Abstract
BackgroundType II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.Methodology/Principal FindingTo profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS). Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin) to simultaneously determine sequence preferences on the N-terminal non-prime (P) and C-terminal prime (P’) sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1′ position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.ConclusionsDegradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1′ positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.
Highlights
Pericellular proteolysis is involved in many important cellular processes such as the transduction of signals across the cell membrane, the release of bioactive growth factors, cytokines and peptide hormones, as well as the interactions with other cells, basement membrane and extracellular matrix proteins [1,2,3]
The Type II transmembrane serine proteases (TTSPs) family is composed of more than 20 members that share a number of structural features including an N-terminal cytoplasmic domain, a transmembrane domain, a central region containing various domains potentially involved in protein-protein interaction, and a C-terminal extracellular serine protease domain (Figure 1A)
Tissue distribution of TTSPs The tissue distribution of TTSP family members was determined by transcript analysis of ten murine tissues using the dedicated murine CLIP-CHIP [1,2,3], with probes for all murine proteases, inactive homologues, and inhibitors (Figure 1B)
Summary
Pericellular proteolysis is involved in many important cellular processes such as the transduction of signals across the cell membrane, the release of bioactive growth factors, cytokines and peptide hormones, as well as the interactions with other cells, basement membrane and extracellular matrix proteins [1,2,3]. The increasing relevance of these processes at the cell surface has focused attention on membrane-associated proteolytic systems, including the family of type II transmembrane serine proteases (TTSPs) [4,5]. The TTSP family is composed of more than 20 members that share a number of structural features including an N-terminal cytoplasmic domain, a transmembrane domain, a central region containing various domains potentially involved in protein-protein interaction, and a C-terminal extracellular serine protease domain (Figure 1A). Type II transmembrane serine proteases (TTSPs) are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors
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