A novel trypsin-like protease was purified to homogeneity from the sputum of patients with chronic airway diseases, by sequential chromatographic procedures. The enzyme migrated on SDS-polyacrylamide gel electrophoresis to a position corresponding to a molecular weight of 28 kDa under both reducing and non-reducing conditions, and showed an apparent molecular weight of 27 kDa by gel filtration, indicating that it exists as a monomer. It had an NH2-terminal sequence of Ile-Leu-Gly-Gly-Thr-Glu-Ala-Glu-Glu-Gly-Ser-Trp-Pro-Trp-Gln-Val-Ser-Leu- Arg-Leu, which differed from that of any known protease. Studies with model peptide substrates showed that the enzyme preferentially cleaves the COOH-terminal side of arginine residues at the P1 position of certain peptides, cleaving Boc-Phe-Ser-Arg-4-methylcoumaryl-7-amide most efficiently and having an optimum pH of 8.6 with this substrate. The enzyme was strongly inhibited by diisopropyl fluorophosphate, leupeptin, antipain, aprotinin, and soybean trypsin inhibitor, but hardly inhibited by secretory leukocyte protease inhibitor at 10 microM. An immunohistochemical study indicated that the enzyme is located in the cells of the submucosal serous glands of the bronchi and trachea. These results suggest that the enzyme is secreted from submucosal serous glands onto the mucous membrane in patients with chronic airway diseases.

Previously we isolated a trypsin-like enzyme designated human airway trypsin-like protease from the sputum of patients with chronic airway diseases. This paper describes the cDNA cloning, characterization of the primary protein structure deduced from the cDNA, and gene expression of this enzyme in various human tissues. We obtained an entire 1517-base pair sequence of cDNA with an open reading frame encoding a polypeptide with 418-amino acid residues. The polypeptide consisted of a 232-residue catalytic region and a 186-residue noncatalytic region with a hydrophobic putative transmembrane domain near the NH 2 terminus. The polypeptide was suggested to be a type II integral membrane protein in which the COOH-terminal catalytic region is extracellular. Therefore, this protein is thought to be synthesized as a membrane-bound precursor and to mature to a soluble and active protease by limited proteolysis. It showed 29 -38% identity in the sequence of the catalytic region with human hepsin, enteropeptidase, acrosin, and mast cell tryptase. The noncatalytic region had little similarity to other known proteins. In Northern blot analysis a transcript of 1.9 kilobases was detectable most prominently in the trachea among 17 human tissues examined.

Human airway trypsin-like protease (HAT) is a serine protease found in sputum of patients with chronic airway diseases and is an agonist of protease-activated receptor-2 (PAR-2). Results from this study show that HAT treatment also enhances mucus production by the airway epithelial cell line NCI-H292 in vitro. Histologic examination showed that HAT enhances mucous glycoconjugate synthesis, whereas the PAR-2 agonist peptide (PAR-2 AP) has no such effect. HAT, but not PAR-2 AP, enhances MUC2 and MUC5AC gene expression 23-fold and 32-fold, respectively. The proteolytic activity of HAT is required to enhance MUC5AC gene expression; the addition of the inhibitors of trypsin-like protease activity of HAT, aprotinin and leupeptin, abolishes its enhancing effect. AG1478, anti-epidermal growth factor receptor (anti-EGFR)-neutralizing antibody, and anti-amphiregulin (AR)-neutralizing antibody all inhibited the stimulatory effect of HAT. Furthermore, HAT increases AR gene expression and subsequent AR protein release, whereas PAR-2 AP shows no such effects. These results indicate that HAT enhances mucin gene expression through an AR-EGFR pathway, and PAR-2 is not sufficient for or does not directly cause HAT-induced mucin gene expression. Thus, HAT might be a possible therapeutic target to prevent excessive mucus production in patients with chronic airway diseases.

