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\begin{document}

\title{Chymotrypsin activity signals to intestinal epithelium by
Protease-Activated Receptor-dependent mechanisms}



\author[1]{Simon Guignard}%
\author[2]{Mahmoud Saifeddine}%
\author[2]{Koichiro Mihara}%
\author[2]{Majid Motahhary}%
\author[3]{Magali Savignac}%
\author[1]{Laura Guiraud}%
\author[1]{David Sagnat}%
\author[4]{mireille sebbag}%
\author[1]{Sokchea Khou}%
\author[5]{Corinne Rolland}%
\author[6]{Barbara Bournet}%
\author[6]{Louis Buscail}%
\author[1]{Etienne Buscail}%
\author[7]{Laurent Alric}%
\author[8]{Caroline Camare}%
\author[1]{Mouna Ambli}%
\author[1]{Nathalie Vergnolle}%
\author[9]{Morley Hollenberg}%
\author[1]{Celine Deraison}%
\author[1]{Chrystelle Bonnart}%
\affil[1]{Inserm U1220}%
\affil[2]{Uinversity of Calgary}%
\affil[3]{Toulouse Institute for Infectious and Inflammatory Diseases (Infinity) INSERM UMR1291 }%
\affil[4]{INSERM UMR 1220}%
\affil[5]{INSERM}%
\affil[6]{Gastroenterology department Toulouse university Hospital}%
\affil[7]{Toulouse University Department of Internal Medicine and Digestive Diseases, Rangueil, Toulouse 3 University Hospital, Toulouse, France.}%
\affil[8]{Toulouse University Hospitals, Department of Clinical Biochemistry, Toulouse, France}%
\affil[9]{University of Calgary}%


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  \date{May 20, 2024}


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\begin{abstract}
Background and purpose Chymotrypsin is a serine protease produced by the
pancreas and secreted into the lumen of the small intestine, where it
digests food proteins. Due to its presence in the gut lumen, we
hypothesized that chymotrypsin activity may be found close to epithelial
cells and signals to them via Protease-activated receptors (PARs). We
deciphered molecular pharmacology mechanisms for chymotrypsin signaling
in intestinal epithelial cells. Experimental approaches The presence and
activity of chymotrypsin were evaluated by western blot (WB) and
enzymatic activity tests in luminal and mucosal compartments of murine
and human gut samples. The ability of chymotrypsin to cleave the
extracellular domain of PAR1 or PAR2 was assessed using cell lines
expressing N-terminally-tagged receptors. The cleavage site of
chymotrypsin on PAR1 and PAR2 was determined by HPLC-MS analysis. To
study the pharmacology of chymotrypsin signals, we investigated calcium
signaling and ERK1/2 activation using calcium mobilization assays and WB
in CMT93 intestinal epithelial cells. Key results We found that
chymotrypsin was present and active in the vicinity of the murine and
human colonic epithelium. Molecular pharmacology studies evidenced that
chymotrypsin cleaved both PARs receptors. While chymotrypsin activated
calcium and ERK1/2 signaling pathways through PAR2, it disarmed PAR1,
preventing further activation by its canonical agonist thrombin.
CONCLUSION Our work suggests that the function of chymotrypsin in the
gut lumen goes well beyond a simple digestive role. Our results
highlight the ability of chymotrypsin to signal to intestinal epithelial
cells via PARs, which may have important physiological consequences in
gut homeostasis.%
\end{abstract}%



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