Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1.
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 (IC50 = 30.5 nm), matriptase (IC50 = 16.5 nm), and trypsin (IC50 = 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 (IC50 = 399 nm) and plasma kallikrein (IC50 = 686 nm). Second, the functions of the KD1 and KD2 domains in sHAI-1B were investigated using P1 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.
Kirchhofer
Daniel
D
Department of Physiology, Genentech, Inc., South San Francisco, California 94080, USA. dak@gene.com
Peek
Mark
M
Li
Wei
W
Stamos
Jennifer
J
Eigenbrot
Charles
C
Kadkhodayan
Saloumeh
S
Elliott
J Michael
JM
Corpuz
Racquel T
RT
Lazarus
Robert A
RA
Moran
Paul
P
D000409:Alanine / Q000737:chemistry; D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D010450:Endopeptidases / Q000737:chemistry*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005091:Exons; D015942:Factor VIIa / Q000737:chemistry; D015956:Factor XIIa / Q000737:chemistry; D015945:Factor XIa / Q000737:chemistry; D015951:Factor Xa / Q000737:chemistry; D005260:Female; D005341:Fibrinolysin / Q000737:chemistry; D015723:Gene Library; D006801:Humans; D020128:Inhibitory Concentration 50; D007438:Introns; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D010051:Ovarian Neoplasms / Q000378:metabolism; D020842:Plasma Kallikrein / Q000737:chemistry; D010957:Plasmids / Q000378:metabolism; D010960:Plasminogen Activators / Q000737:chemistry; D011486:Protein C / Q000737:chemistry; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D053491:Proteinase Inhibitory Proteins, Secretory; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D014018:Tissue Distribution; D014357:Trypsin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D015854:Up-Regulation
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D011486:Protein C; D020033:Protein Isoforms; D053491:Proteinase Inhibitory Proteins, Secretory; D012333:RNA, Messenger; D011994:Recombinant Proteins; C109149:SPINT1 protein, human; D012313:RNA; D010450:Endopeptidases; D010960:Plasminogen Activators; D012697:Serine Endopeptidases; C485595:matriptase; C485596:ST14 protein, human; D015942:Factor VIIa; D015945:Factor XIa; D020842:Plasma Kallikrein; D015956:Factor XIIa; D014357:Trypsin; D015951:Factor Xa; D005341:Fibrinolysin; D000409:Alanine