RESUMO
Elafin is a potent reversible inhibitor of the pro-inflammatory proteases leukocyte elastase and protease 3. It is currently in clinical development for the use in postoperative inflammatory diseases. We investigated the pharmacokinetics of (99m)Tc-labeled elafin ((99m)Tc-Elafin) in blood and individual organs in rat after bolus intravenous injection using the single photon emission tomography (SPECT). (99m)Tc-Elafin predominantly accumulated in the kidney reaching a maximum of 8.5% ± 0.1% of the injected dose per gram (ID/g) at 5 min post injection (p.i) and decreased only slowly during 24 h. In contrast, the initially high radio activity recorded in the other organs rapidly decreased parallel to the radioactivity detected in blood. The blood kinetics fits to a two compartment kinetics model. The radio activity in the dissected kidney was 4.98 ± 1.24%ID/g 24 h p.i, while in other organs, including the brain, no accumulation of (99m)Tc-Elafin was detected. At this time point 30% of the detected radioactivity in the kidney was identified to be not metabolized (99m)Tc-Elafin. In conclusion, the blood and organ-specific kinetic data provide a basis for planning of adequate dosing regimens and the high accumulation of intact elafin in the kidney favors clinical developments targeting inflammatory kidney diseases, such as chronic allograft nephropathy after kidney transplantation.
Assuntos
Elafina/farmacocinética , Inibidores Enzimáticos/farmacocinética , Tecnécio/química , Animais , Elafina/química , Elafina/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Elastase Pancreática/antagonistas & inibidores , Elastase Pancreática/metabolismo , Ratos , Distribuição TecidualRESUMO
Trappin-2 is a serine protease inhibitor with a very narrow inhibitory spectrum and has significant anti-microbial activities. It is a 10 kDa cationic protein composed of two distinct domains. The N-terminal domain (38 residues) named cementoin is known to be intrinsically disordered when it is not linked to the elafin. The C-terminal domain (57 residues), corresponding to elafin, is a cysteine-rich domain stabilized by four disulfide bridges and is characterized by a flat core and a flexible N-terminal part. To our knowledge, there is no structural data available on trappin-2. We report here the complete (1)H, (15)N and (13)C resonance assignment of the recombinant trappin-2 and the (1)H assignments of cementoin and elafin, under the same experimental conditions. This is the first step towards the 3D structure determination of the trappin-2.
Assuntos
Elafina/química , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Sequência de Aminoácidos , Humanos , Domínios ProteicosRESUMO
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.
Assuntos
Anti-Inflamatórios/farmacologia , Elafina/farmacologia , Pulmão/efeitos dos fármacos , Pneumonia/tratamento farmacológico , Inibidores de Proteases/farmacologia , Sequência de Aminoácidos , Animais , Anti-Inflamatórios/química , Líquido da Lavagem Broncoalveolar/química , Fibrose Cística/metabolismo , Fibrose Cística/patologia , Elafina/química , Elafina/genética , Fibronectinas/antagonistas & inibidores , Fibronectinas/metabolismo , Expressão Gênica , Humanos , Cinética , Lipopolissacarídeos , Pulmão/metabolismo , Pulmão/patologia , Masculino , Camundongos , Dados de Sequência Molecular , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Pneumonia/patologia , Inibidores de Proteases/química , Engenharia de Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Transglutaminases/antagonistas & inibidores , Transglutaminases/metabolismoRESUMO
RATIONALE: We hypothesise that elafin levels in acute lung injury (ALI) decrease over time due, in part, to proteolytic degradation as observed in other lung diseases. OBJECTIVES: The aim of this study was to characterise temporal changes in elafin concentration in patients with ALI and to evaluate whether a decrease in elafin levels is due to elevated protease activity. METHODS: Bronchoalveolar lavage fluid (BALF) was obtained from patients with ALI within 48 h of onset of ALI (day 0), at day 3 and at day 7. Elafin levels were quantified by ELISA. Elafin susceptibility to proteolytic cleavage by ALI BALF was assessed by Western blot and by high-performance liquid chromatography-mass spectrometry. MEASUREMENTS AND MAIN RESULTS: Elafin levels were found to be significantly increased at the onset of ALI compared with healthy volunteers and fell significantly by day 7 compared with day 0. In contrast, levels of secretory leukocyte protease inhibitor did not decrease over time. This decrease in elafin was due to cleavage by the 20S proteasome which was significantly increased in ALI BALF. Incubation of ALI BALF with the proteasome inhibitor epoxomicin confirmed that 20S proteasome protease activity was responsible for proteolytic cleavage of elafin, resulting in diminished anti-elastase activity. In addition, free neutrophil elastase activity significantly increased in ALI BALF from day 0 to day 7. CONCLUSIONS: Elafin concentrations fall within the pulmonary compartment over the course of ALI as a result of proteolytic degradation. This loss of elafin may predispose people, in part, to excessive inflammation in ALI.
