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. 2018 May 18;8(1):7834.
doi: 10.1038/s41598-018-26282-y.

Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease

Alexandre Denadai-Souza  1 , Chrystelle Bonnart  1 , Núria Solà Tapias  1 , Marlène Marcellin  2 , Brendan Gilmore  3 , Laurent Alric  4 , Delphine Bonnet  1 , Odile Burlet-Schiltz  2 , Morley D Hollenberg  5 , Nathalie Vergnolle  6   7 , Céline Deraison  1
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Functional Proteomic Profiling of Secreted Serine Proteases in Health and Inflammatory Bowel Disease

Alexandre Denadai-Souza (VSports手机版) et al. Sci Rep. .

Abstract

While proteases are essential in gastrointestinal physiology, accumulating evidence indicates that dysregulated proteolysis plays a pivotal role in the pathophysiology of inflammatory bowel disease (IBD) VSports手机版. Nonetheless, the identity of overactive proteases released by human colonic mucosa remains largely unknown. Studies of protease abundance have primarily investigated expression profiles, not taking into account their enzymatic activity. Herein we have used serine protease-targeted activity-based probes (ABPs) coupled with mass spectral analysis to identify active forms of proteases secreted by the colonic mucosa of healthy controls and IBD patients. Profiling of (Pro-Lys)-ABP bound proteases revealed that most of hyperactive proteases from IBD secretome are clustered at 28-kDa. We identified seven active proteases: the serine proteases cathepsin G, plasma kallikrein, plasmin, tryptase, chymotrypsin-like elastase 3 A, and thrombin and the aminopeptidase B. Only cathepsin G and thrombin were overactive in supernatants from IBD patient tissues compared to healthy controls. Gene expression analysis highlighted the transcription of genes encoding these proteases into intestinal mucosae. The functional ABP-targeted proteomic approach that we have used to identify active proteases in human colonic samples bears directly on the understanding of the role these enzymes may play in the pathophysiology of IBD. .

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Validation of biotin-PK-DPP sensitivity for detection of trypsin-like enzymes (A). 1 µM PK-ABP was incubated with an increasing concentration of trypsin and the biotinylated trypsin product was visualized by electrophoresis followed by detection using streptavidin-linked horseradish peroxidase and ECL. (B) Trypsin was treated first with the broad-spectrum serine protease inhibitor AEBSF (4 mM) (the + condition) prior to its reaction with ABP and ECL detection.
Figure 2
Figure 2
Measurement of trypsin-like activity released by human colonic mucosa. Trypsin-like activity detected in supernatants from colonic tissue samples of control or IBD patients (n = 11–16). Data were analysed by ANOVA followed by the multi comparison test of Holm-Sidak. *P < 0.05 vs. control.
Figure 3
Figure 3
Proteomic profiling of serine proteases released by the human colonic mucosa. (A) Representative ABP proteomic profile, showing the differential repertoire of ABP-labelled serine proteases secreted from control or IBD colonic tissue samples along with the positive trypsin control (20 mU of trypsin). The red arrowheads point to bands corresponding to active proteases, as verified by the inhibitory effects of pre-treatment of the samples with AEBSF (4 mM). (B) Clustering of ABP-labelled serine proteases according to size (kDa). (C) Graphic representation of protease size clusters along with their activity index determined by the impact of enzyme inhibition (−/+AEBSF). The percentage of AEBSF-inhibited bands per patient is represented by the pie graphs. The empty circles represent patients wherein bands within the cluster were not detected (negative), a 0 value was given to these samples as per their activity index. Activity index data were analysed by Kruskal-Wallis followed by the multi comparison test of Dunn. *P < 0.05, **P < 0.01, vs. control; #P < 0.05 vs. CD.
Figure 4
Figure 4
Colonic RNA expression for proteases identified as active. Analytical agarose-gel electrophoresis of RT-PCR products amplified from cDNAs prepared from human colonic mucosa tissue samples are shown with arrows denoting the predicted size (base pairs: bp) of the PCR product. Negative controls (noted -) consisted of RT reactions performed in the absence of enzyme. Positive expression was confirmed using cDNAs prepared from human tissue sources known to express the target proteases.
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