Purification and cDNA cloning of a novel protease inhibitor secreted into culture supernatant by MDCK cells.

The infectivity of influenza viruses to host cells depends on the activation of the viral glycoprotein hemagglutinin (HA) by proteases. Starting from the observation that influenza virus replication in MDCK (Madin Darby canine kidney) cells was impaired by inactivation of trypsin in the culture fluids, we demonstrated that the inhibitory activity was resolved into two Trypsin-inactivating factors (TF), TF A (15 kDa) and TF B (11 kDa). N-terminal protein sequences of the factors revealed that TF A was a known Submandibular Protease Inhibitor (SPI) secreted in dog saliva, while TF B was a novel protein (renamed CKPI; canine kidney protease inhibitor). Following peptide mapping and protein sequencing of CKPI we obtained a 390 bp cDNA encoding a 130-amino-acid protein from MDCK cell total RNA. Protein sequence comparison showed a 63.8% identity with human secretory leukocyte protease inhibitor (SLPI), the molecule containing two conserved whey acidic protein (WAP) motifs, and we suggest that CKPI is thought to be the canine analogue of human SLPI. These results suggest that the inhibitory factors are secreted from MDCK cells, which are involved in prevention of virus replication, and applicable to the protection of host cells from virus infection.     

Secretory leukocyte protease inhibitor mediates proliferation of human endometrial epithelial cells by positive and negative regulation of growth-associated genes

Secretory leukocyte protease inhibitor (SLPI) inhibits chymotrypsin, trypsin, elastase, and cathepsin G. This protein also exhibits proliferative effects, although little is known about the molecular mechanisms underlying this activity. We have generated SLPI-ablated epithelial sublines by stably transfecting the Ishikawa human endometrial cell line with an antisense human SLPI RNA expression vector. We demonstrate a positive correlation between cellular SLPI production and proliferation. We further show that Ishikawa sublines expressing low to undetectable SLPI have correspondingly increased and decreased expression, respectively, of transforming growth factor-beta 1 and cyclin D1 genes, relative to parental cells. SLPI selectively increased cyclin D1 gene expression, with the effect occurring in part at the level of promoter activity. Cellular SLPI levels negatively influenced the anti-proliferative and pro-apoptotic insulin-like growth factor-binding protein-3 expression. We also identified lysyl oxidase, a phenotypic inhibitor of the ras oncogenic pathway and a tumor suppressor, as SLPI-repressed gene, whose expression is up-regulated by transforming growth factor-beta1. Our results suggest that SLPI acts at the node(s) of at least three major interacting growth inhibitory pathways. Because expression of SLPI is generally high in epithelial cells exhibiting abnormal proliferation such as in carcinomas, SLPI may define a novel pathway by which cellular growth is modulated.     

Development of an avidin-biotin competitive inhibition assay and validation of its use for the quantitation of human intervertebral disc serine proteinase inhibitory proteins.

A simple convenient method has been developed for the quantitation of serine proteinase inhibitors (SPIs) in tissue extracts. The method is based on the competitive binding to trypsin and chymotrypsin immobilized using glutaraldehyde on 96-well microtiter plate wells of native SPIs and a biotinylated secretory proteinase inhibitor (SLPI) standard. The bound SLPI standard was visualized using an avidin-alkaline phosphatase conjugate and inhibition curves were determined using absorbancy measurements at 405 nm. The standard assay had a range between 0.02 and 1 microgram SLPI/well and a lower detection limit of 20 ng SLPI/well; an improved microassay had a detection limit of 2 ng SLPI/well. Only active free inhibitor was detected in the assay since denatured and/or enzyme-inhibitor complexes did not bind to the plates. A range of SPI species was demonstrable in human bronchial mucus and intervertebral disc SPI samples using this technique. Quantitation of SPI levels in a number of intervertebral disc samples indicated that the SPIs were depleted in degenerate discs compared to nondegenerate discs (P less than 0.05, n = 12). Since the immobilized trypsin and chymotrypsin microplates used in this assay may be prepared in advance (and are stable at 4 degrees C for at least 1 month) the remaining two steps of the assay (the inhibition step and visualization) may be completed in 2-3 h; thus the assay is simple, convenient, and fast. All reagents (other than the biotinylated SLPI standard) are readily available commercially, and in principle the assay could be adapted to other systems provided defined biotinylated standards were available.     

