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Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety “Golden Valley”
BBRC Biochemical and Biophysical Research Communications 346 (2006) 681–686 www.elsevier.com/locate/ybbrc
Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety ‘‘Golden Valley’’ Mi-Hyun Kim a, Seong-Cheol Park a,e, Jin-Young Kim a, Sun Young Lee a, Hak-Tae Lim d, Hyeonsook Cheong c, Kyung-Soo Hahm a,b,*, Yoonkyung Park a,c,* a
d
Research Center for Proteineous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea b Department of Medicine, Chosun University, Kwangju 501-759, Republic of Korea c Department of Biotechnology, Chosun University, Kwangju 501-759, Republic of Korea Division of Biotechnology, Kangwon National University, 192-1, Hyoja2-Dong, Chunchon, Kangwon-Do 200-701, Republic of Korea e Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Republic of Korea Received 18 May 2006 Available online 9 June 2006
Abstract Potide-G, a small (5578.9 Da) antimicrobial peptide, was isolated from potato tubers (Solanum tuberosum L. cv. Golden Valley) through extraction of the water-soluble fraction, dialysis, ultrafiltration and DEAE-cellulose and C18 reversed-phase high performance liquid chromatography. This antimicrobial peptide was heat-stable and almost completely suppressed the proteolytic activity of trypsin, chymotrypsin and papain, with no hemolytic activity. In addition, potide-G potently inhibited growth of a variety of bacterial (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Clavibacter michiganense subsp. michiganinse) and fungal (Candida albicans and Rhizoctonia solani) strains. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the N-terminal sequence (residues from 1 to 11) of the protein is identical to that of potato proteinase inhibitor, a member of the Kunitz superfamily. And like other members of this class of protease inhibitor, potide-G may have number of beneficial and therapeutic uses. 2006 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Proteinase inhibitor; Inhibition activity; Perianal dermatitis
Plants express a variety of proteins that serve in the defense against pathogens and invading organisms, including ribosome-inactivating proteins [1], lectins [2], protease inhibitors [3] and antifungal proteins [4–6]. Among these, protease inhibitors are believed to play an important role in the defense against attack by both microorganisms and insects, to serve as storage proteins and, perhaps, to be involved in the regulation of endogenous proteases during seed dormancy [7]. Among the different types of protease inhibitors that have been identified in plants [8], particularly high levels of inhibitors of proteinases are found in many Solanaceae family members [9] and account for as much as 20–50% of the water-soluble proteins in potato tubers [10], *
Corresponding authors. Fax: +82 62 2278345. E-mail address: [email protected] (K.-S. Hahm).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.186
which is noteworthy, as increasing levels of protease inhibitor correlates with increases in resistance to pathogens [11]. Indeed, these peptides have recently attracted attention because of their ability to potently inhibit carcinogenesis [12] as well as the growth of pathogenic bacteria and fungus in a number of in vitro systems [13]. In this report, we describe the isolation and characterization of a small antimicrobial peptide, potide-G, from Golden Valley potato tubers. This peptide exhibits potent inhibitory activity against various human or plant pathogenic microbial strains and shows sequence homology with a protease inhibitor that is produced by potato tubers upon fungal attack [13]. Interestingly, the N-terminal sequence of potide-G is identical to the corresponding sequence of proteinase inhibitor, a member of the Kunitz superfamily.
