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Passiflin, a novel dimeric antifungal protein from seeds of the passion fruit.

Abstract

The intent was to isolate an antifungal protein from seeds of the passion fruit (Passiflora edulis) and to compare its characteristics with other antifungal proteins and bovine [beta]-lactoglobulin in view of its N-terminal amino acid sequence similarity to [beta]-lactoglobulin. The isolation procedure entailed ion-exchange chromatography on Q-Sepharose, hydrophobic interaction chromatography on Phenyl-Sepharose, ion-exchange chromatography on DEAE-cellulose, and FPLC-gel filtration on Superdex 75. The isolated 67-kDa protein, designated as passiflin, exhibited an N-terminal amino acid sequence closely resembling that of bovine [beta]-lactoglobulin. It is the first antifungal protein found to have a [beta]-lactoglobulin-like N-terminal sequence. Its dimeric nature is rarely found in antifungal proteins. It impeded mycelial growth in Rhizotonia solani with an [IC.sub.50] of 16 [micro]M and potently inhibited proliferation of MCF-7 breast cancer cells with an [IC.sub.50] of 15 [micro]M. There was no cross-reactivity of passiflin with anti-[beta]-lactoglobulin antiserum. Intact [beta]-lactoglobulin lacks antifungal and antiproliferative activities and is much smaller in molecular size than passiflin. However, it has been reported that hydrolyzed [beta]-lactoglobulin shows antifungal activity. The data suggest that passiflin is distinct from [beta]-lactoglobulin.

[C] 2009 Elsevier GmbH. All rights reserved.

Keywords: Antifungal protein; Passiflora edulis; [beta]-Lactoglobulin

Introduction

Passiflora edulis is a plant belonging to the family Passifloraceae. Various parts of this plant are biologically active. Its leaf extract exerts an antioxidant action (Ferreres et al. 2007) while its rind extract produces an antihypertensive effect (Ichimura et al. 2006; Tapp et al. 2008). The fruit extract displays anti-inflammatory (Vargas et al. 2007; Montanher et al. 2007), anxiolytic (Barbosa et al. 2008; Coleta et al. 2006), and antioxidant (Talcott et al. 2003) activities. In addition, the fruit extract can heal open wounds (Garros Ide et al. 2006), colonic anastomosis (Bezerra et al. 2006), gastric sutures (Silva et al. 2006), abdominal wall wound (Gomes et al. 2006) and bladder wound (Goncalves Filho et al. 2006) in the rat model.

Glycosides (Christensen and Jaroszewski 2001), cycloartane triterpenoids (Yoshikawa et al. 2000a, b), saponins (Yoshikawa et al. 2000a), low-methoxyl pectin (Yapo and Koffi 2006), hydroxynitrilases (Asano et al. 2005), and antifungal proteins resembling 2S albumin in partial amino acid sequence (Agizzio et al. 2003, Pelegrini et al. 2006) have been reported from P. edulis. In view of the relatively scanty information on proteinaceous constituents of P. edulis, we undertook the present investigation to isolate an antifungal protein from the seeds of this plant.

In order to protect themselves from assault of pathogenic fungi, living organisms produce a variety of molecules including antifungal proteins. In plants, antifungal proteins have been isolated from a variety of tissues including fruits (Wang and Ng 2002a), seeds (Wang and Ng 2001a, b), bulbs (Wang and Ng 2002b), rhizomes (Wang and Ng 2005), and roots (Lam and Ng 2001). These antifungal proteins exhibit a wide range of molecular masses and amino acid sequences.

The antifungal protein isolated from P. edulis in this study, designated as passiflin, has an N-terminal sequence closely resembling that of the whey protein [beta]-lactoglobulin. The various biochemical characteristics and biological activities of this novel antifungal protein are presented herein in comparison with other antifungal proteins and bovine [beta]-lactoglobulin.

Materials and methods

Materials

Fresh seeds (100 g) were collected from P. edulis (passion fruits) purchased from a local vendor. Rabbit-anti-bovine-[beta]-lactoglobulin antiserum, bovine [beta]-lactoglobulin, and nystatin were obtained from Sigma Chemical Company, St. Louis, Missouri, USA The fungi were provided by Department of Microbiology, China Agricultural University, China. Q-Sepharose, Phenyl-Sepharose, DEAE-cellulose, and Superdex 75 were from GE Healthcare, Hong Kong.

