ABSTRACT
Membrane-bound proteinase 3 (PR3m) is the main target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis, a systemic small-vessel vasculitis. Binding of ANCA to PR3m triggers neutrophil activation with the secretion of enzymatically active PR3 and related neutrophil serine proteases, thereby contributing to vascular damage. PR3 and related proteases are activated from pro-forms by the lysosomal cysteine protease cathepsin C (CatC) during neutrophil maturation. We hypothesized that pharmacological inhibition of CatC provides an effective measure to reduce PR3m and therefore has implications as a novel therapeutic approach in granulomatosis with polyangiitis. We first studied neutrophilic PR3 from 24 patients with Papillon-Lefèvre syndrome (PLS), a genetic form of CatC deficiency. PLS neutrophil lysates showed a largely reduced but still detectable (0.5-4%) PR3 activity when compared with healthy control cells. Despite extremely low levels of cellular PR3, the amount of constitutive PR3m expressed on the surface of quiescent neutrophils and the typical bimodal membrane distribution pattern were similar to what was observed in healthy neutrophils. However, following cell activation, there was no significant increase in the total amount of PR3m on PLS neutrophils, whereas the total amount of PR3m on healthy neutrophils was significantly increased. We then explored the effect of pharmacological CatC inhibition on PR3 stability in normal neutrophils using a potent cell-permeable CatC inhibitor and a CD34+ hematopoietic stem cell model. Human CD34+ hematopoietic stem cells were treated with the inhibitor during neutrophil differentiation over 10 days. We observed strong reductions in PR3m, cellular PR3 protein, and proteolytic PR3 activity, whereas neutrophil differentiation was not compromised.
Subject(s)
Cathepsin C/antagonists & inhibitors , Cell Membrane/metabolism , Cysteine Proteinase Inhibitors/pharmacology , Granulomatosis with Polyangiitis/pathology , Myeloblastin/metabolism , Adolescent , Adult , Case-Control Studies , Child , Child, Preschool , Female , Granulomatosis with Polyangiitis/drug therapy , Granulomatosis with Polyangiitis/genetics , Granulomatosis with Polyangiitis/metabolism , Humans , Male , Myeloblastin/genetics , Neutrophils/enzymology , Proteolysis , Young AdultABSTRACT
The cysteine protease cathepsin C (CatC) activates granule-associated proinflammatory serine proteases in hematopoietic precursor cells. Its early inhibition in the bone marrow is regarded as a new therapeutic strategy for treating proteolysis-driven chronic inflammatory diseases, but its complete inhibition is elusive in vivo Controlling the activity of CatC may be achieved by directly inhibiting its activity with a specific inhibitor or/and by preventing its maturation. We have investigated immunochemically and kinetically the occurrence of CatC and its proform in human hematopoietic precursor cells and in differentiated mature immune cells in lung secretions. The maturation of proCatC obeys a multistep mechanism that can be entirely managed by CatS in neutrophilic precursor cells. CatS inhibition by a cell-permeable inhibitor abrogated the release of the heavy and light chains from proCatC and blocked â¼80% of CatC activity. Under these conditions the activity of neutrophil serine proteases, however, was not abolished in precursor cell cultures. In patients with neutrophilic lung inflammation, mature CatC is found in large amounts in sputa. It is secreted by activated neutrophils as confirmed through lipopolysaccharide administration in a nonhuman primate model. CatS inhibitors currently in clinical trials are expected to decrease the activity of neutrophilic CatC without affecting those of elastase-like serine proteases.
