Roles of functional and structural domains of hepatocyte growth factor activator inhibitor type 1 in the inhibition of matriptase.
Hepatocyte growth factor activator inhibitor type 1 (HAI-1) is a membrane-bound, Kunitz-type serine protease inhibitor. HAI-1 inhibits serine proteases that have potent pro-hepatocyte growth factor-converting activity, such as the membrane-type serine protease, matriptase. HAI-1 comprises an N-terminal domain, followed by an internal domain, first protease inhibitory domain (Kunitz domain I), low-density lipoprotein receptor A module (LDLRA) domain, and a second Kunitz domain (Kunitz domain II) in the extracellular region. Our aim was to assess the roles of these domains in the inhibition of matriptase. Soluble forms of recombinant rat HAI-1 mutants made up with various combinations of domains were produced, and their inhibitory activities toward the hydrolysis of a chromogenic substrate were analyzed using a soluble recombinant rat matriptase. Kunitz domain I exhibited inhibitory activity against matriptase, but Kunitz domain II did not. The N-terminal domain and Kunitz domain II decreased the association rate between Kunitz domain I and matriptase, whereas the internal domain increased this rate. The LDLRA domain suppressed the dissociation of the Kunitz domain I-matriptase complex. Surprisingly, an HAI-1 mutant lacking the N-terminal domain and Kunitz domain II showed an inhibitor constant of 1.6 pm, and the inhibitory activity was 400 times higher in this HAI-1 mutant than in the mutant with all domains. These findings, together with the known occurrence of an HAI-1 species lacking the N-terminal domain and Kunitz domain II in vivo, suggest that the domain structure of HAI-1 is organized in a way that allows HAI-1 to flexibly control matriptase activity.
Laboratories of Enzyme Chemistry and Nutrition Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto, Japan.
Kenji
K
Kojima
Satoshi
S
Tsuzuki
Tohru
T
Fushiki
Kuniyo
K
Inouye
D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004800:Enzymes, Immobilized; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012995:Solubility
D004800:Enzymes, Immobilized; D008562:Membrane Glycoproteins; D011994:Recombinant Proteins; D015842:Serine Proteinase Inhibitors; C504592:Spint1 protein, rat; D012697:Serine Endopeptidases; C485595:matriptase