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

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Abstract

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).

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Abbreviations

ADAMTS:

A disintegrin and metalloproteinase with thrombospondin motifs

BM:

Basement membrane

BSA:

Bovine serum albumin

CCM:

Cell-conditioned media

Ctsb:

Cathepsin B

Ctsd:

Cathepsin D

Ctsl:

Cathepsin L

Ctsz:

Cathepsin Z

DMEM:

Dulbecco’s modified Eagle's medium

DTT:

Dithiothreitol

E64d:

(2S, 3S)-trans-epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester

ECM:

Extracellular matrix

EDTA:

Ethylene diamine tetraacetic acid

FACS:

Fluorescence-assisted cell sorting

Fc:

Fold change

GFP:

Green fluorescent protein

GO:

Gene ontology

HPLC:

High-performance liquid chromatography

IGF:

Insulin-like growth factor

IGFBP:

Insulin-like growth factor-binding protein

IRES:

Internal ribosomal entry site

Lamp:

Lysosome-associated membrane glycoprotein

LC–MS/MS:

Liquid chromatography tandem mass spectrometry

LDH:

Lactate dehydrogenase

LEF:

Lymphocyte enhancer factor

MEF:

Mouse embryonic fibroblast

MMP:

Matrix metalloprotease

MS:

Mass spectrometry

NCAM:

Neural cell adhesion molecule

ON:

Overnight

PBS:

Phosphate-buffered saline

PICS:

Proteomic identification of protease cleavage sites

PMSF:

Phenylmethanesulfonyl fluoride

POSTN:

Periostin

PVDF:

Polyvinylidene fluoride

SCX:

Strong cation exchange

SDS:

Sodium dodecylsulfate

STRING:

Search tool for the retrieval of interacting genes

SFRP:

Secreted frizzled-related protein

TAILS:

Terminal amine isotopic labeling of substrates

TCF:

T-cell factor

wt:

Wild-type

XIC:

Extracted ions chromatograms

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Acknowledgments

O.S. is supported by an Emmy-Noether grant from the Deutsche Forschungsgemeinschaft (DFG) (SCHI 871/2), a starting grant from the European Research Council (Programme “Ideas”—call identifier: ERC-2011-StG 282111-ProteaSys), and the Excellence Initiative of the German Federal and State Governments (EXC 294, BIOSS). J.N.K. acknowledges the Michael Smith Foundation for the Health Research (MSFHR) career investigator scholar award. T.R. is supported by Deutsche Forschungsgemeinschaft SFB 850 project B7 and grant Re158416-1, and furthermore by the Excellence Initiative of the German Federal and State Governments (EXC 294 and GSC-4, Spemann Graduate School). The authors thank Prof. Dr. Christoph Peters and Florian Christoph Sigloch for critical discussion. Franz Jehle and Bettina Mayer are acknowledged for excellent technical assistance. The authors thank Dr. Dorit Nägler, Munich, for the kind gift of recombinant CTSB and CTSL, Dr. Ulrich Maurer, Freiburg, for assistance with the pMIG system, and Dr. Gill Murphy for kindly providing recombinant human MMP-2. The core facility of the Universitätsklinikum Freiburg (Dr. Marie Follo and Klaus Geiger) is acknowledged for sorting cell lines.

Conflict of interest

The authors declare no conflict of interest.

Author information

Authors and Affiliations

  1. Institute for Molecular Medicine and Cell Research, University of Freiburg, Stefan Meier Strasse 17, 79104, Freiburg, Germany

    Stefan Tholen, Martin L. Biniossek, Martina Gansz, Theresa D. Ahrens, Manuel Schlimpert, Thomas Reinheckel & Oliver Schilling

  2. Faculty of Biology, University of Freiburg, 79104, Freiburg, Germany

    Stefan Tholen, Martina Gansz & Theresa D. Ahrens

  3. Centre for Blood Research, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

    Jayachandran N. Kizhakkedathu

  4. Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

    Jayachandran N. Kizhakkedathu

  5. Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

    Jayachandran N. Kizhakkedathu

  6. BIOSS Centre for Biological Signaling Studies, University of Freiburg, 79104, Freiburg, Germany

    Thomas Reinheckel & Oliver Schilling

Authors
  1. Stefan Tholen
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  2. Martin L. Biniossek
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  3. Martina Gansz
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  4. Theresa D. Ahrens
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  5. Manuel Schlimpert
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  6. Jayachandran N. Kizhakkedathu
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  7. Thomas Reinheckel
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  8. Oliver Schilling
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Corresponding author

Correspondence to Oliver Schilling.

