Biosci. Biotechnol. Biochem., 74 (11), 2320–2322, 2010
Note
Inhibition of Plasma Hyaluronan-Binding Protein Autoactivation by Laccaic Acid Chikako S EKIDO, Naoko N ISHIMURA, Masayuki T AKAI, and Keiji H ASUMIy Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan Received May 13, 2010; Accepted July 26, 2010; Online Publication, November 7, 2010 [doi:10.1271/bbb.100373]
Plasma hyaluronan-binding protein (PHBP) is a serine protease implicated in proteolysis under inflammatory conditions. We identified laccaic acid, a widely used food coloring from scale insects, as a potent inhibitor of the protease in terms of both autoactivation of the PHBP proenzyme (IC50 = 0.35–0.55 g/ml) and the catalytic activity of the active enzyme (IC50 = 1.1 g/ml). Key words:
lac dye; laccaic acid; plasma hyaluronanbinding protein (PHBP); factor VII activating protease (FSAP)
Plasma hyaluronan-binding protein (PHBP),1) alternatively named factor VII activating protease (FSAP), is a serine protease implicated in both coagulation and fibrinolytic systems, as the enzyme catalyzes the activation of blood coagulation factor VII and the proenzyme form of urokinase-type plasminogen activator (u-PA).2,3) PHBP is present in plasma in a singlechain zymogen form (pro-PHBP), a 537-amino acid glycoprotein.1) The zymogen autoproteolytically converts to an active two-chain form via the cleavage at Arg290 –Ile291 .4) Although activation of pro-PHBP in vivo is observed under inflammatory conditions,5,6) no physiologically relevant enzyme responsible for pro-PHBP has been found. Alternatively, negatively charged molecules (such as heparin and RNA) and positively charged molecules (such as polyamines) dramatically promote pro-PHBP autoactivation.7–9) Hence it has been postulated that these molecules contribute to physiological pro-PHBP activation. The mechanism of the action of polyamines is to promote the formation of the proPHBP autoactivation complex,8) while heparin and RNA might act as a scaffold for the accumulation of proPHBP.9) Thus pro-PHBP activation can proceed through multiple mechanisms depending on the kind of pathophysiological stimuli.8,9) It has been suggested that PHBP plays roles in the regulation of inflammation,10) vascular function,11) neointima formation,12) liver fibrosis,13) and atherosclerosis.11,14) Therefore, a compound that regulates pro-PHBP activation might contribute to pharmacological control of the progression of these diseases. In screening for an inhibitor of pro-PHBP autoactivation, we identified bikaverin, a quinone metabolite from a
fungus, as a relatively potent inhibitor.15) However, bikaverin, at concentrations slightly higher than the concentration which affected pro-PHBP autoactivation, inhibited the enzymatic activities of other serine proteases.15) To identify a more selective inhibitor, we screened a panel of compounds related to bikaverin, and identified three anthraquinone compounds, purpurin,15) carminic acid,8) and a lac dye (a mixture of laccaic acids). In this report, we describe the action of laccaic acid. Figure 1 shows some of the results of a screening of compounds structurally related to bikaverin. In this screening, the compound to be tested and pro-PHBP (5 nM), isolated from human plasma,4,8) were incubated with spermidine (5 mM) and 0.1 mM of the substrate SPECTROZYME TH (STH) in buffer A (50 mM Tris–HCl, pH 7.4, 75 mM NaCl, 5 mM CaCl2 , and 0.05% w/v Tween 20) during monitoring A405 at 37 C. In this assay, lac dye showed potent inhibitory activity (IC50 = 0.65 mg/ml), along