Characterization of a Cry1Ac toxin-binding alkaline phosphatase in the midgut from Helicoverpa armigera (Hübner) larvae

Abstract

Midgut membrane-bound alkaline phosphatases (mALP) tethered to the brush border membrane surface by a glycosylphosphatidylinositol (GPI) anchor have been proposed as crucial for Cry1Ac intoxication. In the present work, two full-length cDNAs-encoding alkaline phosphatases in the midgut of Helicoverpa armigera larvae were cloned and named HaALP1 (GenBank accession no. EU729322) and HaALP2 (GenBank accession no. EU729323), respectively. These two clones displayed high identity (above 94%) at the amino acid sequence, indicating that they may represent allelic variants, and were predicted to contain a GPI anchor. Protein sequence alignment revealed that HaALPs were grouped with mALP from the Heliothis virescens midgut. The HaALP1 and HaALP2 (∼68 kDa) proteins were heterologously expressed in Sf9 cells using a baculovirus expression system and purified to homogeneity. Ligand blot and dot blot analysis revealed that the Cry1Ac bound to both denatured and native purified HaALPs. Data from lectin blots, competition assays with soybean agglutinin (SBA) lectin and GalNAc binding inhibition assays were indicative of the presence of GalNAc on HaALPs and binding of Cry1Ac toxin to this residue. This observation was further confirmed through N-glycosidase digestion of HaALPs, which resulted in reduced Cry1Ac binding. Our data represent the first report on HaALPs and their putative role as receptors for Cry1Ac toxin in H. armigera.