Identification of a novel endopeptidase activity from lupin seeds cleaving between arginine pairs of seed storage proteins

Seed protein degradation was originally considered a random process in which the combined action of various proteolytic enzymes just generates free amino acids. Actually, protein cleavage at germination was proposed to occur with a finely tuned mechanism involving first the selective breakdown of specific peptide bonds, altering the storage protein native conformation, and subsequently the massive degradation of the protein large fragments through endo- and exo-peptidase activites. Some of the enzymes involved in the first limited attacks to the storage proteins have been identified: they primarily consist of papain -like cysteine proteinases. In the present work the identification of a novel endopeptidase activity from dry lupin seeds effective on endogenous and exogenous protein substrates containing twin arginine residues is described. The hydrolytic activity was limited and highly specific, giving rise to stable fragments which were characterised by 1- and 2-D electrophoresis and, for the most relevant ones, N-terminal sequencing and mass spectrometry. The lupin endopeptidase activity described was inhibited by typical serine proteinase protein inhibitors. The possible role of the peptidase activity in the liberation of specific functional polypeptides is also proposed