A secretory leukocyte proteinase inhibitor (SLPI)-like protein from Litopenaeus vannamei haemocytes

A partial clone coding for a two-WAP domain protein was isolated from a Litopenaeus vannamei haemocytes cDNA library. The complete sequence was obtained by RACE, and the full-length cDNA sequence is 0.8 Kb long and encodes for a 116-amino acid protein. The domain composition is similar to the mammalian WFDC5 (WAP four disulfide core) and secretory leukocyte proteinase inhibitor (SLPI). Modifications in expression were determined by real-time PCR, after injection of Vibrio alginolyticus, suggesting its participation in the shrimp immune response. Structural and phylogenetic analyses showed close similarity between shrimp and mammalian SLPI, indicating a probable common ancestor. This is the first report of a mammalian SLPI-like protein in an invertebrate.     

Expression and characterization of recombinant human secretory leukocyte protease inhibitor (SLPI) protein from Pichia pastoris

The human secretory leukocyte protease inhibitor (SLPI) has been shown to possess anti-protease, anti-inflammatory and antimicrobial properties. Its presence in saliva is believed to be a major deterrent to oral transmission of human immunodeficiency virus-1. The 11.7 kDa peptide is a secreted, nonglycosylated protein rich in disulfide bonds. Currently, recombinant SLPI is only available as an expensive bacterial expression product. We have investigated the utility of the methylotrophic yeast Pichia pastoris to produce and secrete SLPI with C-terminal c-myc and polyhistidine tags. The post-transformational vector amplification protocol was used to isolate strains with increased copy number, and culturing parameters were varied to optimize SLPI expression. Modification of the purification procedure allowed the secreted, recombinant protein to be isolated from the cell-free fermentation medium with cobalt affinity chromatography. This yeast-derived SLPI was shown to have an anti-protease activity comparable to the commercially available bacterial product. Thus, P. pastoris provides an efficient, cost-effective system for producing SLPI for structure function analysis studies as well as a wide array of potential therapeutic applications.     

Secretory leukocyte protease inhibitor (SLPI), a multifunctional protein in the host defense response

Secretory leukocyte protease inhibitor (SLPI), a ∼12 kDa nonglycosylated cationic protein, is emerging as an important regulator of innate and adaptive immunity and as a component of tissue regenerative programs. First described as an inhibitor of serine proteases such as neutrophil elastase, this protein is increasingly recognized as a molecule that benefits the host via its anti-proteolytic, anti-microbial and immunomodulatory activities. Here, we discuss the diverse functions of SLPI. Moreover, we review several novel layers of SLPI-mediated control that protect the host from excessive/dysregulated inflammation typical of infectious, allergic and autoinflammatory diseases and that support healing responses through affecting cell proliferation, differentiation and apoptosis.     

Identification of SLPI (secretory leukocyte protease inhibitor) in human mast cells using immunohistochemistry and in situ hybridisation.

Recently interest has been focused on secretory leucocyte protease inhibitor (SLPI) and its role in immediate hypersensitive reactions, possibly by inhibiting mast cell chymase. The purpose of this investigation was to show whether or not SLPI is produced in mast cells. Double-immunolabelling revealed that SLPI coexists with mast cell tryptase (60%) and chymase (37%). On the other hand, in situ hybridisation studies demonstrated the expression of SLPI mRNA in all mast cells. The differences in results can be attributed to the fact that in situ hybridisation is a more sensitive method than immunohistochemistry. Hence, we conclude that SLPI is produced in human tonsillar mast cells.     

Characterization of secretory leukocyte protease inhibitor as an inhibitor of implantation serine proteinases

We have recently identified and characterized two implantation serine proteinase genes, ISP1 and ISP2, which give rise to a dimeric proteinase, ISP that facilitates embryo invasion during peri-implantation period. As many proteinases have cognate serpins that regulate their proteolytic activity, we have been investigating anti-tryptases, expressed during this window of implantation. Here, we report the differential expression of secretory leukocyte protease inhibitor (SLPI) in uterine endometrium around the implantation period. The co-localization of SLPI and ISP suggests the possibility that SLPI is an ISP serpin and that expression of SLPI may lead to a reduction in ISP activity. The expression of SLPI is down regulated during the window of embryo-uterine receptivity. Our results are consistent with a model suggesting that the drop in SLPI expression may help to refine the opening of the window of implantation, by allowing the proteolytic activity of embryo invasive serine proteinases such as the ISPs.     

The role of secretory leukocyte proteinase inhibitor and elafin (elastase-specific inhibitor/skin-derived antileukoprotease) as alarm antiproteinases in inflammatory lung disease

Secretory leukocyte proteinase inhibitor and elafin are two low-molecular-mass elastase inhibitors that are mainly synthesized locally at mucosal sites. It is thought that their physicochemical properties allow them to efficiently inhibit target enzymes, such as neutrophil elastase, released into the interstitium. Historically, in the lung, these inhibitors were first purified from secretions of patients with chronic obstructive pulmonary disease and cystic fibrosis. This suggested that they might be important in controlling excessive neutrophil elastase release in these pathologies. They are upregulated by 'alarm signals' such as bacterial lipopolysaccharides, and cytokines such as interleukin-1 and tumor necrosis factor and have been shown to be active against Gram-positive and Gram-negative bacteria, so that they have joined the growing list of antimicrobial 'defensin-like' peptides produced by the lung. Their site of synthesis and presumed functions make them very attractive candidates as potential therapeutic agents under conditions in which the excessive release of elastase by neutrophils might be detrimental. Because of its natural tropism for the lung, the use of adenovirus-mediated gene transfer is extremely promising in such applications.     