Subject(s)
Cathepsin C/metabolism , Lung/enzymology , Neutrophils/enzymology , Pneumonia/enzymology , Animals , Cathepsin C/genetics , Disease Models, Animal , HL-60 Cells , Humans , Lung/pathology , Macaca fascicularis , Mice , Mice, Inbred BALB C , Neutrophils/pathology , Pneumonia/chemically induced , Pneumonia/pathology , Rats, Sprague-Dawley , Sputum/metabolismABSTRACT
Elafin is a serine protease inhibitor produced by epithelial and immune cells with anti-inflammatory properties. Research has shown that dysregulated protease activity may elicit proteolytic cleavage of elafin, thereby impairing the innate immune function of the protein. The aim of this study was to generate variants of elafin (GG- and QQ-elafin) that exhibit increased protease resistance while retaining the biological properties of wild-type (WT) elafin. Similar to WT-elafin, GG- and QQ-elafin variants retained antiprotease activity and susceptibility to transglutaminase-mediated fibronectin cross-linking. However, in contrast to WT-elafin, GG- and QQ-elafin displayed significantly enhanced resistance to degradation when incubated with bronchoalveolar lavage fluid from patients with cystic fibrosis. Intriguingly, both variants, particularly GG-elafin, demonstrated improved lipopolysaccharide (LPS) neutralization properties in vitro. In addition, GG-elafin showed improved anti-inflammatory activity in a mouse model of LPS-induced acute lung inflammation. Inflammatory cell infiltration into the lung was reduced in lungs of mice treated with GG-elafin, predominantly neutrophilic infiltration. A reduction in MCP-1 levels in GG-elafin treated mice compared to the LPS alone treatment group was also demonstrated. GG-elafin showed increased functionality when compared to WT-elafin and may be of future therapeutic relevance in the treatment of lung diseases characterized by a protease burden.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Elafin/pharmacology , Lung/drug effects , Pneumonia/drug therapy , Protease Inhibitors/pharmacology , Amino Acid Sequence , Animals , Anti-Inflammatory Agents/chemistry , Bronchoalveolar Lavage Fluid/chemistry , Cystic Fibrosis/metabolism , Cystic Fibrosis/pathology , Elafin/chemistry , Elafin/genetics , Fibronectins/antagonists & inhibitors , Fibronectins/metabolism , Gene Expression , Humans , Kinetics , Lipopolysaccharides , Lung/metabolism , Lung/pathology , Male , Mice , Molecular Sequence Data , Pneumonia/chemically induced , Pneumonia/metabolism , Pneumonia/pathology , Protease Inhibitors/chemistry , Protein Engineering , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Isoforms/pharmacology , Proteolysis/drug effects , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/pharmacology , Transglutaminases/antagonists & inhibitors , Transglutaminases/metabolismABSTRACT
The function of neutrophil protease 3 (PR3) is poorly understood despite of its role in autoimmune vasculitides and its possible involvement in cell apoptosis. This makes it different from its structural homologue neutrophil elastase (HNE). Endogenous inhibitors of human neutrophil serine proteases preferentially inhibit HNE and to a lesser extent, PR3. We constructed a single-residue mutant PR3 (I217R) to investigate the S4 subsite preferences of PR3 and HNE and used the best peptide substrate sequences to develop selective phosphonate inhibitors with the structure Ac-peptidyl(P)(O-C6H4-4-Cl)2. The combination of a prolyl residue at P4 and an aspartyl residue at P2 was totally selective for PR3. We then synthesized N-terminally biotinylated peptidyl phosphonates to identify the PR3 in complex biological samples. These inhibitors resisted proteolytic degradation and rapidly inactivated PR3 in biological fluids such as inflammatory lung secretions and the urine of patients with bladder cancer. One of these inhibitors revealed intracellular PR3 in permeabilized neutrophils and on the surface of activated cells. They hardly inhibited PR3 bound to the surface of stimulated neutrophils despite their low molecular mass, suggesting that the conformation and reactivity of membrane-bound PR3 is altered. This finding is relevant for autoantibody binding and the subsequent activation of neutrophils in granulomatosis with polyangiitis (formerly Wegener disease). These are the first inhibitors that can be used as probes to monitor, detect, and control PR3 activity in a variety of inflammatory diseases.
