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.

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ABSTRACT. Two different allergy tests, antigen-specific immunoglobulin E quantification (IgE test) and flow cytometric analysis of antigen-specific proliferation of peripheral lymphocytes (lymphocyte proliferation test), were performed to examine differences in allergic reactions to food allergens in dogs with food allergy (FA). Thirteen dogs were diagnosed as FA based on clinical findings and elimination diet trials. Seven dogs clinically diagnosed with canine atopic dermatitis (CAD) were used as a disease control group, and 5 healthy dogs were used as a negative control group. In the FA group, 19 and 33 allergen reactions were identified using the serum IgE test and the lymphocyte proliferation test, respectively. Likewise, in the CAD group, 12 and 6 allergen reactions and in the healthy dogs 3 and 0 allergen reactions were identified by each test, respectively. A significant difference was found between FA and healthy dogs in terms of positive allergen detection by the lymphocyte proliferation test, suggesting that the test can be useful to differentiate FA from healthy dogs but not from CAD. Both tests were repeated in 6 of the dogs with FA after a 1.5-to 5-month elimination diet trial. The IgE concentrations in 9 of 11 of the positive reactions decreased by 20-80%, whereas all the positive reactions in the lymphocyte proliferation test decreased to nearly zero (P<0.05), suggesting that lymphocytes against food allergens may be involved in the pathogenesis of canine FA.