Application of a novel FAM-conjugated activity-based probe to determine cathepsin G activity intracellularly.

Cathepsin G (CatG) is responsible for several distinct immune processes of adaptive and innate immunity depending on extra- or intracellular occurrence of CatG. Recently, we established a method to detect CatG activity at the cell surface of natural killer cells by using the activity-based probe MARS116-Bt in flow cytometry. MARS116-Bt consists of biotin, spacer, amino acid sequence, and a phosphonate warhead which binds covalently to the serine amino acid residue within the active center of CatG. Herein, MARS116 was conjugated to 5(6)-carboxyfluorescein (FAM) in order to limit non-specific signal-to-noise ratio generally resulting from binding of fluorescein-labelled avidin (avidin-FAM) to biotinylated, intracellular proteins; since MARS116-Bt is incubated with avidin-FAM in a second labelling step. MARS116-FAM was capable to detect intracellular CatG activity, in contrast to the control compound MARS116*-FAM which lacks the functional phosphonate warhead crucial for binding to the active-site of CatG and contains a carboxyl group instead. Furthermore, intracellular CatG activity was determined in CD4+ T cells, CD8+ T cells as well as in T regulatory cell (Treg) subsets. Thus, MARS116-FAM is a convenient activity-based probe to detect intracellular CatG activity in a flow cytometry approach.

Mast cell derived carboxypeptidase A3 is decreased among patients with advanced coronary artery disease.

BACKGROUND: Coronary artery disease (CAD) affects milions of people and can result in myocardial infarction (MI). Previously, mast cells (MC) have been extensively investigated in the context of hypersensitivity, however as regulators of the local inflammatory response they can potentially contribute to CAD and/or its progression. The aim of the study was to assess if serum concentration of MC proteases: carboxypeptidase A3, cathepsin G and chymase 1 is associated with the extension of CAD and MI. METHODS: The 44 patients with angiographically confirmed CAD (23 subjects with non-ST-segment elevation MI [NSTEMI] and 21 with stable CAD) were analyzed. Clinical data were obtained as well serum concentrations of carboxypeptidase A3, cathepsin G and chymase 1 were also measured. RESULTS: Patients with single vessel CAD had higher serum concentration of carboxypeptidase than those with more advanced CAD (3838.6 ± 1083.1 pg/mL vs. 2715.6 ± 442.5 pg/mL; p = 0.02). There were no significant differences in levels of any protease between patients with stable CAD and those with NSTEMI. Patients with hypertension had ≈2-fold lower serum levels of cathepsin G than normotensive individuals (4.6 ± 0.9 pg/mL vs. 9.4 ± 5.8 pg/mL; p = 0.001). Cathepsin G levels were also decreased in sera of the current smokers as compared with non-smokers (3.1 ± 1.2 ng/mL vs. 5.8 ± 1.2 ng/mL, p = 0.02). CONCLUSIONS: Decreased serum level of carboxypeptidase is a hallmark of more advanced CAD. Lower serum levels of carboxypeptidase A3 and catepsin G are associated with risk factors of blood vessel damage suggesting a protective role of these enzymes in CAD.

Protective Effects of Chymostatin on Paraquat-Induced Acute Lung Injury in Mice.

This study aims to evaluate the role of chymostatin in paraquat-induced acute lung injury. Institute of Cancer Research mice were randomly distributed into the NS, DMSO, chymostatin, paraquat or chymostatin treatment groups. Six mice from each group were intraperitoneally injected with chloral hydrate at 0, 1, 2, 4, 8, 12, 24 and 48 h after treatment administration. Blood samples were collected through cardiac puncture. Lung tissues were stained with haematoxylin and eosin for the observation of lung histology. The degree of pulmonary oedema was determined on the basis of lung wet-to-dry ratio (W/D). The serum activity of cathepsin G was determined through substrate fluorescence assay. The serum levels of endothelial cell-specific molecule-1 (endocan), tumour necrosis factor-a (TNF-a), interleukin-1ß (IL-1ß), IL-6 and high-mobility group box protein 1 (HMGB1) were determined through enzyme-linked immunosorbent assay. The expression levels of endocan and nuclear NF-κBp65 in the lung were quantified through Western blot. Chymostatin alleviated the pathological changes associated with acute alveolitis in mice; decreased the lung W/D ratio, the activity of cathepsin G and the serum concentrations of TNF-a, IL-1ß, IL-6 and HMGB1; and increased the serum concentration of endocan. Western blot results revealed that chymostatin up-regulated endocan expression and down-regulated nuclear NF-κBp65 expression in the lung. Chymostatin reversed the inflammatory effects of paraquat-induced lung injury by inhibiting cathepsin G activity to up-regulate endocan expression and indirectly inhibit NF-κBp65 activity.

The role and mechanism of cathepsin G in dermatomyositis.

