title: Contribution of cathepsin L to secretome composition and cleavage pattern of mouse embryonic fibroblasts.
abstract: The endolysosomal cysteine endoprotease cathepsin L is secreted from cells in a variety of pathological conditions such as cancer and arthritis. We compared the secretome composition and extracellular proteolytic cleavage events in cell supernatants of cathepsin L-deficient and wild-type mouse embryonic fibroblasts (MEFs). Quantitative proteomic comparison of cell conditioned media indicated that cathepsin L deficiency affects, albeit in a limited manner, the abundances of extracellular matrix (ECM) components, signaling proteins, and further proteases as well as endogenous protease inhibitors. Immunodetection corroborated that cathepsin L deficiency results in decreased abundance of the ECM protein periostin and elevated abundance of matrix metalloprotease (MMP)-2. While mRNA levels of MMP-2 were not affected by cathepsin L ablation, periostin mRNA levels were reduced, potentially indicating a downstream effect. To characterize cathepsin L contribution to extracellular proteolysis, we performed terminal amine isotopic labeling of substrates (TAILS), an N-terminomic technique for the identification and quantification of native and proteolytically generated protein N-termini. TAILS identified >1500 protein N-termini. Cathepsin L deficiency predominantly reduced the magnitude of collagenous cleavage sites C-terminal to a proline residue. This contradicts cathepsin L active site specificity and indicates altered activity of further proteases as a result of cathepsin L ablation.
authors.affiliation: Institute for Molecular Medicine and Cell Research, University of Freiburg, D-79104 Freiburg, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Tholen
authors.forename: Martin L
authors.initials: ML
authors.lastname: Biniossek
authors.forename: Anna-Lena
authors.initials: AL
authors.lastname: Gessler
authors.forename: Sebastian
authors.initials: S
authors.lastname: Müller
authors.forename: Juliane
authors.initials: J
authors.lastname: Weisser
authors.forename: Jayachandran N
authors.initials: JN
authors.lastname: Kizhakkedathu
authors.forename: Thomas
authors.initials: T
authors.lastname: Reinheckel
authors.forename: Oliver
authors.initials: O
authors.lastname: Schilling
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D056668:Cathepsin L / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D005347:Fibroblasts / Q000378:metabolism*; D017353:Gene Deletion; D051379:Mice; D008969:Molecular Sequence Data; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods
chemical_list: D020543:Proteome; D056668:Cathepsin L