Identification and Cloning of Human Placental Bikunin, a Novel Serine Protease Inhibitor Containing Two Kunitz Domains

Abstract

Interrogation of the public expressed sequence tag (EST) data base with the sequence of preproaprotinin identified ESTs encoding two potential new members of the Kunitz family of serine protease inhibitors. Through reiterative interrogation, an EST contig was obtained, the consensus sequence from which encoded both of the novel Kunitz domains in a single open reading frame. This consensus sequence was used to direct the isolation of a full-length cDNA clone from a placental library. The resulting cDNA sequence predicted a 252-residue protein containing a putative NH 2 -terminal signal peptide followed sequentially by each of the two Kunitz domains within a 170-residue ectodomain, a putative transmembrane domain, and a 31-residue hydrophilic COOH terminus. The gene for this putative novel protein was mapped by use of a radiation hybrid panel to chromosome 19q13, and Northern analysis showed that the corresponding mRNA was expressed at high levels in human placenta and pancreas and at lower levels in brain, lung, and kidney. An endogenous soluble form of this protein, which was designated as placental bikunin, was highly purified from human placenta by sequential kallikrein-Sepharose affinity, gel filtration, and C 18 reverse-phase chromatography. The natural protein exhibited the same NH 2 terminus as predicted from the cloned cDNA and inhibited trypsin, plasma kallikrein, and plasmin with IC 50 values in the nanomolar range.The Kunitz (1, 2), Kazal (2), Serpin (3), and mucus (4) families of biological serine protease inhibitors play a vital role in the spatial and temporal regulation of in vivo proteolysis. The prototypical Kunitz inhibitor, bovine aprotinin (2), is a 58-amino acid protein containing three intrachain disulfide bonds in a spacing that is conserved in all family members (1, 2). Although the physiologic function of aprotinin is uncertain, it is a potent inhibitor of several serine proteases, and its potency against kallikrein and plasmin (5) may be relevant to its clinical mode of action (5, 6), particularly in the reduction of perioperative blood loss. A human functional homolog of aprotinin has not been identified, although several larger human proteins containing one or more Kunitz domains are known. These include: tissue factor pathway inhibitor (TFPI), 1 which contains three Kunitz domains (7) and inhibits both factor Xa and the factor VIIa-tissue factor complex (8); TFPI-2 (9), which contains two Kunitz domains (a bikunin) and is a potent inhibitor of the factor VIIa-tissue factor complex, factor XIa, and plasmin (10); and inter-␣-trypsin inhibitor, a plasma-associated bikunin (11). In addition, the following proteins are known to contain a single Kunitz domain: COL␣3/VI, the ␣(3) chain of type VI collagen (12); HKI-B9, a human Kunitz inhibitor (13); the membrane-associated amyloid precursor proteins APP 751 (14) and APP 770 (15); the amyloid precursor-like proteins (APLP) such as APLP2 (16). To identify novel human homologs of aprotinin we employed a bioinformatic approach that exploited the...

Distribution of Hepatocyte Growth Factor Activator Inhibitor Type 1 (HAI-1) in Human Tissues: Cellular Surface Localization of HAI-1 in Simple Columnar Epithelium and Its Modulated Expression in Injured and Regenerative Tissues

“…Another recently identified HGFA inhibitor, HAI-2, may be one of the candidates. However, the levels of HAI-2 mRNA were also low in the liver and lungs Marlor et al 1997). (b) The expression of HAI-1 might be highly situational, depending on the cell type and the presence of other constituents in the intra-or extracellular milieu.…”

“…These inhibitors were designated as HGF activator inhibitor Type 1 and Type 2 (HAI-1 and HAI-2) Shimomura et al 1997). At the same time, HAI-2 was also independently discovered by two other groups as a novel placental bikunin and as a protein overexpressed in pancreatic cancer (Marlor et al 1997;Müller-Pillasch et al 1998).…”

Characterization of Placental Bikunin, a Novel Human Serine Protease Inhibitor

“…In a previous study, we identified and cloned a human cDNA encoding a novel protein containing two Kunitz-like domains which we termed placental bikunin (6). In this study a soluble recombinant fragment of this protein, placental bikunin , 2 was expressed and found to be a potent serine protease inhibitor.…”

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

“…Both KD1 and KD2 can engage in protease inhibition (37), similar to the structurally related but more promiscuous HAI-2 (38, 39) (also referred to as placental bikunin (40) or kop (41)). Three splice variants were reported for HAI-2, designated as HAI-2A, HAI-2B, and HAI-2C (42).…”