Secretory leukocyte protease inhibitor (SLPI) is, like its homologue trappin-2 (pre-elafin), a transglutaminase substrate

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.     

Elafin, an elastase-specific inhibitor, is cleaved by its cognate enzyme neutrophil elastase in sputum from individuals with cystic fibrosis

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.     

The presence of elafin, SLPI, IL1-RA and STNFalpha RI in head and neck squamous cell carcinomas and their relation to the degree of tumour differentiation

Biopsy samples of head and neck carcinomas were investigated with regard to elafin, secretory leukocyte protease inhibitor (SLPI), interleukin 1-receptor antagonist [(IL)1-RA] and soluble tumour necrosis factor alpha receptor antagonist (STNFalpha RI). SLPI and elafin are serine protease inhibitors produced in the serous cells of the upper respiratory airways and in the keratinocytes, respectively. We have now found the presence of elafin and SLPI in squamous cell carcinomas of the upper respiratory tract (tonsillar, hypopharyngeal, tongue, mouth floor, gingival and laryngeal cancer). Significantly higher amounts of SLPI and elafin are present in well-differentiated and moderately differentiated tumours than in poorly differentiated tumours (p < 0.0001 and p < 0.0015). Tumour necrosis factor-alpha and IL-1beta have been shown to stimulate the production of SLPI and elafin. Since these cytokines can both be difficult to detect, we chose to study their inhibitors, STNFalpha RI and IL1-RA, instead. IL1-RA was expressed in highly differentiated tumours as well as in poorly differentiated ones. No significant difference was seen between the groups. STNFalpha RI was only found in very small amounts, sparsely distributed in the tumours, and was not related to the degree of differentiation.     

Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family

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.     

Proteolytic susceptibility of the serine protease inhibitor trappin-2 (pre-elafin): evidence for tryptase-mediated generation of elafin

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.     

Kinetics of the inhibition of neutrophil proteinases by recombinant elafin and pre-elafin (trappin-2) expressed in Pichia pastoris.

Elafin and its precursor, trappin-2 or pre-elafin, are specific endogenous inhibitors of human neutrophil elastase and proteinase 3 but not of cathepsin G. Both inhibitors belong, together with secretory leukocyte protease inhibitor, to the chelonianin family of canonical protease inhibitors of serine proteases. A cDNA coding either elafin or its precursor, trappin-2, was fused in frame with yeast alpha-factor cDNA and expressed in the Pichia pastoris yeast expression system. Full-length elafin or full-length trappin-2 were secreted into the culture medium with high yield, indicating correct processing of the fusion proteins by the yeast KEX2 signal peptidase. Both recombinant inhibitors were purified to homogeneity from concentrated culture medium by one-step cationic exchange chromatography and characterized by N-terminal amino acid sequencing, Western blot and kinetic studies. Both recombinant elafin and trappin-2 were found to be fast-acting inhibitors of pancreatic elastase, neutrophil elastase and proteinase 3 with k(ass) values of 2-4 x 10(6) m(-1).s(-1), while dissociation rate constants k(diss) were found to be in the 10(-4) s(-1) range, indicating low reversibility of the complexes. The equilibrium dissociation constant K(i) for the interaction of both recombinant inhibitors with their target enzymes was either directly measured for pancreatic elastase or calculated from k(ass) and k(diss) values for neutrophil elastase and proteinase 3. K(i) values were found to be in the 10(-10) molar range and virtually identical for both inhibitors. Based on the kinetic parameters determined here, it may be concluded that both recombinant elafin and trappin-2 may act as potent anti-inflammatory molecules and may be of therapeutic potential in the treatment of various inflammatory lung diseases.     

Primary structure of the human elafin precursor preproelafin deduced from the nucleotide sequence of its gene and the presence of unique repetitive sequences in the prosegment.

The human elafin gene was cloned and its entire nucleotide sequence was determined to deduce the amino acid sequence for the precursor of elafin, an elastase-specific inhibitor. The gene spans approximately 1.7 kb and is divided into 3 exons. The gene product preproelafin consists of 117 amino acids: the initiator Met, a putative 25-amino acid signal peptide, a pro-sequence of about 34 amino acids, and the C-terminal 57 amino acids for mature elafin. Possible covalent clotting of the prosegment and its physiological significance have been pointed out based on a remarkable sequence similarity between the pro-sequence and the guinea pig seminal clotting protein SVP-1.     

