Characterization of legumain degradation of intact proteins

Legumain or asparagine endopeptidase is a unique cysteine endopeptidase with a distinctive specificity for the hydrolysis of peptide bonds after asparagine and to a lesser extent after aspartate. In order to improve our understanding of legumain substrate recognition we have performed proteomic profiling of legumain specificity on native proteins using two different N-terminal labelling methodologies (FPPS and ISIL). Our data revealed a strict P1 specificity of legumain with clear cleavage preference for unstructured secondary regions in the substrate proteins. No extended cleavage specificity on native proteins was observed. Moreover, a limited number of identified cleavages on individual substrates suggest its primary role in precision proteolysis and regulatory proteolytic events.