Synthesis and structure-activity relationships of elafin, an elastase-specific inhibitor

doi:10.1016/0006-291X(92)91721-2

Biochemical and Biophysical Research Communications

Volume 185, Issue 3, 30 June 1992, Pages 967-973

https://doi.org/10.1016/0006-291X(92)91721-2 Get rights and content

Abstract

Elafin, an elastase-specific inhibitor isolated from human skin, and its related peptides were synthesized by the solution procedure, and their inhibitory activities were measured against various enzymes. During the oxidative folding reactions of the reduced peptides, the ratio of the active product to the inactive product was varied by changing the concentration of guanidine HCl and the amount of redox reagents. The disulfide structures of fully active synthetic elafin and the inactive product were determined by amino acid analysis, gas-phase sequencing and mass spectrometry of their proteolytic fragments. The relationship between structure and inhibitory activities and/or the folding reaction was examined and the amino terminal part of the elafin molecule was found to have a great influence on the folding reactions, but not on the inhibitory activities.

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Cited by (36)

Characterization of potential elastase inhibitor-peptides regulated by a molecular switch for wound dressings applications
2012, Enzyme and Microbial Technology
Citation Excerpt :
The inhibitor-peptides studied herein were selected from the endogenous elastase inhibitors SLPI and Elafin and from two other endogenous proteins, eosinophil cationic protein (ECP) and surfactant protein D (SP-D). Elafin and SLPI have high cysteine content, 8 and 16 residues respectively, with the correct pairing of disulfide bridges being crucial for elastase inhibition [17–19]. Elafin is a potent inhibitor of both human neutrophil elastase (HNE) and porcine pancreatic elastase (PPE) [18–20], whereas SLPI is a strong HNE inhibitor but a weak PPE inhibitor [18–20].
Elastase plays an important role in wound healing process, degrading damaged tissue and allowing complete tissue recovery. The levels of human neutrophil elastase (HNE) are usually controlled by endogenous inhibitors. However, in the presence of high levels of elastase, like the ones present in chronic wounds, the inhibitors cannot overcome this overproduction and the enzyme starts to degrade the surrounding healthy tissue. In this work we report the development of a molecular switch to control the elastase activity in the exudate of non-healing chronic wounds. A peptide library was generated and screened in a microarray format for protein kinase-mediated phosphorylation. Two peptides were identified as casein kinase Iδ (CKI) substrates: KRCCPDTCGIKCL and its analogous peptide KRMMPDTMGIKML, with cysteine residues replaced by methionine residues. These peptides were studied in solution, both in the phosphorylated and non-phosphorylated forms as potential inhibitors for elastase. The obtained results show that the reversible process of phosphorylation/dephosphorylation results in differential inhibitory activity of the peptides. Thus the reversible process of phosphorylation/dephosphorylation can be used as a kind of molecular switch to control elastase activity. Degradation studies reveal that both the inhibitor-peptides and CKI are degraded by elastase. These results envisage the safe utilisation of these inhibitor-peptides together with CKI in the formulation of wound dressings.
Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis
2008, Journal of Biological Chemistry
Citation Excerpt :
NMR analysis of elafin indicates that the N-terminal extremity of elafin (Ala-1–Pro-13) is a flexible region linked to a rigid and flat structure, also referred as “four-disulfide core” possessing the inhibitory activity (38). A previous structure-activity study of synthetic elafin using variants of the inhibitor demonstrated that the N-terminal part of elafin is not essential for expressing full inhibitory activity (44). Therefore, it is likely that cleavages occurring in the N-terminal part of elafin will not perturb the structure of the four-disulfide core, thus preserving the inhibitory properties of elafin.
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.
Elafin (elastase-specific inhibitor) has anti-microbial activity against Gram-positive and Gram-negative respiratory pathogens
1999, FEBS Letters
Elafin (elastase-specific inhibitor) is a low molecular weight inhibitor of neutrophil elastase which is secreted in the lung. Using synthetic peptides corresponding to full-length elafin (H₂N-¹AVT.....⁹⁵Q-OH), the NH₂-terminal domain (H₂N-¹AVT.....⁵⁰K-OH) and the COOH-terminal domain (H₂N-⁵¹PGS.....⁹⁵Q-OH), we demonstrate that elafin’s anti-elastase activity resides exclusively in the COOH-terminus. Several characteristics of elafin suggest potential anti-microbial activity. The anti-microbial activity of elafin, and of its two structural domains, was tested against the respiratory pathogensPseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 μM elafin and 48% killing of S. aureus by 25 μM elafin. For both organisms, full-length elafin was required to optimise bacterial killing. These findings represent the first demonstration of co-existent anti-proteolytic and anti-microbial functions for elafin.
Synthesis and structure-function study about tenecin 1, an antibacterial protein from larvae of Tenebrio molitor
1998, FEBS Letters
Tenecin 1, an inducible antibacterial protein secreted in the larvae of Tenebrio molitor, has a long N-terminal loop and common structural feature of insect defensin family corresponding to cysteine stabilized α/β motif. To study the function of the N-terminal loop and disulfide bridges, N-terminal loop deleted tenecin 1, reduced tenecin 1 and tenecin 1 were chemically synthesized and their activities were measured. N-terminal loop deleted tenecin and reduced tenecin 1 did not show antibacterial activity. Circular dichroism (CD) spectroscopy data revealed that the α-helical content of tenecin 1 and the other proteins increased in the presence of 50% (v/v) trifluoroethanol (TFE) and the α-helical content of tenecin 1 was much higher than that of the other proteins in buffer with or without 50% (v/v) TFE. These results suggest that disulfide bridges are necessary for the activity structure and the N-terminal loop plays an important role in the increase of α-helix in the membrane mimetic environment and the activity.
Epidermal structural proteins in skin disorders
1997, Journal of Dermatological Science
Solution structure of R-elafin, a specific inhibitor of elastase
1997, Journal of Molecular Biology
The solution structure of r-elafin, a specific elastase inhibitor, has been determined using NMR spectroscopy. Characterized by a flat core and a flexible N-terminal extremity, the three-dimensional structure is formed by a central twisted β-hairpin accompanied by two external segments linked by the proteinase binding loop. A cluster of three disulfide bridges connects the external segments to the central β-sheet and a single fourth disulfide bridge links the binding loop to the central β-turn. The same spatial distribution of disulfide bridges can be observed in both domains of the secretory leukocyte protease inhibitor (SLPI), another elastase inhibitor. The structural homology between r-elafin and the C-terminal domain of SLPI confirms the former as a second member of the chelonianin family of proteinase inhibitors. Based on the homology between the two proteins and recent results obtained for elastase binding mutants of the bovine pancreatic trypsin inhibitor (BPTI), we define the segment 22 to 27 as the binding loop of elafin, with the scissile peptide bond between Ala24 and Met25. In our solution structures, this loop is extended and solvent-exposed, and exhibits a large degree of flexibility. This mobility, already observed for the binding loop in other protease inhibitors in solution, might be an important feature for the interaction with the corresponding protease.

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Synthesis and structure-activity relationships of elafin, an elastase-specific inhibitor

Abstract

J. Biol. Chem

Biochem. Biophys. Res. Commun

Tetrahedron Lett

Thromb. Res