TAILIEUCHUNG - Báo cáo khoa học: Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae

We reportedpreviously a direct correlationbetween reduced soybean agglutinin binding to 63- and 68-kDa midgut gly-coproteins and resistance to Cry1Ac toxin from Bacillus thuringiensisin the tobacco budworm (Heliothis virescens). In the present work we describe the identification of the 68-kDaglycoproteinas amembrane-bound formof alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for [ 125 I]Cry1Ac binding in ligand blots. . | Eur. J. Biochem. 271 3127-3135 2004 FEBS 2004 doi Characterization of a CrylAc-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae Juan L. Jurat-Fuentes1 and Michael J. Adang1 2 Departments of 1 Entomology and 2Biochemistry and Molecular Biology University of Georgia Athens GA USA We reported previously a direct correlation between reduced soybean agglutinin binding to 63- and 68-kDa midgut glycoproteins and resistance to Cry1Ac toxin from Bacillus thuringiensis in the tobacco budworm Heliothis virescens . In the present work we describe the identification of the 68-kDa glycoprotein as a membrane-bound form of alkaline phosphatase we term HvALP. Lectin blot analysis of HvALP revealed the existence of N-linked oligosaccharides containing terminal N-acetylgalactosamine required for 125I Cry1Ac binding in ligand blots. Based on immunoblotting and alkaline phosphatase activity detection reduced soybean agglutinin binding to HvALP from Cry1Ac resist ant larvae of the H. virescens YHD2 strain was attributable to reduced amounts of HvALP in resistant larvae. Quantification of specific alkaline phosphatase activity in brush border membrane proteins from susceptible YDK and F1 generation from backcrosses and YHD2 H. virescens larvae confirmed the observation of reduced HvALP levels. We propose HvALP as a Cry1Ac binding protein that is present at reduced levels in brush border membrane vesicles from YHD2 larvae. Keywords alkaline phosphatase Cry1Ac Heliothis vires-cens resistance N-acetylgalactosamine. Specific binding to insect midgut receptors is a key step in the mode of action of insecticidal Cry toxins from the bacterium Bacillus thuringiensis Bt . Despite exceptions 1 in most cases Cry toxin specificity and potency correlate with the extent of toxin binding to midgut brush border membrane receptors in vitro 2 3 . Effective toxin binding to receptors results in toxin insertion and oligomerization on the

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