Isolation of chymase complexed with physiological inhibitor similar to secretory leukocyte protease inhibitor (SLPI) from hamster cheek pouch tissues

A low molecular weight protein complexed with chymase was isolated from hamster cheek pouch tissues. This protein had an apparent molecular mass of about 10 kDa on SDS-PAGE and the N-terminal sequence showed some homology to secretory leukocyte protease inhibitor (SLPI), which is known as the predominant inhibitor of neutrophil elastase and cathepsin G. Remarkably enhanced inhibition of chymase activity was achieved in the presence of heparin, indicating that the functional property was also similar to SLPI. These findings suggest that this SLPI-like protein is a candidate for a physiological inhibitor of chymase.     

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.     

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.     

Inhibition of human beta-factor XIIa by squash family serine proteinase inhibitors

Many inhibitors of trypsin and human beta-factor XIIa have been isolated from squash and related seeds and sequenced (Wieczorek et al., Biochem. Biophys. Res. Comm. (1985) 126, 646-652). The association equilibrium constants (Ka) of several of these inhibitors have now been determined with human beta-factor XIIa using a modification of the method of Green and Work (Park et al., Fed. Proc. Fed. Am. Soc. Exp. Biol. (1984) 43, 1962). The Ka's range from 7.8 x 10(4) M-1 to 3.3 x 10(8) M-1. Two isoinhibitors from Cucurbita maxima seeds, CMTI-I and CMTI-III, differ in only a single glutamate to lysine change in the P'4 position. This results in a factor of 62 increase in the Ka of the lysine inhibitor, CMTI-III (Ka = 3.3 x 10(8) M-1). To our knowledge, this is the largest effect ever seen for a residue substitution at the P'4 position of a serine proteinase inhibitor. The result is even more surprising because beta-factor XIIa's natural substrate, Factor XI, contains Gly in the P'4 position.     

IgE-mediated histamine release from nasal mucosa is inhibited by SLPI (secretory leukocyte protease inhibitor) to the level of spontaneous release.

The secretory leukocyte protease inhibitor (SLPI) is a low-molecular-weight inhibitor of proteases, such as elastase and cathepsin G which are released from leukocytes during phagocytosis. The purpose of this study was to determine whether or not SLPI is able to inhibit IgE-mediated histamine release. Nasal mucosa from 11 test subjects without atopic disposition was used for this in vitro study. We found that SLPI inhibited histamine release in a dose-dependent way but was without influence on the spontaneous release.     

Suppression of macrophage responses to bacterial lipopolysaccharide (LPS) by secretory leukocyte protease inhibitor (SLPI) is independent of its anti-protease function

Secretory leukocyte protease inhibitor (SLPI), a potent serine protease inhibitor, has been shown to suppress macrophage responses to bacterial lipopolysaccharide (LPS). SLPI contains two topologically superimposable domains. Its C-terminal domain binds and inhibits target proteases. It is not clear whether SLPI's anti-protease function plays a role in the LPS-inhibitory action of SLPI. Four single amino acid substitution mutants of SLPI, M73G, M73F, M73E and M73K, were generated. Wild type SLPI is a potent inhibitor of chymotrypsin and elastase. Mutants M73G and M73F selectively lost inhibitory function towards chymotrypsin and elastase, respectively, whereas mutants M73K and M73E inhibited neither elastase nor chymotrypsin. Macrophage cell lines were established from RAW264.7 cells to stably express each SLPI mutant. Expression of the SLPI protease inhibition mutants suppressed NO and TNF production in response to LPS in a similar fashion as wild type SLPI. Expression of truncated forms of SLPI, containing only its N-terminus or its C-terminus, was similarly sufficient to confer inhibition of LPS responses. Thus, the LPS-inhibitory action of SLPI is independent of its anti-protease function.     

Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles

Serine proteinase inhibitors (Serpins) are irreversible suicide inhibitors of proteases that regulate diverse physiological processes such as coagulation, fibrinolysis, complement activation, angiogenesis, apoptosis, inflammation, neoplasia and viral pathogenesis. The molecular structure and physical properties of serpins permit these proteins to adopt a number of variant conformations under physiological conditions including the native inhibitory form and several inactive, non-inhibitory forms, such as complexes with protease or other ligands, cleaved, polymerised and oxidised. Alterations of a serpin which affect its structure and/or secretion and thus reduce its functional levels may result in pathology. Serpin dysfunction has been implicated in thrombosis, emphysema, liver cirrhosis, immune hypersensitivity and mental disorders. The loss of inhibitory activity of serpins necessarily results in an imbalance between proteases and their inhibitors, but it may also have other physiological effects through the generation of abnormal concentrations of modified, non-inhibitory forms of serpins. Although these forms of inhibitory serpins are detected in tissues and fluids recovered from inflammatory sites, the important questions of which conditions result in generation of different molecular forms of serpins, what biological function these forms have, and which of them are directly linked to pathologies and/or may be useful markers for characterisation of disease states, remain to be answered. Elucidation of the biological activities of non-inhibitory forms of serpins may provide useful insights into the pathogenesis of diseases and suggest new therapeutic strategies.     

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.