Assuntos
Lesão Pulmonar Aguda/fisiopatologia , Elafina/química , Complexo de Endopeptidases do Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Lesão Pulmonar Aguda/metabolismo , Western Blotting , Líquido da Lavagem Broncoalveolar , Elafina/análise , Eletroforese em Gel de Poliacrilamida , Humanos , Proteínas Recombinantes/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/análiseRESUMO
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.
Assuntos
Elafina/farmacologia , Pulmão/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes de Fusão/farmacologia , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Inibidores de Serina Proteinase/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular , Elafina/química , Células Epiteliais/efeitos dos fármacos , Imunofluorescência , Humanos , Pulmão/patologia , Neutrófilos/enzimologia , Proteólise , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Inibidor Secretado de Peptidases Leucocitárias/químicaRESUMO
We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).
Assuntos
Inibidores de Proteases , Estrutura Terciária de Proteína , Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Mapeamento Cromossômico , Elafina/química , Elafina/genética , Elafina/metabolismo , Humanos , Dados de Sequência Molecular , Proteínas/química , Proteínas/metabolismo , Alinhamento de SequênciaRESUMO
WAP (whey acidic protein) is an important whey protein present in milk of mammals. This protein has characteristic domains, rich in cysteine residues, called 4-DSC (four-disulfide core domain). Other proteins, mainly present at mucosal surfaces, have been shown to also possess these characteristic WAP-4-DSC domains. The present review will focus on two WAP-4-DSC containing proteins, namely SLPI (secretory leucocyte protease inhibitor) and trappin-2/elafin. Although first described as antiproteases able to inhibit in particular host neutrophil proteases [NE (neutrophil elastase), cathepsin-G and proteinase-3] and as such, able to limit maladaptive tissue damage during inflammation, it has become apparent that these molecules have a variety of other functions (direct antimicrobial activity, bacterial opsonization, induction of adaptive immune responses, promotion of tissue repair, etc.). After providing information about the 'classical' antiproteasic role of these molecules, we will discuss the evidence pertaining to their pleiotropic functions in inflammation and immunity.
Assuntos
Elafina/imunologia , Imunidade nas Mucosas , Proteínas do Leite/química , Proteínas do Leite/imunologia , Inibidor Secretado de Peptidases Leucocitárias/imunologia , Animais , Elafina/química , Elafina/genética , Humanos , Inflamação/imunologia , Proteínas do Leite/genética , Inibidores de Proteases/química , Inibidores de Proteases/imunologia , Estrutura Terciária de Proteína , Inibidor Secretado de Peptidases Leucocitárias/química , Inibidor Secretado de Peptidases Leucocitárias/genética , Distribuição TecidualRESUMO
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.
