title: Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs) using PICS with proteome-derived peptide libraries.
abstract: BACKGROUND
  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. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.
  
  
  RESULTS
  To 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.
  
  
  CONCLUSIONS
  Degradomics 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.
authors.affiliation: Centre for Blood Research, Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Olivier
authors.initials: O
authors.lastname: Barré
authors.affiliation: Centre for Blood Research, Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Antoine
authors.initials: A
authors.lastname: Dufour
authors.affiliation: Centre for Blood Research, Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Eckhard
authors.affiliation: Centre for Blood Research, Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Reinhild
authors.initials: R
authors.lastname: Kappelhoff
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: François
authors.initials: F
authors.lastname: Béliveau
authors.affiliation: Department of Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.
authors.forename: Richard
authors.initials: R
authors.lastname: Leduc
authors.affiliation: Centre for Blood Research, Department of Oral Biological & Medical Sciences, University of British Columbia, Vancouver, BC, Canada; Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, BC, Canada.
authors.forename: Christopher M
authors.initials: CM
authors.lastname: Overall
mesh_terms: D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000235:genetics; D020134:Catalytic Domain; D006801:Humans; D008239:Lysine / Q000737:chemistry / Q000235:genetics; D019151:Peptide Library; D010449:Peptide Mapping; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D020543:Proteome; D057057:Serine Proteases / Q000737:chemistry* / Q000235:genetics; D012984:Software; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D019151:Peptide Library; D010455:Peptides; D020543:Proteome; D001120:Arginine; D057057:Serine Proteases; D008239:Lysine