A 106-kDa aminopeptidase is a putative receptor for Bacillus thuringiensis Cry11Ba toxin in the mosquito Anopheles gambiae

Abstract

Bacillus thuringiensis (Bt) insecticidal toxins bind to receptors on midgut epithelial cells of susceptible insects, and binding triggers biochemical events that lead to insect mortality. Recently, a 100-kDa aminopeptidase N (APN) was isolated from brush border membrane vesicles (BBMV) of Anopheles quadrimaculatus and shown to bind Cry11Ba toxin with surface plasmon resonance (SPR) detection [Abdullah et al. (2006) BMC Biochem. 7, 16]. In our study, a 106-kDa APN, called AgAPN2, released by phosphatidylinositol-specific phospholipase C (PI-PLC) from Anopheles gambiae BBMV was extracted by Cry11Ba bound to beads. The AgAPN2 cDNA was cloned, and analysis of the predicted AgAPN2 protein revealed a zinc-binding motif (HEIAH), three potential N-glycosylation sites, and a predicted glycosylphosphatidylinositol (GPI) anchor site. Immunohistochemistry localized AgAPN2 to the microvilli of the posterior midgut. A 70-kDa fragment of the 106-kDa APN was expressed in Escherichia coli. When purified, it competitively displaced 125I-Cry11Ba binding to An. gambiae BBMV and bound Cry11Ba on dot blot and microtiter plate binding assays with a calculated K d of 6.4 nM. Notably, this truncated peptide inhibited Cry11Ba toxicity to An. gambiae larvae. These results are evidence that the 106-kDa GPI-anchored APN is a specific binding protein, and a putative midgut receptor, for Bt Cry11Ba toxin.

FIGURE 1

(A) Diagram of the AgAPN2 cDNA, truncated APN2_t clone and primer locations. A grey arrowhead locates the gluzincin aminopeptidase motif. (B) Deduced amino acid sequence of AgAPN2. The putative N-terminal cleavable peptide is underlined. The putative C-terminal GPI-anchor site is in boldface. The conserved gluzincin aminopeptidase motif is in boldface and underlined. The putative N-glycosylation sites are in bold-box. Peptides identified by MS analysis are in boldface and italics. The unique peptide of AgAPN2 is in boldface, italics and underlined. (C) Schematic representation of the APN gene cluster on chromosome 2R. Numbers indicate the position of the starting and the ending nucleotides of each gene. The gene cloned in this research is underlined.

FIGURE 2

Peptide mass fingerprinting identification of a 106-kDa protein from BBMV as AgAPN2. (A) Solubilized proteins from BBMV were fractionated by 0.0-1.0 M NaCl gradient elution. The left side Y-axis indicates the UV absorbance at 280 nm (mAU), and the right side Y-axis indicates the percent conductivity of buffer B (%). Run volumes (ml) and collected fractions are indicated at the bottom. (B) Fractions 26-31 (hatched area) with APN activities were pooled and separated on SDS-PAGE. The Coomassie blue stained band at ~100 kDa (arrowhead, lane 1) corresponds to the band recognized by anti-AgAPN2 serum on a blot (lane 2). The band was excised for mass fingerprinting.

FIGURE 3

Immunolocalization of AgAPN2 on the microvilli of the posterior midgut of An. gambiae larvae. (A) Diagram of a mosquito gut [adapted from (40)]. (B) Sectioned midgut was probed by anti-AgAPN2 serum and detected by Alexa Fluor-488-conjugated goat-anti-rabbit IgG. (C) Control section was probed by pre-immune serum. (Insets) Higher-magnification views correspond to the area of apical membrane of epithelial cells in the lower-magnification image. M, microvilli. L, lumen. An, anterior. Po, posterior. N, nucleus. Scale bar: 100 and 50 μm for the low- and high- magnification images, respectively. All midgut sections were arranged anterior to the left.

FIGURE 4

Solubilization of AgAPN2 from An. gambiae BBMV proteins by PI-PLC digestion and extraction with Cry11Ba-beads. (A) PI-PLC-solubilized proteins were resolved by SDS-PAGE, transferred to PVDF filters and were probed with anti-CRD serum (lane 1) or anti-AgAPN2 antibody (lane 2). (B) Affinity extraction of GPI-anchored proteins with Cry11Ba-bead complex. After being washed, the beads with bound proteins were boiled in SDS-sample buffer, separated by SDS-PAGE, transferred to a PVDF filter and then probed with anti-CRD (lanes 3 and 4) or anti-AgAPN2 antibodies (lanes 5 and 6). A GPI-anchored protein migrating at 106-kDa was extracted by Cry11Ba-beads and detected by anti-AgAPN2 antibodies (arrow). Beads alone extracted no proteins (lanes 3 and 5).