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Materials and methods Potato tubers. Potato tubers (Solanum tuberosum L. cv. Golden Valley) were obtained from the Potatovalley (Kangwon National University, Korea) and were stored at 4 C in the dark at a relative humidity of 95– 100% for up to 6 months. Purification and characterization of potide-G. For purification of potide-G, potato tubers were first soaked in distilled water for several hours and then ground to a fine powder in a coffee grinder. The resulting flour was suspended in extraction buffer containing 100 mM Tris–HCl and 1.5 M LiCl (pH 7.2). After centrifugation, the supernatants were dialyzed in distilled water using a 1000 Da cut-off membrane, and the resulting extract was heated at 70 C for 20 min to obtain the heat-stable peptides. The heat-denatured precipitates were removed by centrifugation for 30 min at 24,000 rpm, after which the supernatant containing the heat-stable proteins was subjected to ultrafiltration through a 10-kDa cut-off membrane, and the extract was loaded onto a DEAE-cellulose reverse-phase column (Vydac, 4.6 · 250 mm) on an HPLC system (Shimadzu, Japan). Peptides that passed through the column unadsorbed were dissolved in 0.1% (v/v) trifluoroacetic acid (TFA) in HPLC grade water (Solvent A) and loaded onto a C18 RPHPLC column in equilibrium with 0.1% TFA. The peptides were eluted with a linear gradient (2% increase/min) of acetonitrile (10–95%) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The effluent was monitored at 230 nm, and the fractions for each peak were pooled and dried in a freeze-dryer. The major peak was then successively resubjected to HPLC using a delayed gradient, and the resulting peak fractions were collected and assayed for antimicrobial activity. The purity and molecular weight of the fractions were confirmed by electrophoresis on a 16.5% tricine acrylamide gel according to the method of Schagger and von Jagow [14]. Mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was carried out in the linear mode using a Voyager DE RP instrument (Perseptive Biosystems, Framingham, MA) as described Pouvreau et al. [10]. Microbial strains. Streptococcus aureus (KCTC 1621),Listeria monocytogenes (KCTC 3710), Clavibacter michiganense subsp. michiganinse (KCTC 9231) and Candida albicans (KCTC 7270) were obtained from the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, Korea. Rhizoctonia solani (KACC 40138) was obtained from the KCTC and Korean Agricultural Culture Collection (KACC). Antibacterial activity. The bacteria were grown to the mid-logarithmic phase in medium containing (g/l) 10 bactotryptone, 5 yeast extract and 10 NaCl (pH 7.0). Peptides were diluted stepwise in 1% bactopeptone. The tested organism (final bacterial suspension: 5 · 103 colony forming units (CFU)/ml) suspended in growth medium (100 ll) was mixed with the test peptide solution in the wells of a microtiter plate such that the final concentration of test peptide was 30 lg/ml. Microbial growth was determined based on the increase in OD620 after incubation for 10 h at 37 C. The minimal inhibitory concentration (MIC) was defined as minimal concentration that prevented any visible growth on the plate. All assays were performed in triplicate. Antifungal activity. Fungal strains were grown at 28 C in potato dextrose broth (PDB) medium. Fungal cells in PDB media were seeded to a density of 2 · 103 cells/well in a 96-well microtiter plate (100 ll per well). Test peptide solution was then added to a final concentration of 5, 30 or 100 lg/ml, and the cell suspension was incubated for 24 h at 28 C. Thereafter, 10 ll of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/ml MTT in phosphate-buffered saline (PBS), pH 7.4) was added to each well, and the plates were incubated for an additional 4 h at 37 C, after which 30 ll of 20% (w/v) SDS solution containing 0.02 M HCl was added, and the plates were incubated for 16 h at 37 C to dissolve the formazan crystals that had formed [15,16]. The turbidity of each well was then measured at 570 nm using a microtiter ELISA reader (Molecular Devices Emax, California, USA). All assays were performed in triplicate.