with related anthraquinones, carminic acid (IC50 = 7.4 mg/ml) and purpurin (IC50 = 1.7 mg/ml). Other quinone compounds tested were inactive (Fig. 1). To identify the lac dye component responsible for the inhibitory activity, we fractionated the dye on reverse phase HPLC. Most of the activity was associated with the major constituent, which was identified as laccaic acid A (LcA; Fig. 1) based on the following data: UV-visible (0.1 M HCl), max nm (") 284 (30,590), 490 (8,710); MALDI-TOF-MS, m=z 538.09 ðM þ HÞþ . The activity of LcA in inhibiting spermidine-induced proPHBP autoactivation (IC50 = 0.35 mg/ml) (Fig. 2A) was comparable to that of the lac dye mixture. Thus the activity of the lac dye was represented by LcA, which amounted to >90% of the dye ingredients, but this does not exclude the possibility that other components have inhibitory activity as well. As described above, pro-PHBP autoactivation proceeds through at least two pathways, autoactivation complex formation triggered by a polyamine (such as spermidine)8) and pro-PHBP accumulation on a scaffold (such as heparin).9) LcA was active in inhibiting heparin-induced autoactivation (IC50 = 0.45 mg/ml) as well as spermidine-induced autoactivation (Fig. 2A). Moreover, LcA inhibited non-induced pro-PHBP autoactivation (spontaneous autoactivation at a high pro-PHBP concentration) (IC50 = 0.55 mg/ml) and the
y To whom correspondence should be addressed. Tel: +81-42-367-5710; Fax: +81-42-367-5708; E-mail:
[email protected] Abbreviations: PHBP, plasma hyaluronan-binding protein; u-PA, urokinase-type plasminogen activator; STH, SPECTROZYME TH (H-D-hexahydrotyrosyl-Ala-Arg-p-nitroanilide); LcA, laccaic acid A
PHBP Inhibition by Laccaic Acid
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Table 1. Summary of Effects of LcA on the Activities of Several Proteases The activity of each protease was determined in triplicate in the presence of varying concentrations of LcA. IC50 values (mM) are shown. Pro-PHBP activation Spermidine- HeparinNoninduced induced induced 0.35 a
0.45
0.55
Metalloprotease
Other serine proteases
Active PHBP
Plasmin u-PA Thrombin
FVIIa
>10 (40%)a
>10 >10 (25%) (35%)
1.1
>10 (4%)
>10 (37%)
FXa
FXIIa Trypsin >10 (7%)
Cysteine protease
Aspartic protease
Bacillo- Cathepsin Cathepsin lysin MA B D
>10 (18%)
>10 (6%)
>10 (19%)
>10 (37%)
Numbers in parentheses represent % inhibition at 10 mg/ml.
O
OH
O
OH
CH3
O
OH
O
OH
O
OH
O
OH
O
OH
O
Cl H3C
O
O O
O
CH3
CH3
OH
O
1 (bikaverin) 0.17
IC50 (µg/ml)
Cl
O
OH
O
OH
O
2
3
4
5
6
> 100
> 100
> 100
> 100
> 100
OH
R OH O
OH
NH2
O
O
OH
CH3
OH
O
OH
O
HOOC
COOH O
OH
O
OH
HOOC OH OH
HO OH
O
O
O
OH
OH O
OH
OH HO
OH
7
8
9 (purpurin)
10 (carminic acid)
11 (lac dye)
> 100
> 100
1.7
7.4
0.65
Fig. 1. Effects of Quinone Compounds on Spermidine-Induced pro-PHBP Autoactivation. A panel of compounds related to bikaverin (1) was screened for inhibition of pro-PHBP autoactivation in the presence of spermidine. The results for the selected compounds are shown: 5-hydroxy-[1,4]naphthoquinone (2); plumbagin (5-hydroxy-2-methyl-[1,4]naphthoquinone) (3); 5,8-dihydroxy-[1,4]naphthoquinone (4); 2,3-dichloro-5,8-dihydroxy-[1,4]naphthoquinone (5); 9,10-dihydroxy-2,3-dihydro-anthracene-1,4-dione (6); 1-amino-anthraquinone (7); 6,11-dihydroxy-naphthacene-5,12-dione (8); purpurin (9); carminic acid (10); lac dye (11). R in the structure of lac dye represents –(CH2 )2 –NH–CO–CH3 (laccaic acid A), –(CH2 )2 –OH (laccaic acid B), or –CH2 –CH(NH2 )–COOH (laccaic acid C). IC50 values were obtained from triplicate determinations.
