Distinct Dibasic Cleavage Specificities of Neuropeptide-Producing Cathepsin L and Cathepsin V Cysteine Proteases Compared to PC1/3 and PC2 Serine Proteases

Neuropeptides, functioning
as peptide neurotransmitters and hormones,
are generated from proneuropeptide precursors by proteolytic processing
at dibasic residue sites (i.e., KR, RK, KK, RR). The cysteine proteases
cathepsin L and cathepsin V, combined with the serine proteases proprotein
convertases 1 and 2 (PC1/3 and PC2), participate in proneuropeptide
processing to generate active neuropeptides. To compare the dibasic
cleavage properties of these proteases, this study conducted global,
unbiased substrate profiling of these processing proteases using a
diverse peptide library in multiplex substrate profiling by mass spectrometry
(MSP-MS) assays. MSP-MS utilizes a library of 228 14-mer peptides
designed to contain all possible protease cleavage sites, including
the dibasic residue sites of KR, RK, KK, and RR. The comprehensive
MSP-MS analyses demonstrated that cathepsin L and cathepsin V cleave
at the N-terminal side and between the dibasic residues (e.g., ↓K↓R,
↓R↓K, and K↓K), with a preference for hydrophobic
residues at the P2 position of the cleavage site. In contrast, the
serine proteases PC1/3 and PC2 displayed cleavage at the C-terminal
side of dibasic residues of a few peptide substrates. Further analyses
with a series of dipeptide-AMC and tripeptide-AMC substrates containing
variant dibasic sites with hydrophobic P2 residues indicated the preferences
of cathepsin L and cathepsin V to cleave between dibasic residue sites
with preferences for flanking hydrophobic residues at the P2 position
consisting of Leu, Trp, Phe, and Tyr. Such hydrophobic amino acids
reside in numerous proneuropeptides such as pro-NPY and proenkephalin
that are known to be processed by cathepsin L. Notably, cathepsin
L displayed the highest specific activity that was 10-, 64-, and 1268-fold
greater than cathepsin V, PC1/3, and PC2, respectively. Peptide-AMC
substrates with dibasic residues confirmed that PC1/3 and P2 cleaved
almost exclusively at the C-terminal side of dibasic residues. These
data demonstrate distinct dibasic cleavage site properties and a broad
range of proteolytic activities of cathepsin L and cathepsin V, compared
to PC1/3 and PC2, which participate in producing neuropeptides for
cell–cell communication.