Antifungal activity against C. albicans also was assayed in 100 · 15 mm Petri dishes containing PDB. After a mycelial colony had developed, sterile blank paper disks (8 mm diameter) were placed 5 mm away from the leading edge of the colony. An aliquot of the test peptide in MES buffer (20 mM, pH 6.0) was added to each disk, and the plates were incubated for 72 h at 28 C. Antifungal activity was reflected by a clear zone of growth inhibition around the disk. Preparation of human red blood cells and assay of hemolytic activity. Human red blood cells (RBCs) were washed by centrifugation and resuspension three times in PBS. The hemolytic activities of potamin-G and melittin (positive control) were evaluated by measuring the release of hemoglobin from fresh RBCs. Aliquots (100 ll) of an 8% suspension of RBCs were transferred to 96-well plates, and hemolysis was determined by measuring the absorbance at 414 nm using the Emax plate reader. No hemolysis (0%) and full hemolysis (100%) were determined in PBS and 0.1% Triton X-100, respectively. Percent hemolysis was calculated using the following equation: % hemolysis = [(Abs414nm with peptide solution Abs414nm in PBS)/(Abs414nm with 0.1% Triton X-100 Abs414nm in PBS)] · 100. Amino acid sequencing of the isolated peptide. Amino acid sequencing of the purified peptide was carried out by the Sequence Centre of Korea Basic Science Institute (Seoul, Korea). Measurement of trypsin, chymotrypsin and papain inhibition. A portion of the inhibitor was incubated for 5 min at 25 C with 25 lg of trypsin, chymotrypsin or papain in 100 ll of 50 mM Tris–HCl buffer (pH 8.0) containing 200 mM CaCl2. Residual trypsin, chymotrypsin or papain activity was determined by adding 300 ll of 1% casein (w/v) and incubating at 25 C. The reaction was terminated after 15 min by adding 1 ml of cold 5% trichloroacetic acid. The reaction mixtures were then centrifuged for 20 min at 32,000g, and the absorbance of the supernatant was determined at 280 nm.
Results and discussion An antimicrobial peptide obtained from potato tubers was purified using ultrafiltration and DEAE-cellulose and C18-reverse-phase HPLC. In the first isolation step, ultrafiltration through a 10-kDa molecular weight cut-off membrane yielded two components: one with molecular masses >10 kDa and one with molecular masses <10 kDa. In the second isolation step, <10-kDa samples were fractionated on DEAE-cellulose into an unadsorbed fraction 1 with antimicrobial activity and an adsorbed fraction 2 without activity (data not shown). Fraction 1 was subjected to further fractionation by C18-reverse-phase HPLC (Fig. 1A). The major single peak (arrow), which we refer to as potide-G, was then further purified through two additional steps of C18-reverse-phase HPLC (Fig. 1B). The protein yields at the various chromatographic steps are shown in Table 1. Upon tricine gel electrophoresis, the isolated potide-G appeared as a single band with a molecular weight of about 6 kDa (Fig. 2). Consistent with that finding, the relative molecular weight calculated based on the protein sequence of potide-G was 5578.9 Da, which was in close agreement with the molecular weight of 5578.9 Da directly determined by MALDI-MS (Fig. 3). We next examined the antimicrobial activity of potide-G against several human and plant pathogenic microbial strains (Table 1). The microbial cells were treated with potide-G and then spread on agar plates (Figs. 4 and 5). Potide-G exerted an antibacterial action against the
M.-H. Kim et al. / Biochemical and Biophysical Research Communications 346 (2006) 681–686
Fig. 1. Elution profile of potide-G. (A) The fraction containing the major peak was separated by reversed-phase HPLC using a C18 column. (B) The purified antimicrobial peptide was then again subjected to reversed-phase HPLC using a C18 column. In (A) and (B) the peptides were eluted with a linear gradient (2% increase/min) of acetonitrile (10–95%) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The effluent was monitored at 214 nm.
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Fig. 2. Tricine gel electrophoresis of the purified potide-G. Lane 1: molecular size markers (in kDa) (26.6, triosephosphate isomerase; 17 kDa, myoglobin; 14.2, a-lactalbumin; 6.5, aprotinin; 3.5, insulin chain B; 1, bradykinin). Lane 2: potide-G.
