Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis

• DOI: 10.1007/s00018-013-1406-1
• Publication Date: 2013-06-28T05:30:59Z
• Authors: Stefan Tholen; Martin L. Biniossek; Martina Gansz; Theresa D. Ahrens; Manuel Schlimpert; Jayachandran N. K...; Thomas Reinheckel; Oliver Schilling
• Keywords: Mice, Knockout; 0303 health sciences; Cell Survival; Cathepsin L; Blotting, Western; Fibroblasts; Hydrogen-Ion Concentration; Chromatography, Ion Exchange; Flow Cytometry; Collagen Type I; Cathepsin B; Mice; 03 medical and health sciences; Gene Expression Regulation; Tandem Mass Spectrometry; Proteolysis; Animals; Matrix Metalloproteinase 2; Cells, Cultured; Chromatography, High Pressure Liquid; Cell Proliferation

Endolysosomal cysteine cathepsins functionally cooperate. Cathepsin B (Ctsb) and L (Ctsl) double-knockout mice die 4 weeks after birth accompanied by (autophago-) lysosomal accumulations within neurons. Such accumulations are also observed in mouse embryonic fibroblasts (MEFs) deficient for Ctsb and Ctsl. Previous studies showed a strong impact of Ctsl on the MEF secretome. Here we show that Ctsb alone has only a mild influence on extracellular proteome composition. Protease cleavage sites dependent on Ctsb were identified by terminal amine isotopic labeling of substrates (TAILS), revealing a prominent yet mostly indirect impact on the extracellular proteolytic cleavages. To investigate the cooperation of Ctsb and Ctsl, we performed a quantitative secretome comparison of wild-type MEFs and Ctsb (-/-) Ctsl (-/-) MEFs. Deletion of both cathepsins led to drastic alterations in secretome composition, highlighting cooperative functionality. While many protein levels were decreased, immunodetection corroborated increased levels of matrix metalloproteinase (MMP)-2. Re-expression of Ctsl rescues MMP-2 abundance. Ctsl and to a much lesser extent Ctsb are able to degrade MMP-2 at acidic and neutral pH. Addition of active MMP-2 to the MEF secretome degrades proteins whose levels were also decreased by Ctsb and Ctsl double deficiency. These results suggest a degradative Ctsl-MMP-2 axis, resulting in increased MMP-2 levels upon cathepsin deficiency with subsequent degradation of secreted proteins such as collagen α-1 (I).