A dimeric high-molecular-weight chymotrypsin inhibitor with antitumor and HIV-1 reverse transcriptase inhibitory activities from seeds of Acacia confusa

ARTICLE IN PRESS Phytomedicine 17 (2010) 621–625

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A dimeric high-molecular-weight chymotrypsin inhibitor with antitumor and HIV-1 reverse transcriptase inhibitory activities from seeds of Acacia confusa S.K. Lam n, T.B. Ng n School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

a r t i c l e in fo

Keywords: Acacia confusa Chymotrypsin inhibitor Antitumor HIV-1 reverse transcriptase inhibitory

abstract A dimeric 70-kDa chymotrypsin inhibitor with substantial N-terminal sequence homology to serine protease inhibitors was isolated from Acacia confusa seeds. The chymotrypsin inhibitor was purified using a protocol that entailed ion exchange chromatography on Q-Sepharose, SP-Sepharose and fast protein liquid chromatography-gel filtration on Superdex 75. The chymotrypsin inhibitor was unadsorbed on both Q-Sepharose and SP-Sepharose. Its chymotrypsin inhibitory activity was stable from pH 3 to 10 and from 0 to 50 1C. It exerted antiproliferative activity toward breast cancer MCF-7 cells with an IC50 of 10.774.2 mM. It inhibited HIV-1 reverse transcriptase with an IC50 of 8 71.5 mM. It was devoid of antifungal activity toward a variety of fungal species. The distinctive features of the chymotrypsin inhibitor included dimeric nature, a high molecular mass, lack of trypsin inhibitory activity, highly potent HIV-1 reverse transcriptase inhibitory activity, specific antitumor activity and relatively high pH-stability. & 2009 Elsevier GmbH. All rights reserved.

Introduction Protease inhibitors have been isolated or found in a diversity of organisms, including human (Lin et al. 1999), other animals (Imamura and Kambara 1989), and plants (Scarafoni et al. 2008). They have attracted the attention of many investigators in view of their importance. The proliferation of over 60 cancer cell lines has been reported to be inhibited by protease inhibitors. Clinical testing of nelfinavir has been performed in cancer patients (Bernstein and Dennis 2008). Populations consuming a diet rich in legumes, which are known to have a high content of trypsin inhibitors, have a reduced incidence of malignant disease (Losso 2008). Furthermore, telaprevir (Gentile et al. 2009) and boceprevir (Mederacke et al. 2009) are used for the treatment of hepatitis C virus infection. Different combinations of protease inhibitors are used for treatment of AIDS: lopinavir/ritonavir (Barragan and Podzamczer 2008), lopinavir/ritonavir or ritonavir-boosted atazanavir (Elliott and Pujari 2008). Boosted saquinavir and atazanavir are generally safe for mother and child during pregnancy (van der Lugt et al. 2008). However, drug resistance to HIV-1 protease inhibitors has been found (Nalam and Schiffer 2008). Toxicity-associated adverse events still remain a major concern when prescribing HIV protease

n

Corresponding authors. Tel.: + 852 26098031; fax: + 852 26035123. E-mail addresses: [email protected] (S.K. Lam), [email protected] (T.B. Ng). 0944-7113/$ - see front matter & 2009 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2009.10.005

inhibitors (Boesecke and Cooper 2008). So, it is important to identify more protease inhibitors for different applications. The existing literature on the constituents of Acacia confusa consists of only a few reports. The bark (Chang et al. 2001; Buchanan et al. 2007; Tung et al. 2007; Tung et al. 2009a, 2009b), heartwood (Chang et al. 2001; Wu et al. 2005; Wu et al. 2008b), leaves, (Lee et al. 2000) and flower (Wu et al. 2008a) contain phytochemicals. A seed trypsin-chymotrypsin inhibitor (Lin et al. 1991) is the only proteincaceous component isolated from A. confusa. In view of the diversity of protease inhibitors and the meager information about the protein constituents of A. confusa seeds that is available, we undertook the present investigation to isolate a protease inhibitor from this source. The results indicate that the isolated chymotrypsin inhibitor possesses some distinctive characteristics. Materials and methods Seeds were collected locally from A. confusa. The seeds have been authenticated by Professor Shiuying Hu, Honorary Professor of Chinese Medicine, The Chinese University of Hong Kong, and kept with the voucher number NG2009008 in Lab 302, Basic Medical Science Building, The Chinese University of Hong Kong. The seeds were blended in distilled water (10 ml/g) using a Waring blender before centrifugation (14000 g, 30 min, 4 1C). NH4HCO3 buffer (pH 8.8) was added to the resulting supernatant until the concentration reached 10 mM. The supernatant was then