Table 1 Steps in the purification of potide-G from potato tubers Fraction
Yield (mg)
Potato tuber Protein extraction buffer Cut-off under 10 kDa DEAE-cellulose C18-HPLC
200,000 83.72 9.8 2.83 0.04
pathogenic bacterial strains S. aureus, L. monocytogenes and C. michiganense subsp. michiganinse; in each case, the MIC was <30 lg/ml (Table 2 and Fig. 4). Potide-G also showed potent antifungal activity against the human and plant pathogenic fungi C. albicans and Saccharomyces cerevisiae, with MICs of <30 lg/ml (Table 2 and Fig. 5). PotideG was somewhat less effective against Escherichia coli and R. solani, with MICs of >30 and 100 lg/ml, respectively. We also evaluated the cytotoxicity of potide-G against mammalian cells by assessing its hemolytic activity using human RBCs. Potide-G showed no hemolytic activity, whereas melittin, which served as a positive control, was strongly hemolytic (Table 3). It thus appears that potide-G possesses remarkable antimicrobial activity against a variety of microbial cells, with no hemolytic activity. When we used the N-terminal sequence of potideG (NH2-Gln-Ile-Cys-Thr-Asn-Cys-Cys-Ala-Gly-Arg-Lys-)
Fig. 3. MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) determination of potide-G molecular mass.
for a homology search, we found that it was 40% identical to potato serine proteinase inhibitor, a member of Kunitz superfamily [17]. Notably, the 11 N-terminal amino acids of potide-G were identical the corresponding amino acids in proteinase inhibitor (Fig. 6), suggesting potide-G from Solanum tuberosum L. cv. Golden Valley also is a member of the Kunitz superfamily. Consistent with that idea, potide-G dose-dependently inhibited the activities of trypsin, chymotrypsin and papain, suggesting that this potently antimicrobial peptide is indeed a serine protease inhibitor (Fig. 7).
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Fig. 4. Antibacterial assays. Potide-G exhibited antibacterial activity against S. aureus, L. monocytogenes, C. michiganense subsp. michiganinse and E. coli: (A) untreated S. aureus; (B) S. aureus + potide-G (30 lg/ml); (C) untreated L. monocytogenes; (D) L. monocytogenes + potide-G (30 lg/ml); (E) untreated C. michiganense subsp. michiganinse; (F) C. michiganense subsp. michiganinse + potide-G (30 lg/ml); (G) untreated E. coli; (H) E. coli + potide-G (30 lg/ ml).
Fig. 5. Antifungal activity of potide-G against C. albicans (a) and S. cerevisiae (b). The white disks were left untreated (A) or were treated with 5 lg of potide-G (B) or 30 lg of potide-G (C). Table 2 Antimicrobial activities of purified potide-G Microorganisms
MIC (lg/ml)
S. aureus L. monocytogenes C. michiganense subsp. michiganinse E. coli
<30 <30 <30
Human pathogenic fungi
C. albicans S. cerevisiae
<30 <30
Plant pathogenic fungi
R. solani
100
Pathogenic bacteria
>30
Table 3 Hemolytic activity of potide-G Peptide (lM)
Potide-G Melittin
% Hemolysis 40
20
10
5
2.5
1.25
0.62
0.31
0 100
0 100
0 100
0 95
0 73
0 36
0 19
0 0
Protease inhibitors in plant tubers and plant seeds are generally thought to serve as storage proteins and to act in the defense against insects and microorganisms [18]. For instance, Conconi et al. [19] showed that protease inhibitors are involved in the wound-induced defense responses of plants against herbivores and pathogens, while Terras et al. [20] suggested that a 2S-albumin-like trypsin inhibitor from barley seeds had some antifungal activity and acted synergistically with thionins to permeabilize fungal membranes [21]. In addition, Chen et al. [22] isolated a 14-kDa trypsin inhibitor from corn that retarded the growth of Aspergillus flavus and was present at high concentrations in Aspergillus-resistant genotypes, but was present at low levels or not at all in Aspergillus-susceptible genotypes. Chen et al. [23] also showed that the resistance of certain corn genotypes to fungal infection is likely related to the action of trypsin inhibitor, which lowers the production and activity of fungal a-amylase, thereby reducing the availability of simple sugars for fungal
M.-H. Kim et al. / Biochemical and Biophysical Research Communications 346 (2006) 681–686
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Fig. 6. N-terminal amino acid sequence of potide-G. Note that the 11 N-terminal amino acids of potide-G are identical to the corresponding sequence in proteinase inhibitor from Solanum melongena.
