title: Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity.
abstract: The interaction between Bacillus thuringiensis Cry toxins and their receptors on midgut cells of susceptible insect larvae is the critical determinant in toxin specificity. Besides GPI-linked alkaline phosphatase in Aedes aegypti mosquito-larval midguts, membrane-bound aminopeptidase N (AaeAPN) is widely thought to serve as a Cry4Ba receptor. Here, two full-length AaeAPN isoforms, AaeAPN2778 and AaeAPN2783, predicted to be GPI-linked were cloned and successfully expressed in Spodoptera frugiperda (Sf9) cells as 112- and 107-kDa membrane-bound proteins, respectively. In the cytotoxicity assay, Sf9 cells expressing each of the two AaeAPN isoforms showed increased sensitivity to the Cry4Ba mosquito-active toxin. Double immunolocalization revealed specific binding of Cry4Ba to each individual AaeAPN expressed on the cell membrane surface. Sequence analysis and homology-based modeling placed these two AaeAPNs to the M1 aminopeptidase family as they showed similar four-domain structures, with the most conserved domain II being the catalytic component. Additionally, the most variable domain IV containing negatively charged surface patches observed only in dipteran APNs could be involved in insect specificity. Overall results demonstrated that these two membrane-bound APN isoforms were responsible for mediating Cry4Ba toxicity against AaeAPN-expressed Sf9 cells, suggesting their important role as functional receptors for the toxin counterpart in A. aegypti mosquito larvae.
authors: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors: Aratee
authors: A
authors: Aroonkesorn
authors: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors: Kusol
authors: K
authors: Pootanakit
authors: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand.
authors: Gerd
authors: G
authors: Katzenmeier
authors: Bacterial Protein Toxin Research Cluster, Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand; Laboratory of Molecular Biophysics and Structural Biochemistry, Biophysics Institute for Research and Development (BIRD), Bangkok 10160, Thailand. Electronic address: chanan.ang@mahidol.ac.th.
authors: Chanan
authors: C
authors: Angsuthanasombat
mesh_terms: D000330:Aedes / Q000737:chemistry / Q000382:microbiology* / Q000502:physiology*; D000595:Amino Acid Sequence; D000818:Animals; D001413:Bacillus thuringiensis / Q000502:physiology*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism*; D018826:CD13 Antigens / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D004731:Endotoxins / Q000378:metabolism*; D006460:Hemolysin Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism*; D007814:Larva / Q000737:chemistry / Q000382:microbiology / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D004731:Endotoxins; D006460:Hemolysin Proteins; D019476:Insect Proteins; D020033:Protein Isoforms; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D018826:CD13 Antigens
keywords: APN structure; Baculovirus expression system; Cry δ-endotoxin; Cytotoxicity assay; Double immunolocalization; Membrane-bound APN