Electronic supplementary material

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18_2013_1406_MOESM1_ESM.pptx

Supplementary Figure S1: Western blot analysis of Ctsb and Ctsl in cell-conditioned media of two different primary MEF populations and the two SV40 immortalized wild-type MEF cell lines. Supplementary Figure S2: Characterization of the wild-type mouse embryonic fibroblast cell lines, Ctsb knockout cell lines, and Ctsb Ctsl knockout cell lines used in this study. (A) Proliferation rate of all MEF cell lines. Proliferation data are presented as mean ± SEM (n = 3). (B) Western blot analysis of Lamp-1. MEF collection a was used for analysis and revealed an increase in Lamp-1 upon single Ctsl as well as double Ctsb and Ctsl deficiency. Lamp-1 signal intensities were quantified and normalized according to tubulin signal intensities. (C) Lactate dehydrogenase (LDH) – release of all cell lines after incubation in serum-free conditions for 24 h. Data are presented as mean normalized to wt a ± SEM (n = 3). Supplementary Figure S3: Distribution profiles of fold change values (log2) for all proteins identified in the quantitative proteome comparisons. (A) Replicate 1 of the secretome comparison of the first wild-type MEF cell line and the first Ctsb-deficient MEF cell line. (B) Replicate 2 of the secretome comparison of the second wild-type MEF cell line and the second Ctsb-deficient MEF cell line. (C) Replicate 1 of the secretome comparison of the first wild-type MEF cell line and the first Ctsb Ctsl double-deficient MEF cell line. (D) Replicate 2 of the secretome comparison of the second wild-type MEF cell line and the second Ctsb Ctsl double-deficient MEF cell line. Fold changes lower than -0.58 and higher than 0.58 represent changes in protein abundance of more than 50 %. A fold change of 0 indicates unaffected protein abundance. (D) Western blot analysis of β-catenin in total cell lysate (TCL) of wild-type, Ctsb-deficient, Ctsl-deficient and Ctsb Ctsl double-deficient MEFs. Tubulin expression was used as loading control. Supplementary Figure S4: Biological processes mainly affected by Ctsb and Ctsl depletion. Results were obtained by analyzing abundance alterations of all extracellular and secreted proteins in the quantitative proteome comparison of wild-type and Ctsb Ctsl-deficient MEFs by STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). Supplementary Figure S5: (A) Western blot analysis of N-cadherin in total cell lysates (TCL) of wild-type, Ctsb-deficient, Ctsl-deficient, and Ctsb Ctsl double-deficient MEFs. Tubulin expression was used as loading control. N-cadherin signal intensities were quantified and normalized according to tubulin signal intensities. (B) Western blot analysis of β-catenin in total cell lysate (TCL) of wild-type, Ctsb-deficient, Ctsl-deficient, and Ctsb Ctsl double-deficient MEFs. Tubulin expression was used as loading control. β-Catenin signal intensities were quantified and normalized according to tubulin signal intensities. Supplementary Figure S6: Western blot analysis of MMP-2 in cell-conditioned media of wild-type, Ctsb-deficient, Ctsl-deficient, and Ctsb Ctsl double-deficient MEFs grown upon different conditions. MEFs were grown on uncoated and coated plates, either coated with fibronectin or collagen type IV. MEFs were cultured in serum-free DMEM, serum-free DMEM without arginine and lysine, or serum-free DMEM containing 0.1 % albumin without arginine and lysine. (PPTX 1041 kb)

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Supplementary material 9 (PDF 547 kb)

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Tholen, S., Biniossek, M.L., Gansz, M. et al. Double deficiency of cathepsins B and L results in massive secretome alterations and suggests a degradative cathepsin-MMP axis. Cell. Mol. Life Sci. 71, 899–916 (2014). https://doi.org/10.1007/s00018-013-1406-1

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  • DOI: https://doi.org/10.1007/s00018-013-1406-1

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