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 chemical proteomic approach to identify novel legumain substrates and improve our understanding of its contribution to health and disease.
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 field asymmetric ion mobility spectrometry (FAIMS). This allowed for assessment of changes in protease cleavage events as well as global protein abundance. We identified 331 N termini that were enriched in wild-type spleens compared to knockout. These included 115 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. >65% of the putative substrates exhibit nuclear localisation, suggesting that legumain is catalytically active in the nucleus.
We also detected enrichment of 210 neo-N termini in legumain-deficient spleens that were cleaved after arginine residues. This signature may reflect cathepsin substrates. Accordingly, we observed increased abundance of cathepsin B and L in knockout spleens by shotgun proteomics. Immunoblotting revealed accumulation of cathepsin B, L and D in their single-chain forms, supporting the hypothesis that legumain may cleave these proteases to their fully mature, two-chain form. By contrast, legumain did not directly cleave cathepsins in vitro cleavage assays. These data confirm legumain as an indirect but critical regulator of the cathepsin proteolytic network, possibly through activation of another as yet unidentified protease.
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.