Functional characterization of Kunitz domains in hepatocyte growth factor activator inhibitor type 1.
Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a Kunitz-type serine protease inhibitor identified as a strong inhibitor of hepatocyte growth factor (HGF) activator and matriptase. HAI-1 is first produced in a membrane-integrated form with two Kunitz domains in its extracellular region, and subsequent ectodomain shedding releases two major secreted forms, one with a single Kunitz domain and one with two Kunitz domains. To determine the roles of the Kunitz domains in the inhibitory activity of HAI-1 against serine proteases, we constructed various HAI-1 mutant proteins and examined their inhibitory activity against HGF activator and trypsin. The N-terminal Kunitz domain (Kunitz I) had potent inhibitory activity against both HGF activator and trypsin, whereas the C-terminal Kunitz domain (Kunitz II) had only very weak inhibitory activity against HGF activator, although its potency against trypsin was equivalent to that of Kunitz I. These results indicate that Kunitz I is the functional domain of HAI-1 for inhibiting the HGF-converting activity of HGF activator. Furthermore, the presence of two Kunitz domains affected the inhibitory activity of HAI-1 against HGF activator, and it showed a similar, but not additive, level of inhibitory activity against trypsin when compared with that of the individual Kunitz domains. These results suggest that serine protease binding sites of Kunitz I and Kunitz II are located close to each other and that proteolytic processing to generate HAI-1 with only one Kunitz domain regulates the activity of HAI-1.
Denda
Kimitoshi
K
Department of Biological Sciences, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Midori-ku, Yokohama 226-8501, Japan.
Shimomura
Takeshi
T
Kawaguchi
Toshiya
T
Miyazawa
Keiji
K
Kitamura
Naomi
N
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D015151:Immunoblotting; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory; D012697:Serine Endopeptidases / Q000378:metabolism; D014162:Transfection; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism
D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D053491:Proteinase Inhibitory Proteins, Secretory; C109149:SPINT1 protein, human; C081092:HGF activator; D012697:Serine Endopeptidases; C485595:matriptase; D014357:Trypsin