Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

Abstract

Background: Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it.

Results: A 100-kDa protein with APN activity (APNAnq 100) was isolated from the brush border membrane of Anopheles quadrimaculatus. Native state binding analysis by surface plasmon resonance shows that APNAnq 100 forms tight binding to a mosquitocidal Bt toxin, Cry11Ba, but not to Cry2Aa, Cry4Ba or Cry11Aa.

Conclusion: An aminopeptidase from Anopheles quadrimaculatus mosquitoes is a specific binding protein for Bacillus thuringiensis Cry11Ba.

Figure 1

Separation of An. quadrimaculatus aminopeptidase N from solubilized BBMV proteins by anion-exchange chromatography. The UV absorbance at 280 nm (mAU) is indicated at the top left corner, and the percent conductivity of buffer B (%) is indicated at the top right corner. Collected fractions are shown at the bottom in 2-ml intervals. Run volume is indicated at the bottom (ml). Fractions 19–21 and 24–34 contain APN activity.

Figure 2

(A) Further purification of APN fractions (fractions 19–21) from anion-exchange chromatography of An. quadrimaculatus BBMV by size-exclusion chromatography. A single peak was eluted at 75 ml elution volume, corresponding to 100 kDa. (B) SDS-PAGE of purified APN (APN_Anq 100) obtained in (A) above. The estimated sizes of the protein bands are indicated on both sides of the gel in kDa.

Figure 3

Real-time binding of Cry11Ba to An. quadrimaculatus APN_Anq 100. Experimental curves (jagged line) are shown overlaid with fitted curves (smooth line) obtained with the 1:1 Langmuir binding with drifting baseline model. The overlaid BIAcore response curves are shown for Cry11Ba toxin injections at 4, 8, 16, 32 μM as indicated.