Fig. 7. Inhibition of trypsin, chymotrypsin and papain by potide-G. Fluorescently labeled casein was incubated for 60 min at room temperature with 25 lg of the indicated enzyme, with or without the indicated concentration of potide-G, after which the fluorescences were read.
growth. A trypsin inhibitor from wheat kernel also elicits a potent antifungal effect [24]. What’s more, another Kunitztype proteinase inhibitor from Prosopis juliflora with activity against papain, trypsin and chymotrypsin exerts insecticide effects against C. maculatus larvae by blocking the affected enzymes in the insect’s digestive system [25]. Finally, protease inhibitors also have recently attracted attention because of their potent anticarcinogenic effect in various in vivo and in vitro systems [26]. Several nontoxic protease inhibitors, mostly of bacterial or plant origin (e.g., from barley seeds, cabbage leaves and Streptomyces), have been purified and are now commercially available for use in preventing protease-induced perianal dermatitis [27]. Potato tuber proteins also have been shown to efficiently inhibit human fecal proteases, and could be useful in the treatment of perianal dermatitis [27]. We found that potide-G exerts an antifungal effect against C. albicans, which is the most common cause of oral, esophageal, vaginal and urinary candidiasis [28], as well as against R. solani, a rice pathogen [29]; C. michiganense subsp. michiganense, a gram-positive coryneform bacterium that is a pathogen in a variety of agriculturally important plants, including tomato, potato and maize [30]; and L. monocytogenes, a Shiga toxin-producing, foodborne pathogen [31]. Thus, like other members of this class of protease inhibitor, potide-G may have a number of beneficial and therapeutic uses. It is also noteworthy that potide-G is heat-stable, which would be expected to enhance the therapeutic utility of this low molecular weight protein. Acknowledgments This work was supported by grant from the Ministry of Science and Technology, Korea and the Korea Science and Engineering Foundation through the Research Center for Proteineous Materials and this study was supported (in
part) by the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture and Forestry. And Y. Park is supported from the BK21 Program, Ministry of Education and Human Resources Development, Korea. References [1] R. Leah, H. Tommerup, I. Svendsen, J. Mundy, Biochemical and molecular characterization of three barley seed proteins with antifungal properties, J. Biol. Chem. 246 (1991) 1564–1573. [2] K. Kamemura, Y. Furuichi, H. Umekawa, H.C. Takahashi, Purification and characterization of novel lectins from Great Northern bean, Phaseolus vulgaris L., Biochim. Biophys. Acta 1158 (1993) 181– 188. [3] Y. Birk, The Bowman–Birk inhibitor, trypsin- and chymotrypsininhibitor from soybeans, Int. J. Pept. Protein Res. 25 (1985) 113– 131. [4] X.Y. Ye, H.X. Wang, T.B. Ng, First chromatographic isolation of an antifungal thaumatin-like protein from French bean legumes and demonstration of its antifungal activity, Biochem. Biophys. Res. Commun. 263 (1999) 130–134. [5] N. Benhamou, K. Broglie, R. Broglie, I. Chet, Antifungal effect of bean endochitinase on Rhizoctonia solani: ultrastructural changes and cytochemical aspects of chitin breakdown, Can. J. Microbiol. 39 (1993) 318–328. [6] R. Vogelsang, W. Barz, Purification, characterization and differential hormonal regulation of a beta-1,3-glucanase and two chitinases from chickpea (Cicer arietinum L.), Planta 189 (1993) 60–69. [7] S. Mazumdar, S.M. Leighton, C.R. Babu, A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties, Phytochemistry 67 (2006) 232–241. [8] F. De Leo, M. Volpicella, F. Licciulli, S. Liuni, R. Gallerani, L.R. Ceci, PLANT-PIs: a database for plant protease inhibitors and their genes, Nucleic Acids Res. 30 (2002) 347–348. [9] N.A. Plate, L.I. Valuev, T.A. Valueva, V.V. Chupov, Biospecific haemosorbents based on proteinase inhibitor. I. Synthesis and properties, Biomaterials 14 (1993) 51–56. [10] L. Pouvreau, H. Gruppen, S.R. Piersma, L.A.M. van den Brock, G.M. van Koningsveld, A.G.J. Voragen, Relative abundance and inhibitory distribution of protease inhibitors in potato juice from cv. Elkana, J. Agric. Food Chem. 49 (2001) 2864–2874.
