The cysteine protease legumain is an attractive drug target in cancer, inflammation, and neurodegenerative diseases. Expressed as an inactive zymogen, legumain is trafficked to lysosomes and activated in acidic conditions. Increasing evidence suggests extra-lysosomal roles for legumain, including in the nucleus, cytoplasm, and extracellular milieu. While legumain exhibits a clear preference to cleave after asparagine residues, its pathophysiological substrates have not been well characterised, especially in extra-lysosomal compartments. We aimed to use a systematic and unbiased approach to identify novel legumain substrates and improve our understanding of its functions.
In lysates from spleens of wild-type and legumain-deficient mice, we dimethylated native and neo-N termini prior to tryptic digest and analysis by high-field asymmetric ion mobility spectrometry (FAIMS)-coupled tandem mass spectrometry. This allowed for sufficient proteome depth to assess changes in both protease cleavage events and protein abundance from a single run without the need for enrichment. We identified 235 N termini that were enriched in wild-type spleens compared to knockout. These included 119 peptides cleaved after asparagine residues, reflecting putative legumain substrates. Surprisingly few proteins with known lysosomal distribution were detected, which may reflect rapid turnover of legumain substrates within lysosomes. 76% of the putative substrates exhibit localisation to nucleus and cytoplasm, suggesting that legumain is catalytically active and mediates limited proteolysis outside of lysosomes. As we continue to validate our data and extend our analyses to disease settings, we expect to improve our biochemical understanding of legumain activity and unravel new and unforeseen functions of this elusive cysteine protease.