AMINOPEPTIDASE N PURIFIED FROM GYPSY-MOTH BRUSH-BORDER MEMBRANE-VESICLES IS A SPECIFIC RECEPTOR FOR BACILLUS-THURINGIENSIS CRYIAC TOXIN

We have evaluated the binding of Bacillus thuringiensis Cry toxins to aminopeptidase N (APN) purified from Lymantria dispar (gypsy moth) bru sh border membrane vesicle (BBMV), CryIAc toxin bound strongly to APN, while either the structurally related CryIAa and CryIAb toxins or Cry IC, CryIIA, and CryIIIA toxins showed weak binding to APN, An in vitro competition binding study demonstrated that the binding of CryIAc to L. dispar BBMV was inhibited by APN, Inhibition of short circuit curre nt for CryIAc, measured by voltage clamping of whole L. dispar midgut, was substantially reduced by addition of phosphatidylinositol-specifi c phospholipase C, which is known to release APN from the midgut membr ane, In contrast, addition of phosphatidylinositol-specific phospholip ase C had only a marginal effect on the inhibition of short circuit cu rrent for CryIAa. These data suggest that APN is the major functional receptor for CryIAc in L. dispar BBMV, A ligand blotting experiment de monstrated that CryIAc recognized a 120-kDa peptide (APN), while CryIA a and CryIAb recognized a 210-kDa molecule in L. dispar BBMV, In contr ast, CryIAa and CryIAb bound to both the 120- and 210-kDa molecules in Manduca sexta BBMV, while CryIAc recognized only the 120-kDa peptide, The 120-kDa peptide (APN) in L. dispar BBMV reacted with soybean aggl utinin, indicating that N-acetylgalactosamine is a component of this g lycoprotein.