SLPI and trappin-2 as therapeutic agents to target airway serine proteases in inflammatory lung diseases: current and future directions

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).     

Protease inhibitors derived from elafin and SLPI and engineered to have enhanced specificity towards neutrophil serine proteases

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_is, 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.     

Elafin (elastase-specific inhibitor) has anti-microbial activity against gram-positive and gram-negative respiratory pathogens.

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 pathogens Pseudomonas aeruginosa and Staphylococcus aureus. Elafin killed both bacteria efficiently, with 93% killing of P. aeruginosa by 2.5 microM elafin and 48% killing of S. aureus by 25 microM 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.     

War and peace between WAP and HIV: role of SLPI, trappin-2, elafin and ps20 in susceptibility to HIV infection

Despite tremendous advances in our understanding of HIV/AIDS since the first cases were reported 30 years ago, we are still a long way from understanding critical steps of HIV acquisition, pathogenesis and correlates of protection. Our new understanding of the importance of the mucosa as a target for HIV infection, as well as our recent observations showing that altered expression and responses of innate pattern recognition receptors are significantly associated with pathogenesis and resistance to HIV infection, indicate that correlates of immunity to HIV are more likely to be associated with mucosal and innate responses. Most of the heterosexual encounters do not result in productive HIV infection, suggesting that the female genital tract is protected against HIV by innate defence molecules, such as antiproteases, secreted mucosally. The present review highlights the role and significance of the serine protease inhibitors SLPI (secretory leucocyte protease inhibitor), trappin-2, elafin and ps20 (prostate stromal protein 20 kDa) in HIV susceptibility and infection. Interestingly, in contrast with SLPI, trappin-2 and elafin, ps20 has been shown to enhance HIV infectivity. Thus understanding the balance and interaction of these factors in mucosal fluids may significantly influence HIV infection.     

中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统的建立及活性检测

中性粒细胞蛋白酶抑制剂elafin是中性粒细胞弹性蛋白酶特异性抑制剂。为建立中性粒细胞蛋白酶抑制剂elafin毕赤酵母表达系统,通过逆转录PCR扩增获取elafin cDNA,双酶切后定向克隆至穿梭质粒pPIC9K上。SalI酶切线性化后,电穿孔法转至毕赤酵母GS115菌株中,PCR筛选出阳性克隆,接种至最小甘油缓冲培养基中,甲醇诱导表达后,取上清阳离子交换层析纯化靶标蛋白。分别以western-blot、弹力纤维蛋白-聚丙烯酰胺凝胶电泳检测重组elafin免疫原性、抗蛋白酶活性。重组elafin在毕赤酵母系统中呈分泌性表达,产量及活性高,分离纯化简便。此表达系统的建立为其后续研究及临床应用奠定了基础。     

Therapeutic potential of human elafin

Elafin is an endogenous human protein composed of an N-terminal transglutaminase substrate motif and a C-terminal WAP (whey acidic protein)-domain with antiproteolytic properties. Elafin is expressed predominantly in epithelial tissue and potently inhibits the neutrophil-derived serine proteases elastase and proteinase-3 by a competitive tight-binding mechanism. Furthermore, it inhibits EVE (endogenous vascular elastase). Studies on several animal models show that antiprotease augmentation with human elafin is an effective strategy in the treatment of inflammatory vascular, systemic and pulmonary diseases and of inflammation triggered by reperfusion injury. This raises the possibility that elafin might be effective in the treatment of a variety of human inflammatory diseases. In a Phase I clinical trial, elafin was well tolerated. Phase II trials are underway to investigate the therapeutic effects of elafin on post-operative inflammation and the clinical consequences of major surgery. Of particular interest is the reduction of post-operative morbidity after oesophagus cancer surgery, coronary artery bypass surgery and kidney transplantation.     

