Regulation Mechanism of the Serine Protease Activity of Plasma Hyaluronan Binding Protein.

Abstract

We earlier reported the purification of a novel hyaluronanbinding protein from human plasma with hyaluronan-conjugated Sepharose which we called plasma hyaluronan binding protein (PHBP).1) The result of the sequence analysis of the PHBP cDNA indicated that it has a similar structure to that of hepatocyte growth factor activator (HGFA), the serine protease which cleaves pro-hepatocyte growth factor (HGF) to the active hetero-dimer form. Indeed, we also detected the serine protease activity of PHBP with Boc-Phe-Ser-Argmethylcoumarine amide (MCA) as the synthetic substrate. PHBP was present in serum as the single-chain precursor form (pro-PHBP) of 70 kDa, however, the purified PHBP from human plasma with AC4 (anti-PHBP mouse monoclonal antibody)-conjugated Sepharose contained the singlechain form and some amount of the hetero-dimer form. The hetero-dimer form was composed of the 50-kDa N-terminal heavy chain and the 27-kDa C-terminal light chain, both of which were bridged by a disulfide linkage. During the incubation of the purified PHBP at 37°C, pro-PHBP completely converted to the hetero-dimer form, which possessed the serine protease activity, by auto-cleavage as well as factor XII of the coagulation system. 2-7) These results suggested that the proteases in the coagulation system were not involved in the activation of pro-PHBP and it was different from the activations of factor XII 8-10) and HGFA, 11) which were cleaved and activated during the coagulation. The results also suggested the presence of protease inhibitor which prevents the fragmentation of pro-PHBP in human serum. In this paper, we describe the purification and identification of the inhibitor for the serine protease activity of PHBP and the activation mechanism of pro-PHBP. MATERIALS AND METHODS MaterialsDextran sulfate (molecular weight of 500 kDa) was purchased from Pharmacia Biotech. Bovine brain phosphatidylethanolamine was obtained from Sigma. Kaolin was from Wako Pure Chemicals.Purification of the Active Form and the Precursor Form of PHBP The active hetero-dimer form of PHBP was purified from human plasma as described previously with AC4 (anti-PHBP mouse monoclonal antibody)-conjugated Sepharose. Briefly, human plasma (500 ml) was applied to the column of AC4-Sepharose (1 mg IgG/ml Sepharose, 50 ml). After washings with 0.5 M NaCl in 10 mM sodium phosphate, pH 7.0 (500 ml) and distilled water (100 ml), the bound proteins were eluted with 0.2 M glycine-HCl, pH 2.5. After neutralization with 1 M Tris, the contaminating IgG was removed by passing it through the column of anti-human IgG rabbit antibody-conjugated Sepharose. The single-chain precursor form of PHBP (pro-PHBP) was purified by the same procedure with the exception that every solution contained 1 mg/ml of FUT-175 (Torii Pharmaceutical Co.). The Pro-PHBP preparation was dialyzed against PBS to remove FUT-175 at 4°C overnight.Measurement of the Inhibitor Activity or the Activator Activity for PHBP To measure the inhibitor activity for PHBP, the active form of PHBP (700 ng) was incuba...

Hepatic Injury-Specific Conversion of Mouse Plasma Hyaluronan Binding Protein to the Active Hetero-Dimer Form.

“…2) and a time-course experiment (Fig. 3) using CCl 4 . The amounts of the 45 and 30-kDa fragments of active PHBP increased CCl 4 -dose dependently (Fig.…”

“…4) C1 inhibitor complexed with active PHBP and inhibited its protease activity effectively. The active PHBP in plasma may be inactivated immediately by C1 inhibitor.…”

Elucidation of Crucial Structures for a Catechol-Based Inhibitor of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease) Autoactivation

“…Alternatively, negatively charged molecules (such as heparin and RNA) and positively charged molecules (such as polyamines and histones) dramatically promote pro-PHBP autoactivation. [9][10][11][12] Hence it has been postulated that these molecules contribute to the regulation of physiological pro-PHBP activation.…”

Inhibitors of Autoactivation of Plasma Hyaluronan-Binding Protein (Factor VII Activating Protease)

“…13,14) Both negatively charged molecules such as heparin and RNA and positively charged molecules such as polyamines dramatically promote pro-PHBP autoactivation. [15][16][17][18] A polyamine such as spermidine induces self-assembly of pro-PHBP (autoactivation complex formation) by binding to and modulating the function of the N-terminal region, which can interact with the third epidermal growth factor domain of pro-PHBP. 16,18) The third epidermal growth factor domain, which has a cluster of basic amino acids, plays a role in binding to anionic substances such as heparin and RNA.…”