Secretory leukocyte protease inhibitor (SLPI) might contaminate murine monoclonal antibodies after purification on protein G

The large scale production of a monoclonal anti-progesterone antibody in serum free medium followed by affinity chromatography on protein G lead to a contamination of the antibody sample with a protein of about 14 kDa. This protein was identified by mass spectrometry as secretory leukocyte protease inhibitor (SLPI). This SLPI contamination lead to a failure of the fiber-optic based competitive fluorescence assay to detect progesterone in milk. Purification of the monoclonal antibody using protein A columns circumvented this problem.     

Secretory leucoprotease inhibitor prevents lipopolysaccharide-induced IkappaBalpha degradation without affecting phosphorylation or ubiquitination

Secretory leucoprotease inhibitor (SLPI) is a non-glycosylated protein produced by epithelial cells, macrophages, and neutrophils and was initially identified as a serine protease inhibitor of the neutrophil proteases elastase and cathepsin G. In addition to its antiprotease activity, SLPI has been shown to exhibit anti-inflammatory properties including down-regulation of tumor necrosis factor-alpha expression by lipopolysaccharide (LPS) in monocytes, inhibition of NF-kappaB activation by IgG immune complexes in a rat model of acute lung injury, and prevention of human immunodeficiency virus infectivity in monocytic cells via as yet unidentified mechanisms. In this report we have shown that SLPI prevents LPS-induced NF-kappaB activation by inhibiting degradation of IkappaBalpha without affecting the LPS-induced phosphorylation and ubiquitination of IkappaBalpha. We have also demonstrated that SLPI prevents LPS-induced interleukin-1 receptor-associated kinase and IkappaBbeta degradation. In addition, we have demonstrated that oxidized SLPI, a variant of SLPI that has diminished antiprotease activity, cannot prevent LPS-induced NF-kappaB activation or Inhibitor kappaB alpha/beta degradation indicating that the anti-inflammatory effect of SLPI on the LPS-signaling pathway is dependent on its antiprotease activity. These results suggest that SLPI may be inhibiting proteasomal degradation of NF-kappaB regulatory proteins, an effect that is dependent on the antiprotease activity of SLPI.     

Secretory leukocyte protease inhibitor: inhibition of human immunodeficiency virus-1 infection of monocytic THP-1 cells by a newly cloned protein

The ability of the salivary protein, secretory leukocyte protease inhibitor (SLPI), to inhibit human immunodeficiency virus-1 (HIV-1) infection in vitro has been reported previously and has led to the suggestion that SLPI may be partially responsible for the low oral transmission rate of HIV-1. However, results contradictory to these findings have also been published. These discrepancies can be attributed to a number of factors ranging from the variability of macrophage susceptibility to HIV infection to the quality of commercially available preparations of SLPI. To resolve these differences and to study further the potential anti-HIV-1 activity of SLPI, the purified and re-folded protein, expressed from a synthetic gene, was examined using human monocytic THP-1 cells. This newly cloned SLPI reduced HIV-1(Ba-L) infection in differentiated THP-1 cells, in contrast to the results observed when using commercially available preparations of SLPI. Interestingly, while the two proteins displayed different anti-HIV effects they had comparable anti-protease activity. The identification of the THP-1 cell line as a system that supports HIV replication, which can be inhibited by a preparation of SLPI now available in large quantities, sets the stage for a thorough investigation of the molecular and structural basis for the anti-HIV activity of SLPI.     

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.     