Elafin expression in human fetal and adult submandibular glands

Elafin, a bifunctional protein, has the NH(2)-terminal domain functions as a transglutaminase substrate for crosslinking to lysine-containing proteins and the COOH-terminal whey acidic protein domain as a potent anti-elastase. Human fetal submandibular glands (n=100) and adult submandibular glands (n=10) were used to elucidate the expression pattern of elafin in the developmental processes of human submandibular gland by immunohistochemistry, in situ hybridization, and western blot analysis. Elafin mRNA was expressed both in the gland epithelium and intralobular mesenchymal tissue of fetal submandibular gland in an early developmental stage (10-18 weeks) and an early intermediate developmental stage (EIDS; 19-24 weeks). The elafin antigen was also found in the intralobular mesenchyme of submandibular gland in the same stages. Thereafter, elafin was transitionally expressed in the ducts and acini of submandibular glands. In the late intermediate developmental stage (LIDS; 25-32 weeks) and the late developmental stage (LDS; 33-40 weeks), elafin became markedly positive in the excretory and striated ducts but weakly positive in the intralobular mesenchymal tissue. The elafin was heavily present in the excretory and striated ducts of adult submandibular gland, while it was sparse in the intralobular mesenchymal tissues. Western blot analysis showed the protein extracts from submandibular glands of EIDS, LIDS, and LDS, adult submandibular gland, fetal tissues (8 weeks), and adult parotid saliva migrated into multiple bands, about 25, 50, 65, and 140 kDa, which were higher than the putative size of elafin protein, 12 kDa. These data suggest that elafin, anti-elastase, is an essential component highly utilized during the morphogenetic processes of fetal salivary gland development and continuously plays a role for the protection of the tubuloalveolar structures of adult salivary gland.     

Elafin prevents lipopolysaccharide-induced AP-1 and NF-kappaB activation via an effect on the ubiquitin-proteasome pathway

The serine anti-protease elafin is expressed by monocytes, alveolar macrophages, neutrophils, and at mucosal surfaces and possesses antimicrobial activity. It is also known to reduce lipopolysaccharide-induced neutrophil influx into murine alveoli as well as to abrogate lipopolysaccharide-induced production of matrix metalloprotease 9, macrophage inhibitory protein 2, and tumor necrosis factor-alpha by as-yet unidentified mechanisms. In this report we have shown that elafin inhibits the lipopolysaccharide-induced production of monocyte chemoattractant protein-1 in monocytes by inhibiting AP-1 and NF-kappaB activation. Elafin prevented lipopolysaccharide-induced phosphorylation of AP-1, c-Jun, and JNK but had no effect on phosphorylation of p38. The lipopolysaccharide-induced degradation of IL-1R-associated kinase 1, IkappaBalpha, and IkappaBbeta was inhibited by elafin but phosphorylation of IkappaBalpha was unaffected. Polyubiquitinated protein including polyubiquitinated IkappaBalpha was shown to accumulate in the presence of elafin. These results suggest that inhibition by elafin of lipopolysaccharide-induced AP-1 and NF-kappaB activation occurs via an effect on the ubiquitin-proteasome pathway.     

Antimicrobial activity of antiproteinases

Low-molecular-mass neutrophil elastase inhibitors have been shown to be important in the control of lung inflammation. In addition to inhibiting the enzyme neutrophil elastase, these low-molecular-mass compounds (10 kDa) have been shown to have other activities. For example, secretory leucocyte proteinase inhibitor (SLPI) and elastase-specific inhibitor/SKALP (skin-derived antileucoproteinase)/elafin have also been shown to have "defensin"-like antimicrobial activities. Indeed, these inhibitors have antimicrobial properties in vitro against bacteria, fungi and, potentially, HIV. In addition, we have shown, using an adenovirus-mediated gene transfer overexpression strategy, that elafin is also active against Pseudomonas aeruginosa infection in mice in vivo. The mechanism of action is currently under investigation. In addition to these direct or indirect effects on microbes, it has been shown that lipopolysaccharide is able to up-regulate SPLI production in macrophages in vitro, and that the addition of recombinant SLPI to human monocytes or the transfection of macrophages with SPLI can down-regulate pro-inflammatory mediators such as tumour necrosis factor, presumably to limit self-damaging excessive inflammation. Using viral gene transfer vectors, we are currently investigating the potential of these inhibitors in various models of inflammation in vivo.     

SLPI and elafin: multifunctional antiproteases of the WFDC family

SLPI (secretory leucoprotease inhibitor) and elafin represent the archetypal members of the WFDC [WAP (whey acidic protein) four disulfide core] family of proteins, and were originally characterized as protease inhibitors but have since been shown to possess a wider repertoire of activities. These functions include antimicrobial and immunomodulatory properties, suggesting that these proteins may play key roles in the innate immune response, and indicate the potential to develop some of these proteins as novel therapeutics. Susceptibility to host and bacterial protease cleavage may, however, limit the efficacy of recombinant protein therapies in diseases with a high protease burden such as CF (cystic fibrosis) lung disease. To overcome this problem, further refinement of the native proteins will be required to provide effective treatment strategies.