Subject(s)
Esters/chemistry , Gene Expression Regulation, Enzymologic/drug effects , Myeloblastin/antagonists & inhibitors , Myeloblastin/chemistry , Oligopeptides/chemistry , Organophosphonates/chemistry , Animals , Apoptosis , Biotinylation , Cell Line , Cell Membrane/metabolism , Humans , Hydrolysis , Inflammation , Insecta , Mass Spectrometry , Models, Chemical , Mutation , Neutrophil Activation , Neutrophils/drug effects , Peptides/chemistry , Proline/chemistry , Protease Inhibitors/chemistry , SolventsABSTRACT
It is now clear that NSPs (neutrophil serine proteases), including elastase, Pr3 (proteinase 3) and CatG (cathepsin G) are major pathogenic determinants in chronic inflammatory disorders of the lungs. Two unglycosylated natural protease inhibitors, SLPI (secretory leucocyte protease inhibitor) and elafin, and its precursor trappin-2 that are found in the lungs, have therapeutic potential for reducing the protease-induced inflammatory response. This review examines the multifaceted roles of SLPI and elafin/trappin-2 in the context of their possible use as inhaled drugs for treating chronic lung diseases such as CF (cystic fibrosis) and COPD (chronic obstructive pulmonary disease).
Subject(s)
Elafin/metabolism , Inflammation/enzymology , Lung Diseases/enzymology , Secretory Leukocyte Peptidase Inhibitor/metabolism , Serine Proteases/metabolism , Serine Proteinase Inhibitors/metabolism , Aerosols , Anti-Bacterial Agents/metabolism , Anti-Bacterial Agents/therapeutic use , Antifungal Agents/metabolism , Antifungal Agents/therapeutic use , Elafin/therapeutic use , Humans , Inflammation/drug therapy , Lung Diseases/drug therapy , Proteinase Inhibitory Proteins, Secretory/metabolism , Proteinase Inhibitory Proteins, Secretory/therapeutic use , Secretory Leukocyte Peptidase Inhibitor/therapeutic use , Serine Proteinase Inhibitors/therapeutic use , Transglutaminases/metabolismABSTRACT
Trappin-2 (also known as pre-elafin) is an endogenous inhibitor of neutrophil serine proteases and is involved in the control of excess proteolysis, especially in inflammatory events, along with the structurally related secretory leucocyte proteinase inhibitor. Secretory leucocyte proteinase inhibitor has been shown to have antibacterial and antifungal properties, whereas recent data indicate that trappin-2 has antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In the present study, we tested the antibacterial properties of trappin-2 towards other respiratory pathogens. We found that trappin-2, at concentrations of 5-20 microm, has significant activity against Klebsiella pneumoniae, Haemophilus influenzae, Streptococcus pneumoniae, Branhamella catarrhalis and the pathogenic fungi Aspergillus fumigatus and Candida albicans, in addition to P. aeruginosa and S. aureus. A similar antimicrobial activity was observed with trappin-2 A62D/M63L, a trappin-2 variant that has lost its antiprotease properties, indicating that trappin-2 exerts its antibacterial effects through mechanisms independent from its intrinsic antiprotease capacity. Furthermore, the antibacterial and antifungal activities of trappin-2 were sensitive to NaCl and heparin, demonstrating that its mechanism of action is most probably dependent on its cationic nature. This enables trappin-2 to interact with the membranes of target organisms and disrupt them, as shown by our scanning electron microscopy analyses. Thus, trappin-2 not only provides an antiprotease shield, but also may play an important role in the innate defense of the human lungs and mucosae against pathogenic microorganisms.
