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Development of a proteomic workflow for the identification of heparan sulfate proteoglycan-binding substrates of ADAM17
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  • Matteo Calligaris,
  • Donatella Spanò,
  • Stephan Mueller,
  • Simone Bonelli,
  • Margot Lo Pinto,
  • Giovanni Zito,
  • Carl Blobel,
  • S Lichtenthaler,
  • Linda Troeberg,
  • Simone Scilabra
Matteo Calligaris
Ri.MED Foundation
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Donatella Spanò
Ri.MED Foundation
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Stephan Mueller
DZNE-Standort München
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Simone Bonelli
Ri.MED Foundation
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Margot Lo Pinto
Ri.MED Foundation
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Giovanni Zito
ISMETT
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Carl Blobel
Hospital for Special Surgery Arthritis and Tissue Degeneration Program
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S Lichtenthaler
DZNE-Standort München
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Linda Troeberg
University of East Anglia Norwich Medical School
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Simone Scilabra
Ri.MED Foundation

Corresponding Author:[email protected]

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Abstract

Ectodomain shedding, which is the proteolytic release of transmembrane proteins from the cell surface, is crucial for cell-to-cell communication and other biological processes. The metalloproteinase ADAM17 mediates ectodomain shedding of over 50 transmembrane proteins ranging from cytokines and growth factors, such as TNF and EGFR ligands, to signaling receptors and adhesion molecules. Yet, the ADAM17 sheddome is only partly defined and biological functions of the protease have not been fully characterized. Some ADAM17 substrates (e.g. HB-EGF) are known to bind to heparan sulfate proteoglycans (HSPG), and we hypothesised that such substrates would be under-represented in traditional secretome analyses, due to their binding to cell surface or pericellular HSPGs. Thus, to identify novel HSPG-binding ADAM17 substrates, we developed a proteomic workflow that involves addition of heparin to solubilize HSPG-binding proteins from the cell layer, thereby allowing their mass spectrometry detection by heparin-secretome (HEP-SEC) analysis. Applying this methodology to murine embryonic fibroblasts stimulated with an ADAM17 activator enabled us to identify 47 transmembrane proteins that were shed in response to ADAM17 activation. This included known HSPG-binding ADAM17 substrates (i.e. HB-EGF, CX3CL1) and 17 novel HSPG-binding putative ADAM17 substrates. Two of these, MHC-I and IL1RL1, were validated as ADAM17 substrates by immunoblotting.
Submitted to PROTEOMICS
08 Mar 2024Submission Checks Completed
08 Mar 2024Assigned to Editor
08 Mar 2024Review(s) Completed, Editorial Evaluation Pending
31 May 2024Editorial Decision: Revise Minor
10 Aug 2024Review(s) Completed, Editorial Evaluation Pending
10 Aug 20241st Revision Received
15 Aug 2024Reviewer(s) Assigned
16 Aug 2024Editorial Decision: Revise Minor
27 Aug 2024Review(s) Completed, Editorial Evaluation Pending
27 Aug 20242nd Revision Received
29 Aug 2024Editorial Decision: Accept