Assuntos
Elafina/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Homologia de Sequência de Aminoácidos , Transglutaminases/metabolismo , Sequência de Aminoácidos , Animais , Cadaverina/análogos & derivados , Cadaverina/metabolismo , Bovinos , Elafina/química , Fator XIIIa/metabolismo , Fibronectinas/química , Fibronectinas/metabolismo , Glutamina , Cobaias , Humanos , Lisina , Espectrometria de Massas , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteína 2 Glutamina gama-Glutamiltransferase , Inibidor Secretado de Peptidases Leucocitárias/químicaRESUMO
Abstract Porphyromonas gingivalis, the major causative bacterium of periodontitis, contributes significantly to elevated proteolytic activity at periodontal pockets owing to the presence of both bacteria and host, predominantly neutrophil-derived, serine proteases. Normally the activity of the latter enzymes is tightly regulated by endogenous proteins, including elafin, a potent neutrophil elastase and proteinase 3 inhibitor released from epithelial cells at sites of inflammation. Here, we report that all three gingipains (HRgpA, RgpB, and Kgp) have the ability to degrade elafin, with RgpB being far more efficient than other gingipains. RgpB efficiently inactivates the inhibitory activity of elafin at subnanomolar concentrations through proteolysis limited to the Arg22-Cys23 peptide bond within the surface loop harboring the inhibitor active site. Notably, elafin resists inactivation by several Staphylococcus aureus-derived serine and cysteine proteases, confirming the high stability of this protein against proteolytic degradation. Therefore, we conclude that elafin inactivation by RgpB represents a specific pathogenic adaptation of P. gingivalis to disturb the protease-protease inhibitor balance in the infected gingival tissue. This contributes to enhanced degradation of host proteins and generation of a pool of peptides serving as nutrients for this asaccharolytic pathogen.
Assuntos
Adesinas Bacterianas/metabolismo , Cisteína Endopeptidases/metabolismo , Elafina/metabolismo , Porphyromonas gingivalis/enzimologia , Adesinas Bacterianas/química , Sequência de Aminoácidos , Cisteína Endopeptidases/química , Cisteína Proteases/metabolismo , Elafina/química , Cisteína Endopeptidases Gingipaínas , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Serina Proteases/metabolismo , Staphylococcus aureus/enzimologia , Especificidade por SubstratoRESUMO
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.
Assuntos
Elafina/química , Neutrófilos/metabolismo , Proteínas Recombinantes de Fusão/química , Inibidor Secretado de Peptidases Leucocitárias/química , Serina Endopeptidases/metabolismo , Sequência de Aminoácidos , Catepsina G , Catepsinas/metabolismo , Relação Dose-Resposta a Droga , Descoberta de Drogas , Elafina/genética , Elafina/metabolismo , Humanos , Pneumopatias , Proteínas de Membrana/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mieloblastina/metabolismo , Elastase Pancreática/metabolismo , Inibidores de Proteases/química , Inibidores de Proteases/metabolismo , Engenharia de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Inibidor Secretado de Peptidases Leucocitárias/genética , Inibidor Secretado de Peptidases Leucocitárias/metabolismo , Alinhamento de Sequência , Eletricidade Estática , Transglutaminases/metabolismoRESUMO
Elafin is a neutrophil serine protease inhibitor expressed in lung and displaying anti-inflammatory and anti-bacterial properties. Previous studies demonstrated that some innate host defense molecules of the cystic fibrosis (CF) and chronic obstructive pulmonary disease airways are impaired due to increased proteolytic degradation observed during lung inflammation. In light of these findings, we thus focused on the status of elafin in CF lung. We showed in the present study that elafin is cleaved in sputum from individuals with CF. Pseudomonas aeruginosa-positive CF sputum, which was found to contain lower elafin levels and higher neutrophil elastase (NE) activity compared with P. aeruginosa-negative samples, was particularly effective in cleaving recombinant elafin. NE plays a pivotal role in the process as only NE inhibitors are able to inhibit elafin degradation. Further in vitro studies demonstrated that incubation of recombinant elafin with excess of NE leads to the rapid cleavage of the inhibitor. Two cleavage sites were identified at the N-terminal extremity of elafin (Val-5-Lys-6 and Val-9-Ser-10). Interestingly, purified fragments of the inhibitor (Lys-6-Gln-57 and Ser-10-Gln-57) were shown to still be active for inhibiting NE. However, NE in excess was shown to strongly diminish the ability of elafin to bind lipopolysaccharide (LPS) and its capacity to be immobilized by transglutamination. In conclusion, this study provides evidence that elafin is cleaved by its cognate enzyme NE present at excessive concentration in CF sputum and that P. aeruginosa infection promotes this effect. Such cleavage may have repercussions on the innate immune function of elafin.