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applied to a 13 cm  5 cm column of Q-Sepharose (GE Healthcare) which had previously been equilibrated with and was then eluted with 10 mM NH4HCO3 buffer (pH 8.8). After removal of unadsorbed proteins, the column was eluted with 1000 mM NaCl added to the NH4HCO3 buffer. The unadsorbed fraction was extensively dialyzed against 10 mM NH4OAc buffer (pH 4.5), then subjected to ion exchange chromatography on a 14 cm  5 cm column of SP-Sepharose (GE Healthcare) in 10 mM NH4OAc buffer (pH 4.5). After removal of unadsorbed proteins, the column was eluted with 1 M NaCl added to the NH4OAc buffer. Again the unadsorbed fraction was collected, lyophilized and further fractionated by gel filtration in a Superdex 75 HR 10/30 column (GE Healthcare) in 10 mM NH4HCO3 buffer (pH 8.8) using an AKTA Purifier (GE Healthcare). The first peak represented purified chymotrypsin inhibitor. Molecular mass determination using sodium dodecyl sulfate– polyacrylamide gel electrophoresis, gel filtration, and N-terminal amino acid sequencing The purified chymotrypsin inhibitor was subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) under reducing conditions (Laemmli and Favre 1973). The gel was stained with Coomassie Blue R-250. Gel filtration on a fast protein liquid chromatography Superdex 75 column HR 10/30 (GE Healthcare) using an AKTA Purifier (GE Healthcare) was conducted to determine the molecular mass of the chymotrypsin inhibitor. The column had been calibrated with molecular mass markers, including Blue Dextran 2000 (to determine void volume), bovine serum albumin (67 kDa), ovalbumin (43 kDa), chymotrypsinogen A (25 kDa), myoglobulin (17.6 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 kDa) and vitamin B12 (1.3 kDa) (GE Healthcare). The N-terminal sequence of the chymotrypsin inhibitor was determined as described by Lam and Ng (2009). Assay for chymotrypsin or trypsin inhibitory activity The assay was modified from Lin et al. (1991). The sample and chymotrypsin or trypsin were mixed in the ratio of 10:1, 5:1, 3:1, 2:1, 1:1, 0.5:1 with a final volume of 40 ml. The mixtures were incubated at room temperature for 15 min. Three hundred and sixty microliters of a 1% casein solution in 0.1 M Tris–HCl buffer (pH 7.4) were added, and then incubated at 37 1C for 15 min before 0.4 ml 10% trichloroacetic acid was added to terminate the reaction. After centrifugation, the absorbance of the supernatant which reflects the amount of casein fragments was measured at 280 nm. Assay of antifungal activity The antifungal activity of chymotrypsin inhibitor was monitored with the agar diffusion assay; 200 mg of protein was added to test the inhibition effect of different fungi. The pathogenic fungi species used included Mycosphaerella arachidicola, Fusarium oxysporum, Helminthosporium maydis, Valsa mali and Rhizoctonia solani (Lam and Ng, 2009). Nystatin (Sigma) was used as a positive control. Assay of HIV-1 reverse transcriptase inhibitory activity. The assay of the chymotrypsin inhibitor for the ability to inhibit HIV-1 reverse transcriptase was carried out by using an enzyme-linked immunosorbent assay kit from Boehringer Mannheim (Germany) (Lam and Ng 2009). Brassica campestris lipid transfer protein (Lin et al. 2007) was used as a positive control.