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Purification and characterization of a heat-stable serine protease inhibitor from the tubers of new potato variety ‘‘Golden Valley’’ Mi-Hyun Kim a, Seong-Cheol Park a,e, Jin-Young Kim a, Sun Young Lee a, Hak-Tae Lim d, Hyeonsook Cheong c, Kyung-Soo Hahm a,b,*, Yoonkyung Park a,c,* a
d
Research Center for Proteineous Materials (RCPM), Chosun University, Kwangju 501-759, Republic of Korea b Department of Medicine, Chosun University, Kwangju 501-759, Republic of Korea c Department of Biotechnology, Chosun University, Kwangju 501-759, Republic of Korea Division of Biotechnology, Kangwon National University, 192-1, Hyoja2-Dong, Chunchon, Kangwon-Do 200-701, Republic of Korea e Division of Applied Life Science, Gyeongsang National University, Chinju 660-701, Republic of Korea Received 18 May 2006 Available online 9 June 2006
Abstract Potide-G, a small (5578.9 Da) antimicrobial peptide, was isolated from potato tubers (Solanum tuberosum L. cv. Golden Valley) through extraction of the water-soluble fraction, dialysis, ultrafiltration and DEAE-cellulose and C18 reversed-phase high performance liquid chromatography. This antimicrobial peptide was heat-stable and almost completely suppressed the proteolytic activity of trypsin, chymotrypsin and papain, with no hemolytic activity. In addition, potide-G potently inhibited growth of a variety of bacterial (Staphylococcus aureus, Listeria monocytogenes, Escherichia coli, and Clavibacter michiganense subsp. michiganinse) and fungal (Candida albicans and Rhizoctonia solani) strains. Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry revealed that the N-terminal sequence (residues from 1 to 11) of the protein is identical to that of potato proteinase inhibitor, a member of the Kunitz superfamily. And like other members of this class of protease inhibitor, potide-G may have number of beneficial and therapeutic uses. 2006 Elsevier Inc. All rights reserved. Keywords: Antimicrobial peptide; Proteinase inhibitor; Inhibition activity; Perianal dermatitis
Plants express a variety of proteins that serve in the defense against pathogens and invading organisms, including ribosome-inactivating proteins [1], lectins [2], protease inhibitors [3] and antifungal proteins [4–6]. Among these, protease inhibitors are believed to play an important role in the defense against attack by both microorganisms and insects, to serve as storage proteins and, perhaps, to be involved in the regulation of endogenous proteases during seed dormancy [7]. Among the different types of protease inhibitors that have been identified in plants [8], particularly high levels of inhibitors of proteinases are found in many Solanaceae family members [9] and account for as much as 20–50% of the water-soluble proteins in potato tubers [10], *
Corresponding authors. Fax: +82 62 2278345. E-mail address: [email protected] (K.-S. Hahm).