Regulation of SLPI and elafin release from bronchial epithelial cells by neutrophil defensins

Secretory leukocyte proteinase inhibitor (SLPI) is a serine proteinase inhibitor that is produced locally in the lung by cells of the submucosal bronchial glands and by nonciliated epithelial cells. Its main function appears to be the inhibition of neutrophil elastase (NE). Recently, NE was found to enhance SLPI mRNA levels while decreasing SLPI protein release in airway epithelial cells. Furthermore, glucocorticoids were shown to increase both constitutive and NE-induced SLPI mRNA levels. In addition to NE, stimulated neutrophils also release alpha-defensins. Defensins are small, antimicrobial polypeptides that are found in high concentrations in purulent secretions of patients with chronic airway inflammation. Like NE, defensins induce interleukin-8 production in airway epithelial cells. This induction is sensitive to inhibition by the glucocorticoid dexamethasone and is prevented in the presence of alpha(1)-proteinase inhibitor. The aim of the present study was to investigate the effect of defensins on the production of SLPI and the related NE inhibitor elafin/SKALP in primary bronchial epithelial cells (PBECs). Defensins significantly increase SLPI protein release by PBECs in a time- and dose-dependent fashion without affecting SLPI mRNA synthesis. In the presence of alpha(1)-proteinase inhibitor, the defensin-induced SLPI protein release is further enhanced, but no effect was observed on SLPI mRNA levels. Dexamethasone did not affect SLPI protein release from control or defensin-treated PBECs. In addition, we observed a constitutive release of elafin/SKALP by PBECs, but this was not affected by defensins. The present results suggest a role for defensins in the dynamic regulation of the antiproteinase screen in the lung at sites of inflammation.     

Conformational stability of secretory leucocyte protease inhibitor: a protein with no hydrophobic core and very little secondary structure

Conformational stability of proteins (including disulfide containing proteins) has been routinely characterized by spectroscopic techniques. Proteins which lack adequate signal of circular dichroism may require unconventional technique. Secretory Leucocyte Protease Inhibitor (SLPI) is a 107 amino acids protein with a high density of disulfide pairing (eight). The native SLPI has no hydrophobic core and contains very little hydrogen bonded secondary structure [Gruetter, M., Fendrich, G., Huber, R., and Bode, W. (1988) The 2.5 A X-ray crystal structure of the acid stable proteinase inhibitor from human mucous secretions analyzed in its complex with bovine alpha-chymotrypsin. The EMBO J. 7, 345-352.]. In this study, conformational stability of SLPI has been investigated by the method of disulfide scrambling, which permits quantification of the native and denatured (scrambled) proteins by HPLC. Due to high heterogeneity of denatured SLPI, the native and scrambled SLPI are extensively overlapped on HPLC. This impediment was further overcome by the development of a novel method which distinguishes the native and scrambled isomers of SLPI by exploiting the relative stability of their disulfide bonds. The study reveals mid-point denaturation of SLPI at 1.36 M of GdmSCN, 4.0 M of GdmCl and >8 M urea. Based on the GdmCl denaturation curve, the unfolding free energy (DeltaG(H20)) of SLPI was estimated to be 4.56 kcal/mol. The results of our studies suggest an alternative strategy for analyzing conformational stability of disulfide proteins that are not suitable to the conventional spectroscopic techniques.     

Construction,non-denaturing affinity purification,and characterization of baculovirally expressed human secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) is a 11.7 kDa mucosal protein with potent anti-microbial, anti-inflammatory, and wound healing activities. Previous efforts to express and purify the non-glycosylated cationic protein as a recombinant protein in bacteria required extensive denaturation and renaturation to refold the disulfide-rich protein into its biologically active form. To overcome this limitation, we have expressed human SLPI as a polyhistidine-tagged protein (bvHisSLPI) using a recombinant baculovirus expression system. Studies were conducted to determine the timing of maximal protein production following baculovirus infection of Sf21 cells. The 16.4kDa-tagged protein was then overexpressed in Sf21 cells following a 48-h infection with bvHisSLPI-encoding baculovirus, purified by nickel-chelating affinity chromatography under non-denaturing conditions, and analyzed by Coomassie-stained SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot. Purified bvHisSLPI was further characterized by enterokinase digestion to remove the polyhistidine tag from its N-terminus. In serine protease inhibition assays, purified bvHisSLPI blocked substrate cleavage by two serine proteases, chymotrypsin and cathepsin G, comparable to bacterially expressed SLPI. The baculovirus expression and affinity purification strategy described here will facilitate further studies of the structural and biological properties of this important multifunctional protein.     