Subject(s)
Anti-Bacterial Agents/metabolism , Antifungal Agents/metabolism , Elafin/metabolism , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Disulfides/chemistry , Dose-Response Relationship, Drug , Elafin/pharmacology , Fibronectins/metabolism , Gram-Negative Bacteria/drug effects , Gram-Negative Bacteria/ultrastructure , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/ultrastructure , Heparin/pharmacology , Kinetics , Models, Chemical , Mutation , Protein Structure, Secondary , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Serine Proteinase Inhibitors/genetics , Serine Proteinase Inhibitors/isolation & purification , Sodium Chloride/pharmacologyABSTRACT
Elafin and SLPI are low-molecular weight proteins that were first identified as protease inhibitors in mucous fluids including lung secretions, where they help control excessive proteolysis due to neutrophil serine proteases (elastase, proteinase 3 and cathepsin G). Elafin and SLPI are structurally related in that both have a fold with a four-disulfide core or whey acidic protein (WAP) domain responsible for inhibiting proteases. Elafin is derived from a precursor, trappin-2 or pre-elafin, by proteolysis. Trappin-2, which is itself a protease inhibitor, has a unique N-terminal domain that enables it to become cross-linked to extracellular matrix proteins by transglutaminase(s). SLPI and elafin/trappin-2 are attractive candidates as therapeutic molecules for inhibiting neutrophil serine proteases in inflammatory lung diseases. Hence, they have become the WAP proteins most studied over the last decade. This review focuses on recent findings revealing that SLPI and elafin/trappin-2 have many biological functions as diverse as anti-bacterial, anti-fungal, anti-viral, anti-inflammatory and immuno-modulatory functions, in addition to their well-recognized role as protease inhibitors.
Subject(s)
Elafin/physiology , Secretory Leukocyte Peptidase Inhibitor/physiology , Amino Acid Sequence , Anti-HIV Agents/pharmacology , Anti-Infective Agents/pharmacology , Anti-Inflammatory Agents/pharmacology , Elafin/chemistry , Elafin/pharmacology , Humans , Lung Diseases/drug therapy , Molecular Sequence Data , Secretory Leukocyte Peptidase Inhibitor/chemistry , Secretory Leukocyte Peptidase Inhibitor/pharmacology , Transglutaminases/metabolismABSTRACT
The neutrophilic serine protease proteinase 3 (PR3) is involved in inflammation and immune response and thus appears as a therapeutic target for a variety of infectious and inflammatory diseases. Here we combined kinetic and molecular docking studies to increase the potency of peptidyl-diphenyl phosphonate PR3 inhibitors. Occupancy of the S1 subsite of PR3 by a nVal residue and of the S4-S5 subsites by a biotinylated Val residue as obtained in biotin-VYDnVP(O-C6H4-4-Cl)2 enhanced the second-order inhibition constant kobs/[I] toward PR3 by more than 10 times ( kobs/[I] = 73000 ± 5000 M-1 s-1) as compared to the best phosphonate PR3 inhibitor previously reported. This inhibitor shows no significant inhibitory activity toward human neutrophil elastase and resists proteolytic degradation in sputa from cystic fibrosis patients. It also inhibits macaque PR3 but not the PR3 from rodents and can thus be used for in vivo assays in a primate model of inflammation.
Subject(s)
Myeloblastin/chemistry , Organophosphonates/antagonists & inhibitors , Animals , Binding Sites , Humans , Inflammation , Kinetics , Macaca , Models, Molecular , Molecular Docking Simulation , Protein Binding , Rodentia , Substrate SpecificityABSTRACT
Cathepsin C (CatC) is a tetrameric cysteine dipeptidyl aminopeptidase that plays a key role in activation of pro-inflammatory serine protease zymogens by removal of a N-terminal pro-dipeptide sequence. Loss of function mutations in the CatC gene is associated with lack of immune cell serine protease activities and cause Papillon-Lefèvre syndrome (PLS). Also, only very low levels of elastase-like protease zymogens are detected by proteome analysis of neutrophils from PLS patients. Thus, CatC inhibitors represent new alternatives for the treatment of neutrophil protease-driven inflammatory or autoimmune diseases. We aimed to experimentally inactivate and lower neutrophil elastase-like proteases by pharmacological blocking of CatC-dependent maturation in cell-based assays and in vivo. Isolated, immature bone marrow cells from healthy donors pulse-chased in the presence of a new cell permeable cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites. These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases, which opens new perspectives for therapeutic applications in humans.