FIGURE 5

Analysis of the interaction between Cry11Ba and AgAPN2 protein. (A) Partially purified recombinant APN2_t or AgCad1 CR11-MPED fragment (1 μg), was resolved by SDS-PAGE and stained with Coomassie blue. (B) Increasing amounts of ¹²⁵I-Cry11Ba were incubated with An. gambiae BBMV (8 μg) with or without 10 μM of each competitor in the binding buffer. (Inset) APN2_t or AgCad1 CR11-MPED (1 μg) was spotted in duplicate on a PVDF membrane and probed with 0.125 nM ¹²⁵I-Cry11Ba. (C) Binding affinity of Cry11Ba to APN2_t. Ninety-six-well microtiter plates coated with 0.5 μg trypsinized Cry11Ba were incubated with increasing nM concentrations of biotinylated APN2_t peptide alone or with 1000-fold molar excess of unlabeled APN2_t peptide to determine specific binding. (D) The 70-kDa APN2_t inclusions were used for toxicity inhibition assay. Cry11Ba toxin or a mixture of Cry11Ba and APN2_t or CR11-MPED (1:100 w/w) were diluted in plastic plates containing 2 ml of deionized water and tested against ten early 4^th instar larvae of An. gambiae. Each treatment was at least in duplicate and the bioassays were conducted three times. Larval mortality was recorded after 48 h. P < 0.01.

FIGURE 6

Phylogenetic tree derived from ClustalX alignment of insect APNs. The tree was constructed by the maximum likelihood method. Classifications are according to (3, 31, 32, 33, 34). GenBank accession numbers are as follows: AaAPNRc2 (AAL85580), AgAPN1(Dinglasan, personal communication), AgAPN2, AgAPN3, AgAPN4, AgAPN5 (this study), AjFbAPN (ABE02186), AjMgAPN (ABH07377), ApAPN (ABD96614), BmAPN1 (AAC33301), BmAPN2 (BAA32140), BmAPN3 (AAL83943) BmAPN4 (BAA33715), CsAPN (ABC69855), EpAPN (AAF99701), HaAPN1 (AAK85538), HaAPN2 (AAK85539), HaAPN3 (AAN04900), HaAPN4 (AAM44056), HaAPN5 (AAW72993), HpAPN1 (AAF37558), HpAPN2 (AAF37559), HpAPN3 (AAF37560), HvAPN110 (AAK58066), HvAMPM (AAC46929), HvAPN170 (AAF08254), LcAPN1 (AAM77681), LdAPN2 (AAD31183), LdAPN2x (AAD31184), LdAPN3 (AAL26894), LdAPN4 (AAL26895), MsAPN1 (CAA61452), MsAPN2 (CAA66466), MsAPN3 (AAM18718), MsAPN4 (AAM13691), OfAPN1 (ABV01346), PiAPN (AAC36148), PxAPN (AAB70755), PxAMPN (CAA66467), PxAPN2 (CAA10950), PxAPN3f (AAF01259), SeAPN1 (AAP44964), SeAPN2 (AAP44965), SeAPN3 (AAP44966), SeAPN4 (AAP44967), SlAPN (AAK69605), TcAPN (XP_972951), TmAPN (AAP94045), TnAPN1 (AAX39863), TnAPN2 (AAX39864), TnAPN3 (AAX39865), TnAPN4 (AAX39866). Species names abbreviations are as follows: Aa, Aedes aegypti; Ag, Anopheles gambiae; Aj, Achaea janata; Ap, Acyrthosiphon pisum; Bm, Bombyx mori; Cs, Chilo suppressalis; Ep, Epiphyas postvittana; Ha, Helicoverpa armigera; Hp, Helicoverpa punctigera; Hv, Heliothis virescens; Lc, Lucilia cuprina; Ld, Lymantria dispar; Ms, Manduca sexta; Of, Ostrinia furnacalis; Pi, Plodia interpunctella; Px, Plutella xylostella; Se, Spodoptera exigua; Sl, Spodoptera litura; Tc, Tribolium castaneum; Tm, Tenebrio molitor; Tn, Trichoplusia ni. Bootstrap values represent the percentage frequency of which sequences resample in 100 replicates.