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\begin{document}

\title{Development of a proteomic workflow for the identification of heparan
sulfate proteoglycan-binding substrates of ADAM17}



\author[1]{Matteo Calligaris}%
\author[1]{Donatella Spanò}%
\author[2]{Stephan Mueller}%
\author[1]{Simone Bonelli}%
\author[1]{Margot Lo Pinto}%
\author[3]{Giovanni Zito}%
\author[4]{Carl Blobel}%
\author[2]{S Lichtenthaler}%
\author[5]{Linda Troeberg}%
\author[1]{Simone Scilabra}%
\affil[1]{Ri.MED Foundation}%
\affil[2]{DZNE-Standort München}%
\affil[3]{ISMETT}%
\affil[4]{Hospital for Special Surgery Arthritis and Tissue Degeneration Program}%
\affil[5]{University of East Anglia Norwich Medical School}%


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  \date{May 23, 2024}


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\begin{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.%
\end{abstract}%



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