Cleaved SLPI, a novel biomarker of chymase activity

Secretory leukocyte protease inhibitor (SLPI) is a protease inhibitor of the whey acidic protein-like family inhibiting chymase, chymotrypsin, elastase, proteinase 3, cathepsin G and tryptase. Performing in vitro enzymatic assays using both Western blotting and liquid chromatography/mass spectrometry techniques we showed that, of the proteases known to interact with SLPI, only chymase could uniquely cleave this protein. The peptides of the cleaved SLPI (cSLPI) remain coupled due to the disulfide bonds in the molecule but under reducing conditions the cleavage can be observed as peptide products. Subsequent ex vivo studies confirmed the presence of SLPI in human saliva and its susceptibility to cleavage by chymase. Furthermore, inhibitors of chymase activity are able to inhibit this cleavage. Human saliva from both normal and allergic individuals was analyzed for levels of cSLPI and a correlation between the level of cSLPI and the extent of allergic symptoms was observed, suggesting the application of cSLPI as a biomarker of chymase activity in humans.     

Secretory leukocyte protease inhibitor plays an important role in the regulation of allergic asthma in mice

Secretory leukocyte protease inhibitor (SLPI) is an anti-inflammatory protein that is observed at high levels in asthma patients. Resiquimod, a TLR7/8 ligand, is protective against acute and chronic asthma, and it increases SLPI expression of macrophages in vitro. However, the protective role played by SLPI and the interactions between the SLPI and resiquimod pathways in the immune response occurring in allergic asthma have not been fully elucidated. To evaluate the role of SLPI in the development of asthma phenotypes and the effect of resiquimod treatment on SLPI, we assessed airway resistance and inflammatory parameters in the lungs of OVA-induced asthmatic SLPI transgenic and knockout mice and in mice treated with resiquimod. Compared with wild-type mice, allergic SLPI transgenic mice showed a decrease in lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), and plasma IgE levels (p < 0.001). Allergic SLPI knockout mice displayed phenotype changes significantly more severe compared with wild-type mice. These phenotypes included lung resistance (p < 0.001), airway eosinophilia (p < 0.001), goblet cell hyperplasia (p < 0.001), cytokine levels in the lungs (p < 0.05), and plasma IgE levels (p < 0.001). Treatment of asthmatic transgenic mice with resiquimod increased the expression of SLPI and decreased inflammation in the lungs; resiquimod treatment was still effective in asthmatic SLPI knockout mice. Taken together, our study showed that the expression of SLPI protects against allergic asthma phenotypes, and treatment by resiquimod is independent of SLPI expression, displayed through the use of transgenic and knockout SLPI mice.     

Potent antimycobacterial activity of mouse secretory leukocyte protease inhibitor

Secretory leukocyte protease inhibitor (SLPI) has multiple functions, including inhibition of protease activity, microbial growth, and inflammatory responses. In this study, we demonstrate that mouse SLPI is critically involved in innate host defense against pulmonary mycobacterial infection. During the early phase of respiratory infection with Mycobacterium bovis bacillus Calmette-Guérin, SLPI was produced by bronchial and alveolar epithelial cells, as well as alveolar macrophages, and secreted into the alveolar space. Recombinant mouse SLPI effectively inhibited in vitro growth of bacillus Calmette-Guérin and Mycobacterium tuberculosis through disruption of the mycobacterial cell wall structure. Each of the two whey acidic protein domains in SLPI was sufficient for inhibiting mycobacterial growth. Cationic residues within the whey acidic protein domains of SLPI were essential for disruption of mycobacterial cell walls. Mice lacking SLPI were highly susceptible to pulmonary infection with M. tuberculosis. Thus, mouse SLPI is an essential component of innate host defense against mycobacteria at the respiratory mucosal surface.