Subject(s)
Cathepsin C/antagonists & inhibitors , Cysteine Proteinase Inhibitors/pharmacology , Neutrophils/enzymology , Serine Proteases/metabolism , Animals , Bronchoalveolar Lavage Fluid , Case-Control Studies , Female , Humans , Leukocyte Elastase/blood , Macaca fascicularis , Papillon-Lefevre Disease/enzymologyABSTRACT
Papillon-Lefèvre syndrome (PLS) (OMIM: 245000) is a rare disease characterized by severe periodontitis and palmoplantar keratoderma. It is caused by mutations in both alleles of the cathepsin C (CatC) gene CTSC that completely abrogate the proteolytic activity of this cysteine proteinase. Most often, a genetic analysis to enable early and rapid diagnosis of PLS is unaffordable or unavailable. In this study, we tested the hypothesis that active CatC is constitutively excreted and can be easily traced in the urine of normal subjects. If this is true, determining its absence in the urine of patients would be an early, simple, reliable, low-cost and easy diagnostic technique. All 75 urine samples from healthy control subjects (aged 3 months to 80 years) contained proteolytically active CatC and its proform, as revealed by kinetic analysis and immunochemical detection. Of the urine samples of 31 patients with a PLS phenotype, 29 contained neither proteolytically active CatC nor the CatC antigen, so that the PLS diagnosis was confirmed. CatC was detected in the urine of the other two patients, and genetic analysis revealed no loss-of-function mutation in CTSC, indicating that they suffer from a PLS-like condition but not from PLS. Screening for the absence of urinary CatC activity soon after birth and early treatment before the onset of PLS manifestations will help to prevent aggressive periodontitis and loss of many teeth, and should considerably improve the quality of life of PLS patients.
Subject(s)
Cathepsin C/urine , Papillon-Lefevre Disease/diagnosis , Papillon-Lefevre Disease/urine , Adolescent , Adult , Aged , Aged, 80 and over , Cathepsin C/genetics , Cathepsin C/metabolism , Child , Child, Preschool , Female , Healthy Volunteers , Humans , Infant , Male , Middle Aged , Phenotype , Young AdultABSTRACT
Neutrophils are among the first cells implicated in acute inflammation. Leaving the blood circulation, they quickly migrate through the interstitial space of tissues and liberate oxidants and other antimicrobial proteins together with serine proteinases. Neutrophil elastase, cathepsin G, proteinase 3 (PR3), and neutrophil serine protease 4 are four hematopoietic serine proteases activated by dipeptidyl peptidase I during neutrophil maturation and are mainly stored in cytoplasmic azurophilic granules. They regulate inflammatory and immune responses after their release from activated neutrophils at inflammatory sites. Membrane-bound PR3 (mbPR3) at the neutrophil surface is the prime antigenic target of antineutrophil cytoplasmic autoantibodies (ANCA) in granulomatosis with polyangiitis (GPA), a vasculitis of small blood vessels and granulomatous inflammation of the upper and/or lower respiratory tracts. The interaction of ANCA with mbPR3 results in excessive activation of neutrophils to produce reactive oxygen species and liberation of granular proteinases to the pericellular environment. In this review, we focus on PR3 and dipeptidyl peptidase I as attractive pharmacological targets whose inhibition is expected to attenuate autoimmune activation of neutrophils in GPA.
