Tissue expression, protease specificity, and Kunitz domain functions of hepatocyte growth factor activator inhibitor-1B (HAI-1B), a new splice variant of HAI-1

J Biol Chem. 2003 Sep 19;278(38):36341-9. doi: 10.1074/jbc.M304643200. Epub 2003 Jun 18.

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 (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.

MeSH terms

  • Alanine / chemistry
  • Alternative Splicing*
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • CHO Cells
  • Cell Line
  • Cloning, Molecular
  • Cricetinae
  • DNA, Complementary / metabolism
  • Dose-Response Relationship, Drug
  • Endopeptidases / chemistry*
  • Enzyme Inhibitors / pharmacology
  • Epithelial Cells / metabolism
  • Escherichia coli / metabolism
  • Exons
  • Factor VIIa / chemistry
  • Factor XIIa / chemistry
  • Factor XIa / chemistry
  • Factor Xa / chemistry
  • Female
  • Fibrinolysin / chemistry
  • Gene Library
  • Humans
  • Inhibitory Concentration 50
  • Introns
  • Membrane Glycoproteins / biosynthesis
  • Membrane Glycoproteins / chemistry*
  • Membrane Glycoproteins / genetics*
  • Models, Genetic
  • Molecular Sequence Data
  • Mutation
  • Ovarian Neoplasms / metabolism
  • Plasma Kallikrein / chemistry
  • Plasmids / metabolism
  • Plasminogen Activators / chemistry
  • Protein C / chemistry
  • Protein Isoforms
  • Protein Structure, Tertiary
  • Proteinase Inhibitory Proteins, Secretory
  • RNA / metabolism
  • RNA, Messenger / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Sequence Homology, Amino Acid
  • Serine Endopeptidases / chemistry
  • Serine Endopeptidases / metabolism
  • Substrate Specificity
  • Tissue Distribution
  • Trypsin / chemistry
  • Trypsin / metabolism
  • Trypsin / pharmacology
  • Up-Regulation

Substances

  • DNA, Complementary
  • Enzyme Inhibitors
  • Membrane Glycoproteins
  • Protein C
  • Protein Isoforms
  • Proteinase Inhibitory Proteins, Secretory
  • RNA, Messenger
  • Recombinant Proteins
  • SPINT1 protein, human
  • RNA
  • Endopeptidases
  • Plasminogen Activators
  • Serine Endopeptidases
  • matriptase
  • ST14 protein, human
  • Factor VIIa
  • Factor XIa
  • Plasma Kallikrein
  • Factor XIIa
  • Trypsin
  • Factor Xa
  • Fibrinolysin
  • Alanine