

Phylogenetic tree describing the sequence similarity of known insect aminopeptidases. The tree was constructed by the neighbor-joining method with the Best Tree mode. A value of 0.1 corresponds to a difference of 10% between two sequences. GenBank accession numbers are as follows: Plutella xylostella APN3 (PxAPN3), AJ222699 (P. Denholf, unpublished data); S. litura APN (SlAPN), AF320764 (this study); H. punctigera APN2 (HpAPN2), AF217249 (5); Epiphyas postvittana APN (EpAPN), AF276241 (35); L. dispar APN1 (LdAPN1), AF126442 (8); H. virescens 120-kDa protein (Hv120kDa), U35096 (10); H. punctigera APN3 (HpAPN3), AF217250 (5); Plodia interpunctella APN1 (PiAPN1), AF034483 (47); P. xylostella APNA (PxAPNA), AF020389 (2); H. virescens 170-kDa protein (Hv170kDa), AF173552 (28); H. punctigera APN1 (HpAPN1), AF217248 (5); M. sexta APN1 (MsAPN1), X89081 (16); B. mori APN1 (BmAPN1), AF084257 (46); P. xylostella APN1 (PxAPN1), X97878 (3); L. dispar APN2 (LdAPN2), AF126443 (8); M. sexta APN2 (MsAPN2), X97877 (3); and B. mori APN2 (BmAPN2), AB011497 (12).

FIG. 3.

Expression of S. litura APN in Sf21 cells. (A) One million uninfected and 72-h-infected cells were harvested, and total cell extract was prepared by resuspending and boiling the cells in SDS sample buffer containing β-mercaptoethanol. (B) Membranes were isolated from both uninfected and infected cells by the procedure described in Materials and Methods. Cell extracts and equal amounts of membrane proteins were resolved by SDS-7.5% PAGE and stained with Coomassie brilliant blue. Lanes 1 and 3, uninfected Sf21 cells; lanes 2 and 4, BV-SlApn-infected cells. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 4.

Time course of S. litura APN expression in Sf21 insect cell culture. U, total cell extract of uninfected cells. BV-SlApn-infected Sf21 cells (10⁶) were harvested at the indicated times. Total cell extract was resolved by SDS-7.5% PAGE and electrotransferred to nitrocellulose membrane. Western blot analysis was carried out with (i) polyclonal antibodies raised against APN and (ii) monoclonal anti-histidine tag antibodies. Bands A and B represent the two forms of APN expressed in the Sf21 cells. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 5.

Flow diagram illustrating the occurrence of a heterogenous population of APN molecules expressed in Sf21 cells as shown in Fig. 4. Population A consists of molecules that have undergone transamidation at the COOH terminus and are transported to plasma membrane; these molecules lose the COOH-terminal His tag and are not visualized with anti-His antibodies. Population B consists of molecules that remain unprocessed and are retained in the endoplasmic reticulum (ER) with the His tag. These molecules are visualized with anti-His antibodies. Details are given in Discussion.

FIG. 6.

Immunolocalization of expressed S. litura APN. Surface expression of the APN was examined at 36 h postinfection by immunofluorescence staining with rabbit polyclonal antibodies raised against APN and FITC-conjugated goat anti-rabbit IgG. (A) Cells overlaid with preimmune serum. (B) Cells overlaid with anti-APN antibodies.

FIG. 7.

Concanavalin A lectin blot detection of glycosylated proteins in Sf21 cell membranes. Lane 1, uninfected cell membranes. Lane 2, infected cell membranes. Lane 3, E. coli-expressed 51-kDa APN. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 8.

Identification of Cry1C toxin-binding protein in S. litura BBMV. (A) Twenty-five micrograms of BBMV proteins was resolved by SDS-PAGE and transferred to nitrocellulose membrane. The membrane strips were incubated with anti-Cry1C antiserum without prior overlaying with Cry1C toxin (lane 1), with Cry1C toxin and anti-Cry1C antiserum (lane 2), and with anti-APN antibodies (lane 3). (B) Sf21-expressed S. litura APN was transferred to the membrane and probed with anti-APN antibodies. The membranes were then incubated with alkaline phosphatase-conjugated secondary antibody and NBT-BCIP substrate as described in Materials and Methods. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 9.

Ligand blot analysis of insect cells expressing S. litura APN. Membrane proteins (2.5 μg) prepared from uninfected Sf21 cells (lane 1) and BV-SlApn-infected cells (lane 2 and 3) were solubilized in SDS sample buffer containing β-mercaptoethanol, separated by SDS-7.5% PAGE, and electrotransferred to nitrocellulose membrane. The nitrocellulose membrane was probed with Cry1C (1 μg/ml) (lanes 1 and 2) and Cry1Ac (1 μg/ml) (lane 3) δ-endotoxins. Binding of the toxin was detected by using antibodies against the toxin followed by alkaline phosphatase-conjugated goat anti-rabbit IgG. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 10.

Immunoprecipitation of APN with Cry1C toxin. CHAPS-solubilized membrane proteins from uninfected cells (lane 1) and infected cells (lane 2) were incubated with purified Cry1C toxin. Using anti-Cry1C antiserum, the protein-toxin complex was pulled down with protein A-Sepharose beads. The beads were boiled in SDS sample buffer containing β-mercaptoethanol, resolved by SDS-PAGE, and electrotransferred to nitrocellulose membrane. The membrane was probed with anti-APN antibodies. The positions of molecular mass markers (in kilodaltons) are indicated on the left.

FIG. 11.

Differential toxin binding to surface-expressed S. litura APN. Sf21 cells infected for 36 h were overlaid with Cry1Ac (A) or Cry1C (B) toxin (1 μg/ml). Binding of the toxin was detected by using antibodies against the toxin followed by FITC-conjugated goat anti-rabbit IgG.