Assuntos
Fibrose Cística/enzimologia , Elafina/química , Elastase de Leucócito/metabolismo , Escarro/enzimologia , Reagentes de Ligações Cruzadas/farmacologia , Fibrose Cística/microbiologia , Relação Dose-Resposta a Droga , Elafina/metabolismo , Fibronectinas/química , Humanos , Lipopolissacarídeos/química , Modelos Biológicos , Ligação Proteica , Estrutura Terciária de Proteína , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/metabolismo , Proteínas Recombinantes/química , Escarro/microbiologiaRESUMO
Since whey-acidic-protein domain (WAP) containing protease inhibitors such as SLPI (secretory leukocyte protease inhibitor) and elafin (elastase-specific inhibitor) have antimicrobial activities and are thought to play critical roles in mucosal defenses, we are interested in these protease inhibitors. By accessing the Novartis mouse expression database, we found that the four WAP family members, SLPI, WFDC2, WFDC5, and WFDC12, are highly expressed in the oral organs, such as the trachea, tongue, and salivary glands. Since their WAP domains play pivotal roles in the antimicrobial and/or antiprotease activities and their application in therapeutics are expected to have practical value, we collected 98 WAP homologues and tried to predict their physiological functions by analyzing their amino acid sequence structures. From the multiple alignments of amino acid sequences, we predicted that most of the mammalian N-terminal WAP domains derived from SLPIs and the WAP domains derived from WFDC12s have antimicrobial activities, whereas most of the mammalian C-terminal WAP domains derived from SLPIs and the WAP domains derived from elafins have antiprotease activities. From the phylogenetic tree, it was revealed that an ancestral WAP protein initially diverged into the WFDC5-C WAP domain and the ancestral protein for the other WAP domains. Subsequently, the ancestral protein for the other WAP domains diverged into two ancestral proteins, one for elafin and SLPI-C WAP domains and the other, for SLPI-N, WFDC15b, WFDC12, and WFDC5-N WAP domains, respectively. Moreover, the tree indicated that the WFDC5-N and WFDC12 WAP domains share a common ancestral protein.
Assuntos
Proteínas do Leite/química , Proteínas do Leite/genética , Inibidores de Proteases/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , DNA Complementar/genética , Elafina/química , Elafina/genética , Humanos , Camundongos , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Proteínas Secretadas Inibidoras de Proteinases/química , Proteínas Secretadas Inibidoras de Proteinases/genética , Inibidor Secretado de Peptidases Leucocitárias/química , Inibidor Secretado de Peptidases Leucocitárias/genética , Alinhamento de SequênciaRESUMO
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.
Assuntos
Elafina/fisiologia , Inibidor Secretado de Peptidases Leucocitárias/fisiologia , Sequência de Aminoácidos , Fármacos Anti-HIV/farmacologia , Anti-Infecciosos/farmacologia , Anti-Inflamatórios/farmacologia , Elafina/química , Elafina/farmacologia , Humanos , Pneumopatias/tratamento farmacológico , Dados de Sequência Molecular , Inibidor Secretado de Peptidases Leucocitárias/química , Inibidor Secretado de Peptidases Leucocitárias/farmacologia , Transglutaminases/metabolismoRESUMO
Pseudomonas aeruginosa is a life-threatening opportunist human pathogen frequently associated with lung inflammatory diseases, namely, cystic fibrosis. Like other species, this gram-negative bacteria is increasingly drug resistant. During the past decade, intensive research efforts have been focused on the identification of natural innate defense molecules with broad antimicrobial activities, collectively known as antimicrobial peptides. Human pre-elafin, best characterized as a potent inhibitor of neutrophil elastase with anti-inflammatory properties, was also shown to possess antimicrobial activity against both gram-positive and gram-negative bacteria, including P. aeruginosa. Its mode of action was, however, not known. Using full-length pre-elafin, each domain separately, and mutated variants of pre-elafin with attenuated antipeptidase activity toward neutrophil elastase, we report here that both pre-elafin domains contribute, through distinct mechanisms, to its antibacterial activity against Pseudomonas aeruginosa. Most importantly, we demonstrate that the whey acidic protein (WAP) domain specifically inhibits a secreted peptidase with the characteristics of arginyl peptidase (protease IV). This is the first demonstration that a human WAP-motif protein inhibits a secreted peptidase to prevent bacterial growth in vitro. Since several WAP-motif proteins from various species demonstrate antimicrobial function with variable activities toward bacterial species, we suggest that this mechanism may be more common than initially anticipated.