Isolation of antifungal protein

The crude extract of P. edulis seeds was chromato-graphed on a 5 cm x 10 cm column of Q-Sepharose in 20 mM Tris-HCl buffer (pH 7.4). Unadsorbed proteins were eluted with the same buffer to yield fraction Ql while adsorbed proteins were eluted stepwise, first with 0.1 M NaCl in the Tris-HCl buffer to yield fraction Q2, and then with 0.5 M NaCl added to the Tris-HCl buffer to yield fractions Q3, Q4, and Q5. Fraction Q4 in 20 mM Tris-HCl buffer (pH 7.4) containing 1.5 M ammonium sulfate was subjected to hydrophobic interaction chromatography on a 2.5 cm x 10 cm column of Phenyl-Sepharose. The column had been equilibrated with 20 mM Tris-HCl buffer (pH 7.4) containing 1.5 M ammonium sulfate. After unadsorbed proteins had come off the column as fraction PS1, the column was eluted with 20 mM Tris-HCl buffer (pH 7.4) to give fraction PS2. Fraction PS2 was further purified on a DEAE-cellulose column in 20 mM Tris-HCl buffer (pH 7.4). After elution of unadsorbed proteins (Dl), the column was eluted with a 0 to 1 M linear NaCl concentration gradient to yield fractions D2 and D3. Fraction D2 was subjected to final purification on a Superdex 75 column in 150 mM [NH.sub.4][HCO.sub.3] buffer (pH 7.4). The first peak (SI) constituted purified antifungal protein which was designated as passiflin.

Protein determination

Protein concentration was determined by the dye-binding method (Bio-Rad) using bovine serum albumin as a standard.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)

It was conducted according to the method of Laemmli and Farve (1973). After electrophoresis, the gel was stained with Coomassie Brilliant Blue. The molecular mass of passiflin was determined by comparison of its electrophoretic mobility with those of molecular mass marker proteins from GE Healthcare.

Determination of native molecular weight by gel filtration

Gel filtration on a fast protein liquid chromatography (FPLC)-Superdex 75 column, which had been calibrated with molecular mass markers, including Blue Dextran 2000, aldolase (158 kDa), BSA (67kDa), ovalbumin (43kDa), chymotrypsinogen A (25 kDa), myoglobulin (17.6 kDa), ribonuclease A (13.7 kDa), aprotinin (6.5 Da), and vitamin B12 (1.3 kDa) (GE Healthcare), was conducted to determine the molecular mass of the protein (Wang and Ng 2001b).

N-terminal amino acid sequence analysis of passiflin

It was conducted by using a Hewlett-Packard HP G1000A Edman degradation unit and an HP 1000 high-performance liquid chromatography (HPLC) system (Wang and Ng 2001b).

Assay of antifungal activity

The assay for antifungal activity was executed using 100 mm x 15 mm petri plates containing 10 ml of potato dextrose agar. After the mycelial colony had developed, sterile blank paper disks (0.625 cm in diameter) were placed around and at a distance of 1 cm away from the rim of the mycelial colony. An aliquot of passiflin, bovine [beta]-lactoglobulin or nystatin (as positive control) in 20 mM phosphate-buffered saline (pH 6.0) was introduced to a disk. The plates were incubated at 23 [degrees]C until mycelial growth had enveloped peripheral disks containing the control (buffer) and had produced crescents of inhibition around disks containing samples with antifungal activity. The fungal species tested included Rhizoctonia solani, Mycosphaerella arachidicola, andFusarium oxysporum. The [IC.sub.50] was determined as described in Wong and Ng (2005b).