Assay of antiproliferative activity. The assay of the antiproliferative activity of the isolated chymotrypsin inhibitor was carried out by testing its inhibitory effect on the growth of human hepatoma HepG2 cells and human breast cancer MCF-7 cells as described by Lam et al. (2009). The cells were cultured in RPMI-1640 medium supplemented with 10% fetal calf serum and 1% penicillin-streptomycin, in a humidified atmosphere of 5% CO2 at 37 1C. The cells (5  103 cells/100 ml/well) were seeded in a 96-well culture plate and serial dilutions of a solution of the chymotrypsin inhibitor, or doxorubicin (as positive control) in 100 ml medium were added. A solution of medium only was added as negative control. After incubation for 48 h, the cells were harvested, standard MTT assay was performed to determine the level of its inhibitory activity. All reported values are the means of triplicate samples.

Results and discussion Isolation of A. confusa chymotrypsin inhibitor Seeds (96 grams) collected from A. confusa trees were homogenized in 1 liter of 10 mM NH4HCO3 buffer. The homogenate was centrifuged and the supernatant saved. Ion exchange chromatography of the seed extract on Q-Sepharose produced an unadsorbed fraction (Q1) and one adsorbed fractions (Q2) (Fig. 1A). The flowthrough fraction, Q1, was separated on SPSepharose into an unadsorbed fraction (SP1) and an adsorbed fraction (SP2) eluted with 1000 mM NaCl (Fig. 1B). SP1 was subsequently chromatographed on Superdex 75. Five fractions, S1, to S5, were obtained (Fig. 1C). A. confusa chymotrypsin inhibitor resided in fraction S1. Fraction S1 was re-chromatographed on Superdex 75. It was eluted as a single homogeneous peak with the same elution volume as before corresponding to a molecular mass of 70 kDa (not shown). It demonstrated a single 38-kDa band in SDS-PAGE (Fig. 2). Earthworm (Wojtaszek et al. 2006) and Schizolobium parahyba (Teles et al., 2004) chymotrypsin inhibitors are adsorbed on an anion exchanger (Mono Q) and a cation exchanger (SP-Sephadex), respectively. A. confusa trypsinchymotrypsin inhibitor has been purified by gel filtration (Sephadex G-50) and affinity chromatography (TrypsinSepharose 4B) (Lin et al. 1991). It is interesting that A. confusa chymotrypsin inhibitor is unabsorbed on both a strong anion (Q-Sepharose) and a strong cation exchanger (SP-Sepharose). A summary of the purification of A. confusa chymotrypsin inhibitor is included in Table 1. The N-terminal sequence of A. confusa chymotrypsin inhibitor is homologous to other protease inhibitors (Table 2). The molecular masses of chymotrypsin is 25 kDa. The molecular masses of most chymotrypsin inhibitors are less than 10 kDa. The molecular mass of chymotrypsin inhibitors from Bombyx mori (Zhao et al., 2007) and Naja atra venom (Zhou et al. 2004) are 7.2 and 6.4 kDa, respectively. Some chymotrypsin inhibitors have a molecular mass between 10 and 25 kDa. Some examples are from Lens culinaris (16 kDa) (Cheung and Ng 2007), Schizolobium parahyba (20 kDa) (Teles et al. 2004), and Psophocarpus tetragonolobus (21 kDa) (Kortt, 1980). To the best of our knowledge, only Oesophagostomum radiatum chymotrypsin inhibitor (32 kDa) (Willadsen 1977), Erythrina variegata chymotrypsin inhibitor (40 kDa) (Iwanaga et al. 1998), and A. confusa trypsin-chymotrypsin inhibitors (42 kDa) (Lin et al. 1991) have a molecular mass larger than chymotrypsin (25 kDa). The A. confusa chymotrypsin inhibitor isolated in the present study is the largest (70 kDa) encountered thus far. Amongst all the chymotrypsin inhibitors, only a few of them are dimeric. Ecotin, from E. coli., is homodimeric (Chung et al. 1983). A. confusa