Subject(s)
Cathepsin C/antagonists & inhibitors , Enzyme Inhibitors/pharmacology , Granulomatosis with Polyangiitis/enzymology , Myeloblastin/antagonists & inhibitors , Animals , Antibodies, Antineutrophil Cytoplasmic/immunology , Autoimmunity , Cathepsin C/metabolism , Cell Membrane/immunology , Cell Membrane/metabolism , Enzyme Inhibitors/therapeutic use , Granulomatosis with Polyangiitis/drug therapy , Granulomatosis with Polyangiitis/immunology , Humans , Myeloblastin/metabolism , Neutrophils/drug effects , Neutrophils/immunology , Neutrophils/metabolismABSTRACT
Neutrophil serine proteases (NSPs), including elastase, proteinase 3 and cathepsin G, play critical roles in the pathogenesis of chronic inflammatory lung diseases. The release of excess NSPs leads to the destruction of lung tissue and an overexuberant, sustained inflammatory response. Antiproteases could be valuable tools for controlling these NSP-mediated inflammatory events. We have examined the capacity of trappin-2 A62L, a potent engineered inhibitor of all three NSPs, to protect human lung A549 epithelial cells from the deleterious effects of NSPs. Trappin-2 A62L, significantly inhibited the detachment of A549 cells and the degradation of the tight-junction proteins, E-cadherin, ß-catenin and ZO-1, induced by each individual NSP and by activated neutrophils. Trappin-2 A62L also decreased the release of the pro-inflammatory cytokines IL-6 and IL-8 from A549 cells that had been stimulated with elastase or LPS. Trappin-2 A62D/M63L, a trappin-2 variant that has no antiprotease activity, has similar properties, suggesting that the anti-inflammatory action of trappin-2 is independent of its antiprotease activity. Interestingly, we present evidence that trappin-2 A62L, as well as wild-type trappin-2, enter A549 cells and move rapidly to the cytoplasm and nucleus, where they are likely to exert their anti-inflammatory effects. We have also demonstrated that trappin-2 A62L inhibits the early apoptosis of A549 cells mediated by NSPs. Thus, our data indicate that trappin-2 A62L is a powerful anti-protease and anti-inflammatory agent that could be used to develop a treatment for patients with inflammatory lung diseases.
Subject(s)
Elafin/pharmacology , Lung/drug effects , Neutrophils/drug effects , Recombinant Fusion Proteins/pharmacology , Secretory Leukocyte Peptidase Inhibitor/pharmacology , Serine Proteinase Inhibitors/pharmacology , Apoptosis/drug effects , Cell Line , Elafin/chemistry , Epithelial Cells/drug effects , Fluorescent Antibody Technique , Humans , Lung/pathology , Neutrophils/enzymology , Proteolysis , Recombinant Fusion Proteins/chemistry , Recombinant Proteins/chemistry , Recombinant Proteins/pharmacology , Secretory Leukocyte Peptidase Inhibitor/chemistryABSTRACT
Human lungs contain secretory leukocyte protease inhibitor (SLPI), elafin and its biologically active precursor trappin-2 (pre-elafin). These important low-molecular weight inhibitors are involved in controlling the potentially deleterious proteolytic activities of neutrophil serine proteases including elastase, proteinase 3 and cathepsin G. We have shown previously that trappin-2, and to a lesser extent, elafin can be linked covalently to various extracellular matrix proteins by tissue transglutaminases and remain potent protease inhibitors. SLPI is composed of two distinct domains, each of which is about 40% identical to elafin, but it lacks consensus transglutaminase sequence(s), unlike trappin-2 and elafin. We investigated the actions of type 2 tissue transglutaminase and plasma transglutaminase activated factor XIII on SLPI. It was readily covalently bound to fibronectin or elastin by both transglutaminases but did not compete with trappin-2 cross-linking. Cross-linked SLPI still inhibited its target proteases, elastase and cathepsin G. We have also identified the transglutamination sites within SLPI, elafin and trappin-2 by mass spectrometry analysis of tryptic digests of inhibitors cross-linked to mono-dansyl cadaverin or to a fibronectin-derived glutamine-rich peptide. Most of the reactive lysine and glutamine residues in SLPI are located in its first N-terminal elafin-like domain, while in trappin-2, they are located in both the N-terminal cementoin domain and the elafin moiety. We have also demonstrated that the transglutamination substrate status of the cementoin domain of trappin-2 can be transferred from one protein to another, suggesting that it may provide transglutaminase-dependent attachment properties for engineered proteins. We have thus added to the corpus of knowledge on the biology of these potential therapeutic inhibitors of airway proteases.