Assay of antiproliferative activity on tumor cell lines

Breast cancer MCF-7 cells (ATCC) and HepG2 cells (ATCC) were suspended in RPMI medium and adjusted to a cell density of 5 x [10.sup.4] cells/ml. A 100 [micro]l aliquot of this cell suspension was seeded in a well of a 96-well plate, followed by incubation at 37 [degrees]C for 24 h. Different concentrations of passiflin, bovine [beta]-lactoglobulin, or doxorubicin (as positive control) in 100 [micro]l RPMI medium were then added to the wells and incubated for 48 h. Thirty microliters of 5 mg/ml [3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide] [MTT] (Sigma) in phosphate-buffered saline were spiked into each well, and the plates were incubated for 4h. After removal of the solution in each well, dimethyl sulfoxide (150 [micro]l) was added to each well to dissolve the MTT-formazan at the bottom of the well. After 10 min, absorbance at 595 nm was measured by using a microplate reader (Lam and Ng 2001).

Assay of hemagglutinating and ribonuclease activities

The assays were conducted as described in Lam and Ng (2001), Wong and Ng (2005a). Concanavaiin A and porcine pancreatic RNase (Sigma) were used as positive controls in hemagglutination and ribonuclease assays, respectively.

Assay for HIV-1 reverse transcriptase inhibitory activity

The assay for HIV reverse transcriptase inhibitory activity was carried out according to instructions supplied with the ELISA kit from Boehringer Mannheim (Germany) (Wong and Ng 2005b). Brassica campestris lipid transfer protein (Lin et al. 2007) was used as positive control.

Western blot analysis

Passiflin and [beta]-lactoglobulin were subjected to electrophoresis, and then transferred to an immobilon-P transfer membrane (Millipore, MA, USA) using a semidry transfer system (Bio-Rad). The membrane was blocked with 5% skim milk in TBS-T (Tris-buffered saline with 0.1% Tween 20). The membrane was incubated with a rabbit-anti-bovine-[beta]-lactoglobulin polyclonal antiserum (1:500 dilution) overnight at 4[degrees]C, followed by several washes with TBS-T. The signal was developed by incubation with anti-rabbit horseradish peroxidase-conjugated secondary antibodies (GE Healthcare) for 1.5 h, followed by several washes with TBS-T. Enhanced chemiluminescence detection reagents (GE Healthcare) were used for detecting the signal (Lai et al. 2006).

Results and discussion

Ion-exchange chromatography of P. edulis seed extract on Q-Sepharose produced a very large unad-sorbed fraction (Ql) and two adsorbed fractions (Q2 eluted with 0.1 M NaCl, and Q3, Q4, and Q5 eluted with 0.5 M NaCl). Antifungal activity resided only in fraction Q4 (Fig. 1A). This fraction was separated on Phenyl-Sepharose into an unadsorbed fraction (PS1) devoid of antifungal activity and an adsorbed fraction (PS2) with antifungal activity (Fig. IB). Fraction PS2 was subsequently resolved on DEAE-cellulose into a large unadsorbed fraction (Dl) and two smaller adsorbed fractions (D2 and D3). Antifungal activity was confined to the adsorbed fraction D2 eluted within the 0-0.6 M NaCl gradient (Fig. 1C). This active fraction D2 was subjected to final purification on Superdex 75. Four fractions, S1-S4, were obtained (Fig. ID). Antifungal activity resided in the first fraction (SI). The first fraction demonstrated a single 34-kDa band in SDS-PAGE (Fig. 2) and a single 67-kDa peak upon rechromatography on Superdex 75 (not shown). A summary of purification of the antifungal protein is presented in Table 1. The N-terminal amino acid sequence of the antifungal protein was highly homologous to bovine [beta]-lactoglobulin, but was distinct from sequences of published antifungal proteins (Table 2).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]
Table 1. Yields of antifungal protein from 100g fresh Passiflora edulis
seeds at different stages of purification.

Column            Chromatographic fraction  Yield (mg)

_                      Crude Extract           2050
Q-Sepharose            Q4                       830
Phenyl-Sepharose       PS2                      340
DEAE-cellulose         D2                        32
Superdex 75            SI (passiflin)             5

Table 2. Comparison of N-terminal amino acid sequence of passiflin with
mammalian [beta]-lactoglobulins (results of BLAST search) and
previously isolated antifungal peptide and protein from passion fruit.