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Fig. 1. (1A) Ion exchange chromatography of A. confusa extract on Q-Sepharose. The chymotrypsin inhibitor was confined to the unbound fraction Q1. (1B) Ion exchange chromatography of fraction Q1 on SP-Sepharose. The chymotrypsin inhibitor was found in the unbound fraction SP1. (1C) Size exclusion chromatography of fraction SP1 on Superdex 75. The fraction S1 represented the chymotrypsin inhibitor.

Table 1 Yields of various active chromatographic fractions obtained at different stages of purification (from 96 g seeds). Column

Q-Sepharose SP-Sepharose Superdex 75

Chromatographic fraction Crude Extract Q1 SP1 S1 (chymotrypsin inhibitor)

Yield (mg protein) 780 170 80 9

trypsin-chymotrypsin inhibitor is heterodimeric (Lin et al. 1991). The N-terminal amino acid sequences of A. confusa trypsinchymotrypsin inhibitor (Lin et al. 1991) and A. confusa chymotrypsin inhibitor are completely different (Table 2).

Biological activities of A. confusa chymotrypsin inhibitor

Fig. 2. SDS-polyacrylamide gel electrophoresis. Left lane: Fraction S1 representing A. confusa chymotrypsin inhibitor. Right lane: Molecular mass markers from GE Healthcare including phosphorylase b (94 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa) and soybean trypsin inhibitor (20 kDa), and a-lactalbumin (14.4 kDa).

The molar ratio of inhibition between A. confusa chymotrypsin inhibitor and alpha-chymotrypsin was 2:1, which gave about 100% inhibition (Fig. 3). The molar ratio of chymotrypsin inhibitor to chymotrypsin is usually 1:1, Schizolobium parahyba chymotrypsin inhibitor (da Silva et al. 2008) is one of the examples. Psophocarpus tetragonolobus inhibitor has a ratio 1:2 (Kortt, 1980). Both A. confusa trypsin-chymotrypsin inhibitor (Lin et al. 1991) and A. confusa chymotrypsin inhibitor have an inhibitor to chymotrypsin ratio of 2:1. This is is probably due to their dimeric structure. As the inhibition is not affected by an excess amount of substrate, the mode of inhibition is non-competitive. A. confusa chymotrypsin inhibitor did not have inhibitory effect on trypsin up to a molar ratio of 10:1 (Fig. 3), which is similar to

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Table 2 Comparison of N-terminal amino acid sequence of A. confusa chymotrypsin inhibitor with other related proteins (Results of BLAST search). Residue no.

A. confusa chymotrypsin inhibitor Taeniopygia guttata serine (or cysteine) protease inhibitor Bos taurus serpin peptidase inhibitor A. confusa trypsin- chymotrypsin inhibitor (A chain) (B chain)

1 74 86 1 1

Sequence

AMMKAEVDEMLPGAA NMMKAEVDLYLPKLK NMGKREVDLYLPRFK KELLDADGDILRNGG DDESCKDLGISIDDE

% Homology

Total no. of residues in protein

– 60 40 – –

 720 189 394 135  128

100 90

Trypsin

80 % Inhibition

Chymotrypsin

Chymotrypsin inhibitor DOX

70 60 50 40 30

0.5 to 1

1 to 1

2 to 1

3 to 1

20

5 to 1

100 90 80 70 60 50 40 30 20 10 0

10 to 1

% Inhibition

Residues identical to those of A. confusa chymotrypsin inhibitor are underlined.

