Tissue Expression, Protease Specificity, and Kunitz Domain Functions of Hepatocyte Growth Factor Activator Inhibitor-1B (HAI-1B), a New Splice Variant of HAI-1

Abstract

Hepatocyte growth factor activator inhibitor-1 (HAI-1) is an integral membrane protein expressed on epithelial cells and contains two extracellular Kunitz domains (N-terminal KD1 and C-terminal KD2) known to inhibit trypsin-like serine proteases. In tumorigenesis and tissue regeneration, HAI-1 regulates the hepatocyte growth factor (HGF)/c-Met pathway by inhibiting the activity of HGF activator (HGFA) and matriptase, two serine proteases that convert pro-HGF into its biologically active form. By screening a placental cDNA library, we discovered a new splice variant of HAI-1 designated HAI-1B that contains an extra 16 amino acids adjacent to the C terminus of KD1. To investigate possible consequences on Kunitz domain function, a soluble form of HAI-1B (sHAI-1B) comprising the entire extracellular domain was produced. First, we found that sHAI-1B displayed remarkable enzyme specificity by potently inhibiting only HGFA (IC 50 ‫؍‬ 30.5 nM), matriptase (IC 50 ‫؍‬ 16.5 nM), and trypsin (IC 50 ‫؍‬ 2.4 nM) among 16 serine proteases examined, including plasminogen activators (urokinase-and tissue-type plasminogen activators), coagulation enzymes thrombin, factors VIIa, Xa, XIa, and XIIa, and activated protein C. Relatively weak inhibition was found for plasmin (IC 50 ‫؍‬ 399 nM) and plasma kallikrein (IC 50 ‫؍‬ 686 nM). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P 1 residue-directed mutagenesis to show that inhibition of HGFA, matriptase, trypsin, and plasmin was due to KD1 and not KD2. Furthermore, analysis by reverse transcription-PCR demonstrated that HAI-1B and HAI-1 were co-expressed in normal tissues and various epithelial-derived cancer cell lines. Both isoforms were up-regulated in eight examined ovarian carcinoma specimens, three of which had higher levels of HAI-1B RNA than of HAI-1 RNA. Therefore, previously demonstrated roles of HAI-1 in various physiological and pathological processes likely involve both HAI-1B and HAI-1.Hepatocyte growth factor activator inhibitor-1 (HAI-1)1 is an integral cell surface protein of 66 kDa expressed on epithelial cells (1-3). HAI-1 is known to inhibit the enzymatic activity of HGF activator (HGFA) (1, 4) and matriptase (5-9), two trypsinlike serine proteases capable of converting the inactive singlechain form of hepatocyte growth factor (pro-HGF) (10 -14) into its biologically active two-chain form (HGF). When activated HGF binds to its receptor c-Met, it promotes phospho-transfer activity of the intracellular tyrosine kinase domain leading to activation of multiple intracellular signaling pathways. Therefore, as an inhibitor of HGFA and matriptase, HAI-1 may control the local generation of HGF and thus modulate the activity of the HGF/c-Met receptor system, which is involved in such biological processes as tissue regeneration, morphogenesis, and tumorigenesis (reviewed in Refs. 15-18).The activation of the HGF-converting enzymes represents yet another level of HGF/c-Met pathway regulation. Similar to the coagulation factors, HGFA is ma...

Crystal Structure of a Two-domain Fragment of Hepatocyte Growth Factor Activator Inhibitor-1

“…Mouse studies also support a tight regulatory relationship between matriptase HAI-1 and prostasin (11). In vitro inhibition of additional membrane-associated serine proteases hepsin (12), TMPRSS13 (13), HAT (14), and HATL5 (15) as well as the soluble proteases trypsin, plasmin, and plasma kallikrein have also been reported (16).…”

Structural insight into distinct mechanisms of protease inhibition by antibodies

“…Alternatively, could antibodies inhibit catalysis through other mechanisms? In this study, we attempted to answer these questions by using hepatocyte growth-factor activator (HGFA) as a model system, because structures of this serine protease (family S1) as well as sensitive substrate assays were available (14,15). The serum-derived 34-kDa active HGFA consists of a protease domain disulfide linked to the 35-residue light-chain (16).…”

“…It efficiently cleaves prohepatocyte growth factor (pro-HGF) into the functionally competent two-chain hepatocyte growth factor (HGF) leading to activation of the HGF/Met signaling pathway during tissue regeneration and in cancer growth (17)(18)(19). The N-terminal Kunitz domain (KD1) of the endogenous HGFA inhibitor-1 (HAI-1) (15,20) binds into the HGFA active site in a substratelike manner (14).…”

“…Identical conditions were used for experiments with active-site inhibitors. KD1 was expressed and purified as described (15). The Hfac-221 is a reversible HGFA inhibitor obtained from Alan Olivero (Genentech).…”

“…Pro-HGF activation assays with active sitetitrated HGFA (Val-373-Ser-655) were carried out essentially as described (15) by using serial dilutions of antibody incubated with 0.8 nM HGFA and 25 g/ml of 125 I-pro-HGF. For chromogenic substrate assays with Spectrozyme FVIIa (Methanesulfonyl-D-cyclohexylalanyl-butyl-arginine-paranitroanilide) (American Diagnostica), 3.9 nM HGFA was incubated for 40 min in 96-well plates with increasing concentrations of antibodies in HBSA buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 5 mM CaCl 2 , 0.5 mg/ml BSA].…”