                             Residue no.  Sequence

Passiflin                        1        AFLDIQKVAGTWYSLA

Bovine [beta]-lactoglobulin      8        KGLDIQKVAGTWYSLA

Bubalus bubalis [beta]-          8        KGLDIQKVAGTWYSLA
lactoglobulin

Capra hircus [beta]-             8        KGLDIQKVAGTWYSLA
lactoglobulin

Rangifer tarandus [beta]-        8        KDLDVQKVAGTWYSLA
lactoglobulin

Mouflon [beta]-                  8        KGLDIQKVAGTWYHLA
lactoglobulin

2S albumin-like antifungal       1        QSERFEQQMQGQDFSHDERFLSQAA
peptide [from reference 20]

2S albumin-like antifungal       1        PSERCRRQMQGDFS
protein [from reference 19]

Residue number 1 in passiflin is "A".


It inhibited mycelial growth in R. solani (Fig. 3A) with an [IC.sub.50] value of 16 [+ or -] 0.9 [micro]M (n = 3) (Fig. 3B), but not in M. arachidicola and F. oxysporum when tested up to 100 [micro]M. The antifungal protein inhibited proliferation of MCF-7 tumor cells with an [IC.sub.50] near 15 [+ or -] 1.2 [micro]M (n = 3) (Fig. 4), but there was no inhibition toward HepG2 cells when tested up to 100 [micro]M. Both the antifungal protein and bovine [beta]-lactoglobulin were devoid of haemagglutinating activity, ribonuclease activity, and inhibitory activity on HIV-1 reverse transcriptase when tested up to 100 [micro]M (not shown). For comparison, bovine [beta]-lactoglobulin was tested for the various aforementioned activities and found to be lacking in these activities (Figs. 3A and 4). The comparison of pharmacological activities of passiflin to various positive controls used in this study, including doxorubicin, nystatin and B. campestris lipid transfer protein, is summarized in Table 3. Western blotting of bovine [beta]-lactoglobulin using a rabbit-anti-bovine-[beta]-lactoglobulin antiserum yielded positive results. In contrast, there was no cross-reactivity of passiflin with the same antiserum (Fig. 5).

[FIGURE 3 OMITTED]

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]
Table 3. Comparison of biological potencies of passiflin, doxorubicin,
nystatin, and B. campestris lipid transfer protein.

                                    Passiflin        Doxorubicin
                                    ([IC.sub.50]     ([IC.sub.50]
                                    in [micro]M)     in [micro]M)

Antiproliferative activity against --               11.5 [+ or -] 2.1
HepG2 cells

Antiproliferative activity against  15 [+ or -] 1.2   4.3 [+ or -] 1.6
MCF-7 cells

Antifungal activity against         16 [+ or -] 0.9  -
Rhizoctonia solani

Inhibitory activity against        --               -
HIV-1 reverse transcriptase

                     Nystatin ([IC.sub.50]  B. campestris lipid
                     in [micro]M)           transfer protein
                                            ([IC.sub.50] in [micro]M)

Antiproliferative   --                       6.2 [+ or -] 5.4
activity
against HepG2 cells

Antiproliferative   --                        38 [+ or -] 3.6
activity
against MCF-7 cells

Antifungal           10.2 [+ or -] 2.7         -
activity against
Rhizoctonia
solani

Inhibitory          --                        4.6 [+ or -] 1.2
activity against
HIV-1 reverse
transcriptase


The antifungal protein purified in the present study is unique in its possession of an N-terminal amino acid sequence with remarkable homology to bovine [beta]-lactoglobulin. Antifungal proteins with such a structure have not been encountered before. It is extremely interesting since passiflin is of plant origin while [beta]-lactoglobulin is a mammalian whey protein. Despite this structural resemblance, there are many points of dissimilarities between the two proteins indicating that they are distinct proteins. The molecular mass of passiflin (67 kDa) is higher than that of [beta]-lactoglobulin (36.6 kDa). In addition, intact [beta]-lactoglobulin does not demonstrate antifungal or antiproliferative activity whereas passiflin is endowed with these activities, indicating differences in biological activities between the two proteins. However, [beta]-lactoglobulin hydrolysate manifested antifungal activity (Hernandez-Ledesma et al. 2008). Furthermore, passiflin shows no cross-reactivity with a rabbit-anti-bovine-[beta]-lactoglobulin antiserum suggesting immunological distinctiveness between them.

