Activation of bean (Phaseolus vulgaris) α-amylase inhibitor requires proteolytic processing of the proprotein

Abstract

Seeds of the common bean (Phaseolus vulgaris) contain a plant defense protein that inhibits the α-amylases of mammals and insects. This α-amylase inhibitor (αAI) is synthesized as a proprotein on the endoplasmic reticulum and is proteolytically processed after arrival in the protein storage vacuoles to polypeptides of relative molecular weight (Mr) 15,000 to 18,000. We report two types of evidence that proteolytic processing is linked to activation of the inhibitory activity. First, by surveying seed extracts of wild accessions of P. vulgaris and other species in the genus Phaseolus, we found that antibodies to αAI recognize large (Mr 30,000-35,000) polypeptides as well as typical αAI processing products (Mr 15,000-18,000). αAI activity was found in all extracts that had the typical αAI processed polypeptides, but was absent from seed extracts that lacked such polypeptides. Second, we made a mutant αAI in which asparagine-77 is changed to aspartic acid-77. This mutation slows down the proteolytic processing of pro-αAI when the gene is expressed in tobacco. When pro-αAI was separated from mature αAI by gel filtration, pro-αAI was found not to have α-amylase inhibitory activity. We interpret these results to mean that formation of the active inhibitor is causally related to proteolytic processing of the proprotein. We suggest that the polypeptide cleavage removes a conformational constraint on the precursor to produce the biochemically active molecule.