10 0 0.2

Inhibitor to protease ratio

earthworm protease inhibitor (Wojtaszek et al. 2006). Caesalpinia bonduc (Bhattacharyya et al., 2007), broad bean (Ye et al. 2001) and A. confusa (Lin et al., 1991) trypsin-chymotrypsin inhibitors are able to inhibit both trypsin and chymotrypsin. Other inhibitors like wheat protease inhibitor can inhibit both subtilisin and chymotrypsin (Di Gennaro et al. 2005). The activity of the A. confusa chymotrypsin inhibitor was stable throughout the pH range 3–10. Only 60% activity remained at pH 2 and pH 11. No activity remained at pH 0, 1, 12–14. It was stable in the temperature range 0–50 1C. Only 70%, 40% and 0% activity remained at 60 1C, 70 1C, and 80 1C–100 1C, respectively. A. confusa chymotrypsin inhibitor has a relatively high pH stability, albeit modest thermostability. Schizolobium parahyba chymotrypsin inhibitor (Teles et al. 2004) shows a higher pH-stability (from pH 2–pH 12) and thermostability (up to 70 1C). It is probably due to that fact that they have a smaller molecular size than A. confusa chymotrypsin inhibitor. A. confusa chymotrypsin inhibitor and doxorubicin inhibited proliferation of MCF-7 tumor cells with an IC50 of 10.7 74.2 mM and 8.772.2 mM, respectively (Fig. 4). But A. confusa chymotrypsin inhibitor could not inhibit the proliferation of HepG2 cells when tested up to 100 mM (not shown). A. confusa chymotrypsin inhibitor displays specific antitumor activity, it is similar to other protease inhibitors which can inhibit the proliferation of different cancer cell lines (Bernstein and Dennis 2008), although some inhibitors, e.g. Vigna mungo protease inhibitor (Cheung and Ng 2009), are devoid of this activity. A. confusa trypsin-chymotrypsin inhibitor has not been tested for such biological activity (Lin et al. 1991). A. confusa chymotrypsin inhibitor inhibited HIV-1 reverse transcriptase with an IC50 of 871.5 mM (Fig. 5). There was almost complete inhibition at 100 mM. The inhibitory activity is highly potent when compared with other protease inhibitors.

0.6

0.8 1.0 1.2 1.4 log concentration (µM)

1.6

1.8

Fig. 4. Effects of A. confusa chymotrypsin inhibitor on viability of breast cancer MCF-7 cells. The viability of MCF-7 cells after incubation for 48 h in the presence of various concentrations of A. confusa chymotrypsin inhibitor in RPMI medium was determined as described in Materials and methods. Viability of cells in RPMI medium only was taken as negative control. Values are expressed as mean 7 SD (n =3). The IC50 of A. confusa chymotrypsin inhibitor and doxorubicin (DOX) were 10.7 74.2 mM and 8.7 72.2 mM, respectively (mean 7SD, n =3). The results shown here were from one of the three experiments.

100 90 80 % Inhibition

Fig. 3. Inhibitory effect of A. confusa chymotrypsin inhibitor toward chymotrypsin and trypsin. The minimum molar ratio of chymotrypsin inhibitor to alphachymotrypsin to produce 100% inhibition was 2:1. It did not have inhibitory effect on trypsin up to a molar ratio of 10:1.

0.4

70 60 50 40 30 20 10 0 0.0

0.5 1.0 1.5 2.0 log concentration of chymotrypsin inhibitor (µM)

Fig. 5. The dose-dependent inhibitory effect of A. confusa chymotrypsin inhibitor on HIV-1 reverse transcriptase indicated as percent inhibition as compared to a control without control. Values are represented as the mean7 SD (n =3). The IC50 was 8 71.5 mM (mean 7SD, n= 3). The results shown here were from one of the three experiments.

Some serine protease inhibitors have only slight anti-HIV-1 reverse transcriptase activity, e.g. IC50 of V. mungo inhibitors is in the millimolar range (Cheung and Ng 2009). A. confusa trypsinchymotrypsin inhibitor has not been tested for such biological activity (Lin et al. 1991). A. confusa chymotrypsin inhibitor could not inhibit any fungi studied when tested up to 200 mg in the agar diffusion assay (not shown). On the contrary, some serine proteases inhibitors exert an

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