Subject(s)
Elafin/metabolism , GTP-Binding Proteins/metabolism , Secretory Leukocyte Peptidase Inhibitor/metabolism , Sequence Homology, Amino Acid , Transglutaminases/metabolism , Amino Acid Sequence , Animals , Cadaverine/analogs & derivatives , Cadaverine/metabolism , Cattle , Elafin/chemistry , Factor XIIIa/metabolism , Fibronectins/chemistry , Fibronectins/metabolism , Glutamine , Guinea Pigs , Humans , Lysine , Mass Spectrometry , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Glutamine gamma Glutamyltransferase 2 , Secretory Leukocyte Peptidase Inhibitor/chemistryABSTRACT
The secretory leukocyte protease inhibitor (SLPI), elafin, and its biologically active precursor trappin-2 are endogeneous low-molecular weight inhibitors of the chelonianin family that control the enzymatic activity of neutrophil serine proteases (NSPs) like elastase, proteinase 3, and cathepsin G. These inhibitors may be of therapeutic value, since unregulated NSP activities are linked to inflammatory lung diseases. However SLPI inhibits elastase and cathepsin G but not proteinase 3, while elafin targets elastase and proteinase 3 but not cathepsin G. We have used two strategies to design polyvalent inhibitors of NSPs that target all three NSPs and may be used in the aerosol-based treatment of inflammatory lung diseases. First, we fused the elafin domain with the second inhibitory domain of SLPI to produce recombinant chimeras that had the inhibitory properties of both parent molecules. Second, we generated the trappin-2 variant, trappin-2 A62L, in which the P1 residue Ala is replaced by Leu, as in the corresponding position in SLPI domain 2. The chimera inhibitors and trappin-2 A62L are tight-binding inhibitors of all three NSPs with subnanomolar K(i)s, similar to those of the parent molecules for their respective target proteases. We have also shown that these molecules inhibit the neutrophil membrane-bound forms of all three NSPs. The trappin-2 A62L and elafin-SLPI chimeras, like wild-type elafin and trappin-2, can be covalently cross-linked to fibronectin or elastin by a tissue transglutaminase, while retaining their polypotent inhibition of NSPs. Therefore, the inhibitors described herein have the appropriate properties to be further evaluated as therapeutic anti-inflammatory agents.
Subject(s)
Elafin/chemistry , Neutrophils/metabolism , Recombinant Fusion Proteins/chemistry , Secretory Leukocyte Peptidase Inhibitor/chemistry , Serine Endopeptidases/metabolism , Amino Acid Sequence , Cathepsin G , Cathepsins/metabolism , Dose-Response Relationship, Drug , Drug Discovery , Elafin/genetics , Elafin/metabolism , Humans , Lung Diseases , Membrane Proteins/metabolism , Models, Molecular , Molecular Sequence Data , Myeloblastin/metabolism , Pancreatic Elastase/metabolism , Protease Inhibitors/chemistry , Protease Inhibitors/metabolism , Protein Engineering , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Secretory Leukocyte Peptidase Inhibitor/genetics , Secretory Leukocyte Peptidase Inhibitor/metabolism , Sequence Alignment , Static Electricity , Transglutaminases/metabolismABSTRACT
Elafin and its precursor trappin-2 (also called pre-elafin) are potent protein inhibitors of neutrophil serine proteases such as leukocyte elastase and proteinase 3. Trappin-2 has unique conserved sequence motifs rich in Gln and Lys residues. These motifs are substrates for transglutaminases that may enable trappin-2 to be cross-linked to extracellular matrix proteins, thus anchoring the inhibitor at its site of action. We have used Western blotting and ELISA-based assays to demonstrate that both elafin and trappin-2 can be conjugated to various extracellular matrix proteins in vitro by a type 2 transglutaminase. Cross-linked elafin and trappin-2 still inhibited their target proteases. Surface plasmon resonance studies allowed the determination of the kinetic constants governing the interaction of fibronectin-bound elafin and trappin-2 with neutrophil elastase and proteinase 3. Both inhibitors were potent inhibitors when cross-linked to fibronectin by transglutamination, with equilibrium dissociation constants K(i) for their interaction with target proteases of 0.3 nM (elastase-elafin), 20 nM (proteinase 3-elafin), 0.3 nM (elastase-trappin-2), and 12 nM (proteinase 3-trappin-2). The conjugated inhibitors reacted more slowly with their target enzymes than did the soluble inhibitors, perhaps due to their immobilization, with association rate constants of 2-7 x 10(5) M(-)(1) s(-)(1) for elastase and 1-4 x 10(4) M(-)(1) s(-)(1) for proteinase 3. We believe this is the first demonstration that transglutaminase-mediated cross-linking of serine protease inhibitors to proteins preserves their inhibitory capacities.