Passiflin is devoid of ribonuclease and hemagglutinat-ing (lectin) activities. This observation deserves attention since some ribonucleases like the aforementioned ginseng ribonucleases (Lam and Ng 2001; Ng and Wang 2001; Gozia et al. 1993) and some lectins (Gozia et al. 1993; Yan et al. 2005) exhibit antifungal activity. Its lack of inhibitory activity toward HIV-1 reverse transcriptase is also noteworthy since some plant proteins comprising protease inhibitors (Ye et al. 2001), lectins (Wong and Ng 2005a) and antifungal proteins (Wong and Ng 2005b) display this antiretroviral activity. [beta]-Lactoglobulin lacks ribonuclease and HIV-1 reverse transcriptase inhibitory activities.

The antifungal activity of passiflin is species specific. It impedes mycelial growth in R. solani, but not in other fungi such as F. oxysporum and M. arachidicola. This observation is reminiscent of similar findings in case of antifungal proteins from asparagus seeds (Wang and Ng 2001b) and shallot bulbs (Wang and Ng 2002b). These two antifungal proteins inhibit only one out of the several fungal species tested.

Passiflin manifests a potent inhibitory action on breast cancer cells with an [IC.sub.50] value of 15 [micro]M. This finding is in keeping with previous demonstrations of the antiproliferative action of some antifungal proteins including ribosome-inactivating proteins (Tsao et al. 1990) and defensins (Wong and Ng 2005b). Interestingly, passiflin has no inhibitory activity toward hepatoma HepG2 cells, illustrating a specificity of action. Likewise, the ribosome-inactivating proteins trichosanthin and momorcharin exert highly potent inhibitory activity against choriocarcinoma but are much less active toward hepatoma cells (Tsao et al. 1990).

Another distinctive feature of passiflin is its chromatographic behavior on Q-Sepharose and DEAE-cellu-lose. Most of the antifungal proteins are unadsorbed on these anion exchangers whereas passiflin is adsorbed. The chromatographic procedure employed for purification of passiflin is highly efficient since it removes a considerable amount of materials without antifungal activity at each step.

Passiflin is distinct from the 2S albumin-like antifungal protein and peptide previously isolated from seeds of passion fruit (Pelegrini et al. 2006; Agizzio et al. 2003), as evidenced by differences in molecular mass, N-terminal amino acid sequence, and species specificity of antifungal activity. Passiflin is devoid of antifungal activity toward F. oxysporum which, however, is susceptible to the 2S albumin-like antifungal protein and peptide.

In summary, passiflin isolated from P. edulis is a distinctive dimeric antifungal protein. To date, only a small number of antifungal proteins have been shown to be dimeric, e.g. those from sanchi ginseng (Lam and Ng 2001), Chinese ginseng (Ng and Wang 2001), and American ginseng (Wang and Ng 2000). Passiflin possesses a [beta]-lactoglobulin-like N-terminal sequence. However, it does not cross-react with an anti-[beta]-lactoglobulin antiserum. It exhibits antiproliferative and antifungal activities which are missing in [beta]-lactoglobulin. Thus, passiflin is biologically and immunologically unrelated to [beta]-lactoglobulin. In this context, it is worth mentioning that thaumatin-like proteins have antifungal activity but no sweet taste while the converse is true of thaumatin although they are highly homologous in structure (Ye et al. 1999).

Acknowledgements

We thank the Medicine Panel, CUHK Research Committee, for award of a direct grant.

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S.K. Lam *, T.B. Ng

Department of Biochemistry, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China

* Corresponding author.

E-mail address: lamszekwani@yahoo.com.hk (S.K. Lam).
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Author:Lam, S.K.; Ng, T.B.
Publication:Phytomedicine: International Journal of Phytotherapy & Phytopharmacology
Article Type:Report
Geographic Code:9CHIN
Date:Mar 1, 2009
Words:4157
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