|Willis, Jonathan - UNIVERSITY OF TENNESSEE|
|Adang, Michael - UNIVERSITY OF GEORGIA|
|Jurat-Fuentes, Juan Luis - UNIVERSITY OF TENNESSEE|
Submitted to: Insect Biochemistry and Molecular Biology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 14, 2009
Publication Date: February 6, 2009
Citation: Perera, O.P., Willis, J.D., Adang, M.J., Jurat-Fuentes, J. 2009. Cloning & Characterization of the Cry1Ac-binding Alkaline Phosphatase (HvALP) from Heliothis virescens. Insect Biochemistry and Molecular Biology.39:294-302. Interpretive Summary: Primary function of alkaline phosphatases (ALP) is the removal of phosphate groups from biological molecules. Membrane bound forms of ALP (mALP) in tobacco budworm, Heliothis virescens, have been identified as receptors for Cry toxins from Bacillus thuringiensis. We characterized the mALPs from the midgut of H. virescens by testing the Cry1Ac binding capacity of proteins produced from cloned mALP genes. mALP proteins expressed in bacterial cells, which lack glycosylation, failed to bind to Cry 1Ac. This report highlights the importance of glycosylation of mALPs in binding of Cry1Ac toxin.
Technical Abstract: Membrane bound alkaline phosphatases (mALPs) in the insect midgut have been reported as functional receptors for Cry toxins from the bacterium Bacillus thuringiensis. We previously reported the identification of HvALP in the midgut of Heliothis virescens larvae as a Cry1Ac binding protein that is down-regulated in Cry1Ac-resistant insects. To further characterize HvALP, we localized mALP protein to foregut and midgut tissues using anti-mALP serum and then cloned five mALPs from H. virescens larval midgut. All five clones displayed high levels of sequence identity (above 90%), suggesting that they may represent allelic variants, and grouped with alternative lepidopteran mALPs in sequence alignments. All these cloned ALPs were predicted to contain a glycosylphosphatidylinositol (GPI) anchor and were named HvmALP1 to 5. We expressed two of the most diverse HvmALPs in a heterologous system to test binding of Cry1Ac and recognition by HvALP-cross reacting antiserum. Our data highlight the importance of glycosylation for Cry1Ac binding to HvALP and suggest that, depending on glycosylation, all the identified HvmALPs may be synonymous with HvALP, the Cry1Ac-binding phosphatase identified in H. virescens midgut epithelium.