Dermatomyositis (DM) is an idiopathic inflammatory myopathy characterized by CD4+ T cells and B cells infiltration in perivascular and muscle tissue. Although the infiltration of inflammatory cells plays a key role in muscle damage, the exact mechanism is not clear. Cathepsin G (CTSG) is a member of the serine proteases family and can increase the permeability of vascular endothelial cells and the chemotaxis of inflammatory cells. In this study, we found that the expression of CTSG was increased in peripheral blood mononuclear cells and muscle tissues of DM patients. The activity of CTSG was significantly increased in DM patients and correlated with disease activity. Serum CTSG induced the expression of protease activated receptor 2 (PAR2) and altered the cytoskeleton of human dermal microvascular endothelial cells. Our studies indicate, for the first time, that CTSG may play an important role in muscle inflammatory cells infiltration by increasing the permeability of vascular endothelial cells.

Elevated Leukocyte Azurophilic Enzymes in Human Diabetic Ketoacidosis Plasma Degrade Cerebrovascular Endothelial Junctional Proteins.

OBJECTIVE: Diabetic ketoacidosis in children is associated with vasogenic cerebral edema, possibly due to the release of destructive polymorphonuclear neutrophil azurophilic enzymes. Our objectives were to measure plasma azurophilic enzyme levels in children with diabetic ketoacidosis, to correlate plasma azurophilic enzyme levels with diabetic ketoacidosis severity, and to determine whether azurophilic enzymes disrupt the blood-brain barrier in vitro. DESIGN: Prospective clinical and laboratory study. SETTING: The Children's Hospital, London Health Sciences Centre. SUBJECTS: Pediatric type 1 diabetes patients; acute diabetic ketoacidosis or age-/sex-matched insulin-controlled. MEASUREMENTS AND MAIN RESULTS: Acute diabetic ketoacidosis in children was associated with elevated polymorphonuclear neutrophils. Plasma azurophilic enzymes were elevated in diabetic ketoacidosis patients, including human leukocyte elastase (p < 0.001), proteinase-3 (p < 0.01), and myeloperoxidase (p < 0.001). A leukocyte origin of human leukocyte elastase and proteinase-3 in diabetic ketoacidosis was confirmed with buffy coat quantitative real-time polymerase chain reaction (p < 0.01). Of the three azurophilic enzymes elevated, only proteinase-3 levels correlated with diabetic ketoacidosis severity (p = 0.002). Recombinant proteinase-3 applied to human brain microvascular endothelial cells degraded both the tight junction protein occludin (p < 0.05) and the adherens junction protein VE-cadherin (p < 0.05). Permeability of human brain microvascular endothelial cell monolayers was increased by recombinant proteinase-3 application (p = 0.010). CONCLUSIONS: Our results indicate that diabetic ketoacidosis is associated with systemic polymorphonuclear neutrophil activation and degranulation. Of all the polymorphonuclear neutrophil azurophilic enzymes examined, only proteinase-3 correlated with diabetic ketoacidosis severity and potently degraded the blood-brain barrier in vitro. Proteinase-3 might mediate vasogenic edema during diabetic ketoacidosis, and selective proteinase-3 antagonists may offer future vascular- and neuroprotection.

Neutrophil serine proteases and their endogenous inhibitors in coronary artery ectasia patients.

OBJECTIVE: Proteolytic enzymes possibly contribute to coronary artery ectasia (CAE). This study aimed to determine whether neutrophils, neutrophil serine proteases (NSPs), and their endogenous inhibitors participated in the pathological process of CAE. METHODS: The study consisted of 30 patients with CAE, 30 patients with coronary artery disease (CAD), and 29 subjects with normal coronary arteries (Control). The following circulating items were measured: the main NSPs, including human neutrophil elastase (HNE), cathepsin G (CG), and proteinase 3 (PR3); soluble elastin (sElastin), which was a degradation product of elastin fibres; NSP inhibitors such as α1-protease inhibitor (α1-PI), α2-macroglobulin (α2-MG), secretory leucoprotease inhibitor (SLPI), and elafin; as well as two neutrophil activation markers (myeloperoxidase and lactoferrin) and three classic neutrophil activators [tumor necrosis factor-α (TNF-α), interleukin-8 (IL-8), and bacterial endotoxin]. RESULTS: The levels of HNE, CG, and sElastin were elevated in the CAE group. The levels of α1-PI and α2-MG were also significantly increased in the CAE group. The levels of myeloperoxidase and lactoferrin were higher in the CAE group. The levels of TNF-α, IL-8, and endotoxin were unchanged in the CAE group compared with those in the CAD group. CONCLUSION: Neutrophils may participate in the process of vessel extracellular matrix destruction and coronary ectasia by releasing NSPs in a non-classical manner.

Quantitative Proteomic Analysis of Peripheral Blood Mononuclear Cells in Ankylosing Spondylitis by iTRAQ.