B
120 No inducer SPD-induced Heparin-induced Active PHBP
100 80
500
1 / v (nmol pNA -1 min ng)
PHBP autoactivation or PHBP activity (% of control)
A
60 40 20
0.8 400
0.4
300 200
0
100
LcA (µg/ml)
0
0 0
2
4
6
LcA (µg/ml)
8
10
-40
-20
0
20
40
60
1 / [STH] (mM-1)
Fig. 2. Inhibition of pro-PHBP Autoactivation and the Active Form of PHBP by LcA. A, Autoactivation of pro-PHBP was assayed by incubating 5 nM pro-PHBP in 50 ml of buffer A at 37 C in the presence of 5 mM of spermidine (SPD-induced) or 0.5 mg/ml of heparin (Heparininduced) as well as LcA at the indicated concentrations. After incubation for 20 min to allow pro-PHBP autoactivation, the reaction mixture received 5 ml of chromogenic substrate STH (1.1 mM) to measure kinetically the initial velocity of its hydrolysis by the resulting PHBP as the change in absorbance at 405 nm. The initial velocity was calculated from plots of absorbance versus time, which gave a straight line up to about 5 min. Non-induced pro-PHBP autoactivation was assayed by incubating 20 nM of the zymogen. Active PHBP was assayed by incubating the active form of PHBP (2 nM), prepared as described previously,10) with 0.1 mM STH in buffer A at 37 C in the presence of the indicated concentrations of LcA. B, PHBP inhibition by LcA was analyzed kinetically with varying concentrations of the substrate. The concentrations of LcA are shown to the right of each line. Values represent the mean SD for triplicate incubations.
activity of the active form of PHBP (IC50 = 1.1 mg/ml) (Fig. 2A). The dose-response curve for active PHBP inhibition was distinct from those for autoactivation. Full inhibition was not achieved even at 10 mg/ml, whereas nearly complete inhibition of autoactivation was observed at 3–5 mg/ml. Kinetic analysis suggested that LcA noncompetitively inhibited the active form of PHBP with respect to the substrate STH (Fig. 2B), with a Ki of 0:60 0:05 mg/ml. These results can be interpreted as indicating that LcA binds to an allosteric site of the enzyme, resulting in the formation of an enzyme-inhibitor complex that shows reduced activity toward the synthetic substrate STH, while the complex is completely inactive in the activation of pro-PHBP. To assess the selectivity of the action of LcA, its effects on the activities of a variety of proteases were examined (Table 1). The enzymes tested were serine proteases and other functionally different classes of proteases. LcA showed little inhibition of plasminogen activator u-PA, coagulation factor XIIa, and bacillolysin MA, a metalloprotease. Inhibition of plasmin, thrombin, coagulation factors VIIa and Xa, trypsin, cathepsin B, and cathepsin D was in a range of 18–40% at a LcA concentration of 10 mg/ml, 18 to 29 times as high as the IC50 values for pro-PHBP autoactivation (Table 1). It has been postulated that PHBP acts as a mediator of inflammation and vascular responses.10,11) It is likely that multiple mediators account for pro-PHBP activation in such complicated responses. Heparin and polyamines are
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candidate mediators, while the two kinds of mediators utilize different mechanisms. Polyamine (spermidine) induces self-assembly of pro-PHBP (autoactivation complex formation) by binding to its N-terminal anionic amino acid-rich region (NTR) and modulating the function of NTR, which can interact with the third epidermal growth factor domain (E3) of pro-PHBP.8) E3, which has a cluster of basic amino acids, plays a role in binding to anionic substances such as heparin.8,9) Heparin provides a scaffold for pro-PHBP accumulation via interaction with E3, and thus facilitates molecular recognition between pro-PHBP on the scaffold.9) Selective inhibitors such as carminic acid inhibit spermidineinduced formation of the autoactivation complex, but does not affect autoactivation induced by heparin.8) Thus carminic acid does not affect the execution of proteolytic activation, but rather abolishes prerequisite autoactivation complex formation. LcA inhibits the autoactivation induced by spermidine and by heparin. Therefore, it should inhibit the process of proteolysis. This is confirmed by the results of kinetic analysis using the active form of PHBP and a synthetic substrate (Fig. 2B). The results suggest that LcA-induced conformational change decreases the turnover rate of the enzyme. It should be noted that LcA causes complete inhibition of pro-PHBP autoactivation (proteolysis), in contrast with the partial inhibition of activity toward the synthetic substrate (amidolysis) (Fig. 2A). Thus LcA-induced conformational change might greatly affect catalysis on the protein substrate. Like LcA, an agent that can inhibit both processes of pro-PHBP autoactivation would be of use in suppressing the pro-PHBP activation induced by diverse pathophysiological stimuli.
Acknowledgments We thank Mr. Eisaku Yamamoto and Dr. Shingo Yamamichi for discussion. Human plasma for proPHBP isolation was provided by the Japanese Red Cross Society of Tachikawa.
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