Isolation and characterization of a novel trypsin-like protease found in rat bronchiolar epithelial Clara cells. A possible activator of the viral fusion glycoprotein.
A novel trypsin-like protease associated with rat bronchiolar epithelial Clara cells, named Tryptase Clara, was purified to homogeneity from rat lung by a series of standard chromatographic procedures. The enzyme has apparent molecular masses of 180 +/- 16 kDa on gel filtration and 30 +/- 1.5 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Its isoelectric point is pH 4.75. Studies with model peptide substrates showed that the enzyme preferentially recognizes a single arginine cleavage site, cleaving Boc-Gln-Ala-Arg-4-methylcoumaryl-7-amide most efficiently and having a pH optimum of 7.5 with this substrate. The enzyme is strongly inhibited by aprotinin, diisopropylfluorophosphate, antipain, leupeptin, and Kunitz-type soybean trypsin inhibitor, but inhibited only slightly by Bowman-Birk soybean trypsin inhibitor, benzamidine, and alpha 1-antitrypsin. Immunohistochemical studies indicated that the enzyme is located exclusively in the bronchiolar epithelial Clara cells and colocalized with surfactant. An immunoreactive protein with a molecular mass of 28.5 kDa was also detected in airway secretions by Western blotting analyses, suggesting that the 30-kDa protease in Clara cells is processed before or after its secretion. Proteolytic cleavage of the hemagglutinin of influenza virus is a prerequisite for the virus to become infectious. Tryptase Clara was shown to cleave the hemagglutinin and activate infectivity of influenza A virus in a dose-dependent way. These results suggest that the enzyme is a possible activator of inactive viral fusion glycoprotein in the respiratory tract and thus responsible for pneumopathogenicity of the virus.
Kido
H
H
Division of Enzyme Chemistry, University of Tokushima, Japan.
Yokogoshi
Y
Y
Sakai
K
K
Tashiro
M
M
Kishino
Y
Y
Fukutomi
A
A
Katunuma
N
N
D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D015153:Blotting, Western; D001980:Bronchi / Q000166:cytology / Q000201:enzymology*; D002478:Cells, Cultured; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D006868:Hydrolysis; D007150:Immunohistochemistry; D009980:Influenza A virus / Q000276:immunology; D008297:Male; D008969:Molecular Sequence Data; D051381:Rats; D012697:Serine Endopeptidases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D053802:Tryptases; D014760:Viral Fusion Proteins / Q000378:metabolism*
D014760:Viral Fusion Proteins; D012697:Serine Endopeptidases; C075431:tryptase Clara; D053802:Tryptases