Production of Bioactive Peptides by Neutrophil Elastase Cleavage of E‐cadherin

Abstract

BACKGROUNDThe inflammatory microenvironment in the gut contains a variety of proteases from numerous sources including inflammatory cells. We have been studying the ability of proteases to induce a switch in the colonic epithelium from a barrier to a repair phenotype (epithelial to mesenchymal transition [EMT]) characterized by increased migration and disrupted homeostasis. EMT involves the degradation of junctional proteins such as E‐cadherin [Ecad], but the functional consequences of Ecad loss remain incompletely understood.AIMTo test the hypothesis that inflammatory proteases cleave Ecad to produce peptides that alter epithelial homeostasis by altering cell migration, proliferation, or cell death.METHODSRecombinant Ecad was incubated with neutrophil elastase [NE] in a minimized cell‐free system and 24 peptides identified by mass spectrometry were chosen for high‐throughput screening based on frequency, p‐value scoring, and accessibility of peptide cleavage site. Synthesized peptides were tested for biological activities including proliferation, cell death, and cell migration. Murine CMT‐93 intestinal epithelial cells were cultured in 96 well plates and wounded 5 days post‐confluency using the EssenBio WoundMaker™ tool. Plates were imaged with the IncuCyte™ live‐cell imaging system for 24–48 hours following exposure to 1, 10, and 100 mg/mL concentrations of peptides. Cells were transfected with a GFP marker for automated cell counting to measure proliferation. Cytotoxicity was determined using Cytotox dye entering cells undergoing membrane degradation. All analysis was done using the IncuCyte™ ZOOM platform in conjunction with ImageJ software.RESULTSBasolateral stimulation of human Caco2 and murine CMT93 cells with NE confirmed the ability of NE to produce C‐ and N‐terminal Ecad fragments in vitro. From our synthesized peptides, we identified 10 Ecad peptides that significantly influenced wound closure rates, both positively and negatively. Two peptides (100 mg/mL) significantly inhibited wound healing rate by 13–26% compared to vehicle controls. Six peptides (100 μg/mL) significantly increased wound healing rates by 7–11% and three peptides (10 μg/mL) significantly increased wound healing rates by 6–13% compared to vehicle controls. Preliminary results suggest that several of these peptides also have cytostatic or pro‐proliferative activity.CONCLUSIONSOur results suggest that degradation of cell junction proteins by inflammatory proteases can create bioactive peptides that alter epithelial homeostasis and alter cell migration. Our data reveal a novel and complex pathway whereby epithelia respond to inflammatory tissue damage at the cellular level to alter a barrier phenotype to a more dynamic epithelium.Support or Funding InformationFunding: Supported by the Canadian Institutes of Health Research (CIHR)