Subject(s)
Extracellular Matrix Proteins/metabolism , GTP-Binding Proteins/metabolism , Leukocyte Elastase/metabolism , Protein Precursors/metabolism , Proteins/metabolism , Transglutaminases/metabolism , Elafin , Fibronectins/metabolism , Humans , Kinetics , Leukocyte Elastase/antagonists & inhibitors , Myeloblastin , Protein Glutamine gamma Glutamyltransferase 2 , Protein Interaction Mapping , Proteinase Inhibitory Proteins, Secretory , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Surface Plasmon ResonanceABSTRACT
A number of serine, cysteine, metallo- and acid proteases were evaluated for their ability to proteolytically cleave the serine protease inhibitor trappin-2, also known as pre-elafin, and to release elafin from its precursor. None of the metalloproteases or acid proteases examined cleaved trappin-2, while serine and cysteine proteases preferentially cleaved trappin-2 within its non-inhibitory N-terminal moiety. Cathepsin L, cathepsin K, plasmin, trypsin and tryptase were able to release elafin by cleaving the Lys 38 -Ala 39 peptide bond in trappin-2. However, purified tryptase appeared to be efficient at releasing elafin. Incubation of trappin-2 with purified mast cells first challenged with anti-immunoglobulin E or calcium ionophore A23187 resulted in the rapid generation of elafin. This proteolytic release of elafin from trappin-2 was inhibited in the presence of a tryptase inhibitor, suggesting that this mast cell enzyme was involved in the process. Finally, ex vivo incubation of trappin-2 with sputum from cystic fibrosis patients indicated the production of a proteolytic immunoreactive fragment with the same mass as that of native elafin. This cleavage did not occur when preincubating the sputum with polyclonal antibodies directed against tryptase. Taken together, these findings indicate that tryptase could likely be involved in the maturation of trappin-2 into elafin under physiological conditions.
Subject(s)
Peptide Hydrolases/metabolism , Proteins/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/metabolism , Serine Proteinase Inhibitors/metabolism , Amino Acid Sequence , Animals , Cattle , Cells, Cultured , Humans , Hydrolysis , Molecular Sequence Data , Pichia/genetics , Proteinase Inhibitory Proteins, Secretory , Serine Proteinase Inhibitors/genetics , TryptasesABSTRACT
The process of pressure-induced modification of horse liver alcohol dehydrogenase (HLADH) was followed by measuring in situ catalytic activity (up to 250 MPa), intrinsic fluorescence (0.1-600 MPa) and modifications of FTIR spectra (up to 1000 MPa). The tryptophan fluorescence measurements and the kinetic data indicated that the pressure-induced denaturation of HLADH was a process involving several transitions and that the observed transient states have characteristic properties of molten globules. Low pressure (< 100 MPa) induced no important modification in the catalytic efficiency of the enzyme and slight conformational changes, characterized by a small decrease in the centre of spectral mass of the enzyme's intrinsic fluorescence: a native-like state was assumed. Higher pressures (100-400 MPa) induced a strong decrease of HLADH catalytic efficiency and further conformational changes. At 400 MPa, a dimeric molten globule-like state was proposed. Further increase of pressure (400-600 MPa) seemed to induce the dissociation of the dimer leading to a transition from the first dimeric molten globule state to a second monomeric molten globule. The existence of two independent structural domains in HLADH was assumed to explain this transition: these domains were supposed to have different stabilities against high pressure-induced denaturation. FTIR spectroscopy was used to follow the changes in HLADH secondary structures. This technique confirmed that the intermediate states have a low degree of unfolding and that no completely denatured form seemed to be reached, even up to 1000 MPa.