A novel influenza A virus activating enzyme from porcine lung: purification and characterization.
Proteolytic activation of hemagglutinin, an envelope glycoprotein of the influenza virus, by host proteases is essential for infection and proliferation of the virus. However, there is no well-defined, inherent source of host proteases in man or swine, both of which are natural hosts for human influenza viruses. We have recently isolated a 32 kDa protein in a high salt extract from porcine lungs, which possess the hemagglutinin processing activity. In this study, we attempted to purify another hemagglutinin processing enzyme from porcine lung. The purified enzyme, named tryptase TC30, exhibited a molecular mass of about 30 kDa by SDS-PAGE and 28.5 kDa by gel filtration chromatography, suggesting that it is a monomer. Tryptase TC30 cleaved peptide substrates with Arg at the P1 position, and preferentially substrates with the Ser-Ile-Gin-Ser-Arg sequence corresponding to the HA cleavage site sequence of the A/PR/8/34 influenza virus. Among various inhibitors tested, trypsin-type serine protease inhibitors, such as aprotinin, antipain, benzamidine and leupeptin, efficiently inhibited the proteolytic activity of the enzyme. The N-terminal 40 amino acid sequence of tryptase TC30 exhibits more than 60% homology to mast cell tryptases from mice MCP-6 and human tryptase-alpha and -beta. These data indicate that tryptase TC30, the 30 kDa enzyme from porcine lung, is a novel hemagglutinin-cleaving enzyme.
Sato
Masayuki
M
Biological Research Laboratories, Sankyo Co., Ltd., 1-2-58 Hiromachi, Shinagawa-ku, Tokyo 140-8710, Japan.
Yoshida
Shuku
S
Iida
Kenji
K
Tomozawa
Takanori
T
Kido
Hiroshi
H
Yamashita
Makoto
M
D000595:Amino Acid Sequence; D000818:Animals; D004285:Dogs; D006388:Hemagglutinins / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000235:genetics / Q000378:metabolism*; D008168:Lung / Q000201:enzymology*; D008407:Mast Cells / Q000201:enzymology; D051379:Mice; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010455:Peptides / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D051381:Rats; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000145:classification / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine; D053802:Tryptases
D006388:Hemagglutinins; D010455:Peptides; D011480:Protease Inhibitors; C509190:Tpsb2 protein, mouse; D012697:Serine Endopeptidases; C509222:Tpsab1 protein, mouse; C509223:Tpsab1 protein, rat; C509221:Tpsb2 protein, rat; D053802:Tryptases