MOLECULAR-CLONING OF A GPI-ANCHORED AMINOPEPTIDASE-N FROM BOMBYX-MORIMIDGUT - A PUTATIVE RECEPTOR FOR BACILLUS-THURINGIENSIS CRYIA TOXIN

An aminopeptidase N (APN) with a molecular weight of 110 kDa was relea sed from the midgut membrane of Bombyx mori by phosphatidylinositol-sp ecific phospholipase C (PI-PLC), and purified to a homogeneous state. This 110-kDa APN was different from the 100-kDa APN that we previously reported, in chromatographic behaviors, substrate specificity, and N- terminal and internal amino acid sequences. However, the N-terminal se quence of 110-kDa APN, DPAFRLPTTTRPRHYQVTLT, was highly homologous wit h those of Manduca sexta and Heliothis virescens APNs, which were iden tified as a receptor for an insecticidal toxin of Bacillus thuringiens is. From a B. mori midgut cDNA library, we cloned the 110-kDa APN cDNA that possessed a 2958-bp open reading frame encoding a 111 573-Da pol ypeptide of 986 residues. The sequence of the eicosa-peptide Asp(42)-T hr(61) deduced from the cDNA was completely matched with the N-termina l sequence of the mature 110-kDa APN. One potential N-glycosylation si te, HEXXHXW zinc-binding motif and characteristic proline-rich repeats were observed in the ORF. Moreover, the primary sequence contained tw o hydrophobic peptides on N- and C-termini. The N-terminal peptide seq uence showed characteristics of leader peptide for secretion and the C -terminal peptide contained a possible glycosylphosphatidylinositol (G PI) anchoring site. Taken together, the deduced amino acid sequence su ggests that the 110-kDa APN is a GPI-anchored protein and a specific r eceptor protein for B. thuringiensis CryIA delta-endotoxin. (C) 1998 E lsevier Science B.V. All rights reserved.