This study was designed to identify and quantify the different proteins expression levels in ankylosing spondylitis (AS) and to explore the pathogenesis of AS. We performed isobaric tags for relative and absolute quantitation (iTRAQ) coupled with multiple chromatographic fractionation and tandem mass spectrometry to detect the proteins profiling in peripheral blood mononuclear cells (PBMCs) from AS patients and healthy controls. Mascot software and the International Protein Index and the Gene Ontology (GO) database were used to conduct the bioinformatics analysis. The differentially expressed proteins were validated by enzyme-linked immunosorbent assay (ELISA). A total of 1,232 proteins were identified by iTRAQ, of which 183 showed differential expression and 18 differentially expressed proteins were acute phase reactants. Upon mapping of the differentially expressed proteins to GO database, we found four differentially expressed proteins involved in the biological process of cell killing, including up-regulated cathepsin G (CTSG), neutrophil defensin3 (DEFA3), protein tyrosine phosphatase receptor type C (PTPRC), and down-regulated peroxiredoxin-1(PRDX1),which were consistent with the verified results of ELISA. Our proteomic analyses suggested that the proteins involved in the biological process of cell killing might play an important role in the pathogenesis of AS.

Identification of a 14 kDa endocan fragment generated by cathepsin G, a novel circulating biomarker in patients with sepsis.

Severe septic syndrome, which is the most prevalent and lethal cause of acute respiratory distress syndrome, remains one of the most frequent causes of admission and death in intensive care units (ICU). Inflammatory phenomenon leading to severe sepsis are multiple and not yet completely understood. The main target damage during severe sepsis is the endothelium. Endocan, specifically secreted by activated-pulmonary vascular endothelial cells, is thought to play a key role in the control of the lung inflammatory reaction. A recent clinical investigation found that a low plasma endocan level was predictive of respiratory failure. In this study, the hypothesis that low levels of endocan may result from proteolysis was tested. We demonstrate that cathepsin G (CG), neutrophil elastase (NE), and to a lesser extent proteinase 3 (PR3), degrade endocan. Interestingly, a novel endocan peptide fragment of 14 kDa, named p14, was identified, resulting from the specific cleavage of endocan by CG, corresponding to the N-terminal 111-116 amino acids of the endocan polypeptide. An immunoassay specific for p14 endocan fragment was then developed, and revealed increased plasma levels of p14 in 20 out of 55 severe septic patients, ranging from 0.52 to 10.40 ng/mL versus undetectable p14 in plasma from 32 control subjects (p=0.0011). No correlations were found between p14 and endocan blood levels in severe septic patients. Taken together, the p14 endocan fragment represents a novel interesting biomarker which could participate to the pathogenesis of sepsis.

Surface plasmon resonance imaging biosensor for cathepsin G based on a potent inhibitor: development and applications.

A specific surface plasmon resonance imaging (SPRI) array biosensor for the determination of the enzymatically active cathepsin G (CatG) has been developed. For this purpose, a specific interaction between an inhibitor immobilized onto a chip surface and CatG in an analyzed solution was used. The MARS-115 CatG peptidyl inhibitor containing the 1-aminoalkylphosphonate diaryl ester moiety at the C terminus and N-succinamide with a free carboxylic function was synthesized and covalently immobilized onto the gold chip surface via the thiol group (cysteamine). Atomic force microscopy was used for the observation of surface changes during the subsequent steps of chip manufacture. Optimal detection conditions were chosen. High specificity of synthesized inhibitor to CatG was proved. The precision, as well as the accuracy, was found to be well suited to enzyme determination. The sensor application for the determination of CatG in white blood cells and saliva was shown for potential diagnosis of leukemia and oral cavity diseases during the early stages of those pathological states.

In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential.

Cathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel's Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

Proteomic analysis of plasma from patients undergoing coronary artery bypass grafting reveals a protease/antiprotease imbalance in favor of the serpin alpha1-antichymotrypsin.

We used proteomics to identify systematic changes in the plasma proteins of patients undergoing coronary artery bypass grafting (CABG) by means of cardiopulmonary bypass surgery. It is known that, after CABG, a complex systemic inflammatory responses ensues that favors the occurrence of adverse postoperative complications frequently recognizing inflammation itself and/or thrombosis as the underlying mechanism. We found a marked and persistent postoperative increase in the levels of the serpin-protease inhibitor alpha(1)-antichymotrypsin (alpha(1)-ACT) that fully maintains the inhibitory activity blunting its protease substrate cathepsin G. An intraoperative increase followed by a rapid decline in proteases activation was documented, accompanied by a substantial induction of leucine-rich-alpha-2-glycoprotein, a protein involved in neutrophilic granulocyte differentiation. Finally, a time-dependent alteration in the expression of haptoglobin, transthyretin, clusterin, and apoE was observed. In conclusion, we showed that after CABG, a protease/antiprotease imbalance occurs with early cathepsin G activation and a more delayed increase in alpha(1)-ACT. As cathepsin G is a serpin involved both in inflammation and coagulation activation, this confirms and expands the concept of a marked dysregulation of both inflammatory and hemostatic balances occurring after CABG. The pharmacologic modulation of this imbalance may be a new therapeutic target to reduce postoperative complications.