Purification and Characterization of a Novel Hyaluronan-Binding Protein (PHBP) from Human Plasma: It Has Three EGF, a Kringle and a Serine Protease Domain, Similar to Hepatocyte Growth Factor Activator

Abstract

A novel hyaluronan-binding protein (PHBP) was purified from human plasma by affinity chromatography on hyaluronan-conjugated Sepharose. The contaminating IgM and albumin in the partially purified preparation were removed with anti-IgG antibody-conjugated Sepharose and anti-albumin antibody-conjugated Sepharose, respectively, and no other contaminant was observed. Finally, 800 micrograms of PHBP was isolated from 500 ml of human plasma. PHBP gave a single 70-kDa band on SDS-PAGE under non-reducing conditions, and 50-kDa and 17-kDa bands under reducing conditions. Thus, PHBP was a heterodimer composed of 50-kDa and 17-kDa subunits, bridged by a disulfide linkage. Both subunits had novel N-terminal amino acid sequences, indicating that PHBP was a novel hyaluronan-binding protein in human plasma. The amino acid sequence deduced from the nucleotide sequence of the cloned PHBP cDNA exhibited significant homology to that of hepatocyte growth factor activator (HGFA). The results of Northern blot analysis indicated that liver, kidney, and pancreas expressed PHBP mRNA. The predicted structure of PHBP showed three epidermal growth factor (EGF) domains, a kringle domain and a serine protease domain, from its N-terminus, although HGFA has a fibronectin type II domain, an EGF domain, a fibronectin type I domain, an EGF domain, a kringle domain, and a serine protease domain, from its N-terminus.

Activation of Hepatocyte Growth Factor (HGF) by Endogenous HGF Activator Is Required for Metanephric Kidney Morphogenesis in Vitro

“…A human protein similar to HGFA has recently been identified (22,23) (GenBank TM accession number AAB46909). This molecule, hyaluronin-binding protein, was only 38% identical to murine HGFA at the amino acid level.…”

“…However, other members of the plasminogen activator family, coagulation factor XII, tissue plasminogen activator (tPA), and urokinase (uPA), are also weak activators of HGF (11,15,16). An HGFAlike molecule, PHBP, has recently been identified and could potentially, based on its sequence, activate HGF (22,23). Two of these enzymes, tPA and uPA, are also expressed in developing kidney (29).…”

Marburg I Polymorphism of Factor VII–Activating Protease

“…In the remaining population, follow-up was 96.5% (nϭ826) and 95.6% (nϭ684) complete, respectively. [1][2][3] Blood specimens for DNA extraction were drawn as part of the 1995 follow-up. Adequate polymerase chain reaction products were not obtainable in 16 samples, which left 810 (1995) and 678 (2000) subjects for the main analysis.…”

“…The plasmatic serine protease factor seven-activating protease (FSAP) has been recognized as a novel potent activator of prourokinase-dependent fibrinolysis. [1][2][3][4][5][6] We recently characterized a single nucleotide polymorphism (SNP) of FSAP, termed "Marburg I polymorphism," which impairs the capacity of FSAP to activate prourokinase without attenuating its potential contribution to the extrinsic coagulation pathway. 4 -6 This may drive hemostasis toward a prothrombotic state.…”

Factor VII-Activating Protease

“…FSAP was identified by a number of investigators and isolated either because of its affinity for immobilized hyaluronic acid 7 or its presence in commercial prothrombin complex concentrates. 8 The protein contains domain structures frequently associated with coagulation and fibrinolytic enzymes, including three "EGF"-like domains, a "kringle," and a serine protease domain.…”