Cathepsin G Activity as a New Marker for Detecting Airway Inflammation by Microscopy and Flow Cytometry

Abstract

Muco-obstructive lung diseases feature extensive bronchiectasis due to the uncontrolled release of neutrophil serine proteases into the airways. To assess if cathepsin G (CG) is a novel key player in chronic lung inflammation, we developed membrane-bound (mSAM) and soluble (sSAM) FRET reporters. The probes quantitatively revealed elevated CG activity in samples from 46 patients. For future basic science and personalized clinical applications, we developed a rapid, highly informative, and easily translatable small-molecule FRET flow cytometry assay for monitoring protease activity including cathepsin G. We demonstrated that mSAM distinguished healthy from patient cells by FRET-based flow cytometry with excellent correlation to confocal microscopy data.

Neutrophil Adaptations upon Recruitment to the Lung: New Concepts and Implications for Homeostasis and Disease

“…Importantly, they have been shown to develop a unique inflammatory phenotype after recruitment into the CF airway lumen, where they maintain viability and exocytose their primary granules but have reduced ability to phagocytose bacteria in a distinct fate now called GRIM (granule releasing, immunomodulatory, and metabolically active) neutrophils [105,116,117]. Exocytosis of the primary granules, which are usually sequestered in the cytoplasm, results in release of effectors including NE, Cathepsin G, MPO and Arg1, which has been found to correlate with disease progression in mice with CF-like lung disease and young children and older patients with CF [118][119][120]. This pro-inflammatory phenotype with reduced ability to clear pathogens poses an intriguing paradox and is becoming a focal point in addressing CF lung disease.…”

Peptidyl Activity-Based Probes for Imaging Serine Proteases

“…These linkers are equipped with additional oxygen groups in the structure increasing reporter solubility and permeability throughout the cell membrane. The classic examples of polyethylene glycols (PEGs) are PEG (4) and PEG (2), which have been applied in HNE and CatG probes ( Gehrig et al, 2012 ; Kasperkiewicz et al, 2014 ; Guerra et al, 2019 ).…”

“…The anchoring of the hydrolysis product is not the only advantage of probe lipidation. This strategy has also been used to monitor protease activity at the cell surface (CatG) ( Guerra et al, 2019 ) or membrane-bound enzymes (PR3). In another strategy, the fluorophore can be attached to the C-terminal end of the probe while the quencher is attached at the N-terminus.…”

“…In this example of FRET transfer, the hydrolysis of peptide sequence leads to a difference in fluorescence emission; an approximately 8.9-fold increase in donor emission and a 0.9-fold decrease in acceptor fluorescence. The exact characterization of the TAMRA-coumarin 343 pair leads to the conclusion that this donor-acceptor pair can be successfully applied to basic diagnostic applications, as it has been used to monitor CatG activity over time ( Guerra et al, 2019 ). When two fluorophores are used, they both contribute to an overall background due to their fluorescent nature.…”

The Life Science Toolbox Provided by Chemical Biology

“…In the last decade, several labs have developed low molecular weight fluorescent probes to detect tumor cells during surgery in the operating theater ,. My own lab prepared low molecular weight FRET reporters for detecting protease activity from cells in sputum samples of cystic fibrosis and COPD patients …”