Assuntos
Antibacterianos/farmacologia , Elafina/farmacologia , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/enzimologia , Inibidores de Serina Proteinase/farmacologia , Antibacterianos/química , Antibacterianos/metabolismo , Meios de Cultura , Elafina/química , Elafina/genética , Elafina/metabolismo , Humanos , Testes de Sensibilidade Microbiana , Mutação , Pseudomonas aeruginosa/crescimento & desenvolvimento , Serina Endopeptidases/efeitos dos fármacos , Serina Endopeptidases/metabolismo , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/metabolismoRESUMO
Pre-elafin is a tight-binding inhibitor of neutrophil elastase and myeloblastin; two enzymes thought to contribute to tissue damage in lung emphysema. Previous studies have established that pre-elafin is also an effective anti-inflammatory molecule. However, it is not clear whether both functions are linked to the antipeptidase activity of pre-elafin. As a first step toward elucidating the structure/function relationship of this protein, we describe here the construction and characterization of pre-elafin variants with attenuated antipeptidase potential. In these mutants, the P1' methionine residue of the inhibitory loop is replaced by either a lysine (pre-elafinM25K) or a glycine (pre-elafinM25G) residue. Both mutated variants are stable and display biochemical properties undistinguishable from WT (wild-type) pre-elafin. However, compared with WT pre-elafin, their inhibitory constants are increased by one to four orders of magnitude toward neutrophil elastase, myeloblastin and pancreatic elastase, depending on the variants and enzymes tested. As suggested by molecular modelling, this attenuated inhibitory potential correlates with decreased van der Waals interactions between the variants and the enzymes S1' subsite. In elastase-induced experimental emphysema in mice, only WT pre-elafin protected against tissue destruction, as assessed by the relative airspace enlargement measured using lung histopathological sections. Pre-elafin and both mutants prevented transient neutrophil alveolitis. However, even the modestly affected pre-elafinM25K mutant, as assayed in vitro with small synthetic substrates, was a poor inhibitor of the neutrophil elastase and myeloblastin elastolytic activity measured with insoluble elastin. We therefore conclude that full antipeptidase activity of pre-elafin is essential to protect against lung tissue lesions in this experimental model.
Assuntos
Elafina/genética , Elafina/metabolismo , Enfisema/patologia , Pulmão/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Inibidores de Serina Proteinase/metabolismo , Animais , Elafina/química , Enfisema/metabolismo , Feminino , Regulação da Expressão Gênica , Cinética , Pulmão/citologia , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Neutrófilos/metabolismo , Conformação Proteica , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/genéticaRESUMO
Elafin has a primary structure with two functional domains; a transglutaminase substrate domain at the N-terminus and a protease inhibitor domain at the C-terminus. Elafin expression has so far been reported only for epithelial tissues. Accumulation of elafin was immunohistochemically detected in the actinic elastosis of sun-damaged skin. Exposure of normal skin to UVA induced elafin expression that colocalized with elastic fibers. Incubation of synthetic transglutaminase substrate domain of elafin and elastin molecules in the presence of tissue transglutaminase in vitro resulted in the formation of a higher molecular complex on SDS-PAGE. Elafin expression was not detected in normal cultured skin fibroblasts, but was induced by UVA irradiation at both messenger RNA and protein levels. When radiolabeled insoluble elastin was incubated with recombinant full-length elafin and tissue transglutaminase, insoluble elastin became more resistant to neutrophil elastase digestion. These results indicate that (1) dermal fibroblasts potentially express elafin on UV irradiation, (2) UV-mediated elafin interacts with elastin, and (3) the elafin-elastin complex protects elastic fibers from elastolytic degradation, leading to the accumulation of elastic fibers in the actinic elastosis of sun-damaged skin. The transglutaminase substrate moiety of elafin plays an important role in anchoring elafin at its proper sites of action during UV-induced aging processes.