The effect of biochemical components on the viscoelasticity of nasal mucus from 24 patients with chronic sinusitis (CS) was investigated by multiple stepwise regression analysis. The dynamic viscosity (eta') and the elastic modulus (G') of nasal mucus were determined with an oscillating sphere magnetic rheometer at oscillatory frequencies of 1 and 10 Hz. The eta' and G' values of mucus determined at 1 Hz were 1.6 +/- 1.5 Pa/s and 31.8 +/- 31.0 Pa, respectively, and these values were much higher than optimal viscoelasticity for mucociliary transport. The concentrations of fucose, N-acetyl neuraminic acid, albumin, IgG, secretory-IgA, and lysozyme were measured in the same mucus samples. The multiple regression analysis showed that the concentration of fucose, a marker of mucous glycoproteins, was the most important determinant of eta' and G'. The analysis also revealed that the level of IgG was the next important determinant. The coefficients of multiple determination for fucose and IgG were 0.732 and 0.733 when the response variables were eta' and G', respectively. The results indicate that locally produced mucous glycoproteins may largely contribute to the high viscoelasticity of nasal mucus in CS.

. Human airway trypsin-like protease stimulates human bronchial fibroblast proliferation in a protease-activated receptor-2-dependent pathway.

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The objective of this study is to examine the differential expression of mast cell tryptase and its receptor, protease-activated receptor-2 (PAR-2), in the synovium and synovial fluid of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). Biochemical and immunohistochemical analyses were performed to determine whether the trypsin-like protease in the synovium is identical to mast cell tryptase. The effects of mast cell tryptase on the proliferation of synovial fibroblast-like cells (SFCs) and the release of IL-8 thereof were evaluated by the [3H]-thymidine incorporation and ELISA, respectively. The trypsin-like protease in the synovium of RA patients was identical to human mast cell tryptase, which was composed of two subunits: 33 and 34 kDa. The 33- and 34-kDa proteins are different glycosylated forms of the 31-kDa protein, which was unglycosylated. Mast cell tryptase activity in RA synovial fluid was significantly higher than that in OA synovial fluid, while their activities and expression in the synovium were similar. Expression of PAR-2 mRNA in the synovium was higher in RA than in OA. Mast cell tryptase containing the unglycosylated 31-kDa subunit was the predominant form in synovial fluid. RA patients had higher amounts of this subunit in their synovial fluid than OA patients. Mast cell tryptase and PAR-2 activating peptide stimulated the proliferation of SFCs and release of IL-8 from these cells. Mast cell tryptase secretion into RA synovial fluid is higher than OA synovial fluid. Mast cell tryptase in synovial fluid stimulates the proliferation of SFCs and the release of pro-inflammatory cytokines via PAR-2, which may contribute to exacerbation of synovitis in RA.

Human airway trypsin-like protease (HAT), a novel serine protease in the airways, enhances cell growth and IL-8 production. The expression and role of HAT in the skin however, is unknown. Immunofluorescence staining and reverse transcription (RT)-PCR were done to know HAT production in normal and psoriatic tissues and keratinocyte cell lines. Cell growth and/or IL-8 release analyses were made by bromo-deoxyuridine (BrdU) uptake and ELISA. Psoriatic epidermis showed more extensive immunofluorescence expression of HAT, and less extensive expression of protease-activated receptor (PAR)-2. RT-PCR demonstrated a higher HAT and a lesser PAR-2 mRNA expressions in psoriatic epidermis. Normal keratinocyte and epidermoid carcinoma cell lines expressed HAT and PAR-2 mRNA, and immortalized keratinocytes (HaCaT) expressed PAR-2, but not HAT mRNA. PAR-2 was detected along the keratinocyte surface in culture and became invisible upon HAT stimulation, suggesting a process of its internalization. HAT or PAR-2 activating peptide did not enhance BrdU uptake, but induced an IL-8 release. Treatment with HAT and IL-1beta synergistically increased the effect of IL-8 release. Inhibition of PAR-2 resulted in a decreased HAT-induced IL-8 release. Thus, HAT might promote PAR-2-mediated IL-8 production to accumulate inflammatory cells in the epidermal layer of psoriasis.