title: Synergistic and inhibitory effects of aminopeptidase peptides on Bacillus thuringiensis Cry11Ba toxicity in the mosquito Anopheles gambiae.
abstract: Cry11Ba produced by Bacillus thuringiensis subsp. jegathesan is an active toxin for larvae of the mosquito Anopheles gambiae. A 106-kDa aminopeptidase N (APN), called AgAPN2, was previously identified as a Cry11Ba receptor in A. gambiae. A 70-kDa fragment of AgAPN2 expressed in Escherichia coli binds Cry11Ba with high affinity (K(d) = 6.4 nM) and inhibits Cry11Ba activity by 98% in bioassays [Zhang et al. (2008) Biochemistry 47, 11263-11272]. To identify regions involved in toxicity, we truncated the 70-kDa APN fragment into peptides of 28- and 30-kDa ta and tb, respectively, and tested their abilities to mediate toxicity and bind Cry11Ba. While AgAPN2ta reduced Cry11Ba toxicity by 85%, AgAPN2tb showed a significant enhancement effect on Cry11Ba toxicity. The purified peptides showed evidence of structural folding and bound the same site(s) on Cry11Ba with high affinity. The inhibitory AgAPN2ta blocked Cry11Ba binding to brush border membrane vesicles (BBMV) of A. gambiae whereas the toxicity enhancing AgAPN2tb increased Cry11Ba binding on BBMV. A deletion at the N-terminus ((336)S-P(420)) of AgAPN2ta significantly reduced AgAPN2ta binding to Cry11Ba and its inhibitory effect. Deletion of the central region ((676)I-W(760)) of AgAPN2tb eliminated its increased toxin binding and toxicity enhancement effect without affecting Cry11Ba binding. A "bridge" model is proposed for AgAPN2tb action whereby the peptide binds Cry11Ba and vectors it to sites on the larval midgut.
authors.affiliation: Department of Entomology, University of Georgia, Athens, Georgia 30602-2603, USA.
authors.forename: Rui
authors.initials: R
authors.lastname: Zhang
authors.forename: Gang
authors.initials: G
authors.lastname: Hua
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Urbauer
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Adang
mesh_terms: D000818:Animals; D000852:Anopheles / Q000201:enzymology* / Q000382:microbiology*; D001413:Bacillus thuringiensis / Q000378:metabolism*; D001427:Bacterial Toxins / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000633:toxicity*; D018826:CD13 Antigens / Q000737:chemistry* / Q000378:metabolism*; D007814:Larva / Q000382:microbiology / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding
chemical_list: D001427:Bacterial Toxins; D010455:Peptides; D018826:CD13 Antigens