0006-291X/$ - see front matter 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2006.05.186
which is noteworthy, as increasing levels of protease inhibitor correlates with increases in resistance to pathogens [11]. Indeed, these peptides have recently attracted attention because of their ability to potently inhibit carcinogenesis [12] as well as the growth of pathogenic bacteria and fungus in a number of in vitro systems [13]. In this report, we describe the isolation and characterization of a small antimicrobial peptide, potide-G, from Golden Valley potato tubers. This peptide exhibits potent inhibitory activity against various human or plant pathogenic microbial strains and shows sequence homology with a protease inhibitor that is produced by potato tubers upon fungal attack [13]. Interestingly, the N-terminal sequence of potide-G is identical to the corresponding sequence of proteinase inhibitor, a member of the Kunitz superfamily.
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Materials and methods Potato tubers. Potato tubers (Solanum tuberosum L. cv. Golden Valley) were obtained from the Potatovalley (Kangwon National University, Korea) and were stored at 4 C in the dark at a relative humidity of 95– 100% for up to 6 months. Purification and characterization of potide-G. For purification of potide-G, potato tubers were first soaked in distilled water for several hours and then ground to a fine powder in a coffee grinder. The resulting flour was suspended in extraction buffer containing 100 mM Tris–HCl and 1.5 M LiCl (pH 7.2). After centrifugation, the supernatants were dialyzed in distilled water using a 1000 Da cut-off membrane, and the resulting extract was heated at 70 C for 20 min to obtain the heat-stable peptides. The heat-denatured precipitates were removed by centrifugation for 30 min at 24,000 rpm, after which the supernatant containing the heat-stable proteins was subjected to ultrafiltration through a 10-kDa cut-off membrane, and the extract was loaded onto a DEAE-cellulose reverse-phase column (Vydac, 4.6 · 250 mm) on an HPLC system (Shimadzu, Japan). Peptides that passed through the column unadsorbed were dissolved in 0.1% (v/v) trifluoroacetic acid (TFA) in HPLC grade water (Solvent A) and loaded onto a C18 RPHPLC column in equilibrium with 0.1% TFA. The peptides were eluted with a linear gradient (2% increase/min) of acetonitrile (10–95%) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The effluent was monitored at 230 nm, and the fractions for each peak were pooled and dried in a freeze-dryer. The major peak was then successively resubjected to HPLC using a delayed gradient, and the resulting peak fractions were collected and assayed for antimicrobial activity. The purity and molecular weight of the fractions were confirmed by electrophoresis on a 16.5% tricine acrylamide gel according to the method of Schagger and von Jagow [14]. Mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) was carried out in the linear mode using a Voyager DE RP instrument (Perseptive Biosystems, Framingham, MA) as described Pouvreau et al. [10]. Microbial strains. Streptococcus aureus (KCTC 1621),Listeria monocytogenes (KCTC 3710), Clavibacter michiganense subsp. michiganinse (KCTC 9231) and Candida albicans (KCTC 7270) were obtained from the Korean Collection for Type Cultures (KCTC), Korea Research Institute of Bioscience and Biotechnology (KRIBB), Taejon, Korea. Rhizoctonia solani (KACC 40138) was obtained from the KCTC and Korean Agricultural Culture Collection (KACC). Antibacterial activity. The bacteria were grown to the mid-logarithmic phase in medium containing (g/l) 10 bactotryptone, 5 yeast extract and 10 NaCl (pH 7.0). Peptides were diluted stepwise in 1% bactopeptone. The tested organism (final bacterial suspension: 5 · 103 colony forming units (CFU)/ml) suspended in growth medium (100 ll) was mixed with the test peptide solution in the wells of a microtiter plate such that the final concentration of test peptide was 30 lg/ml. Microbial growth was determined based on the increase in OD620 after incubation for 10 h at 37 C. The minimal inhibitory concentration (MIC) was defined as minimal concentration that prevented any visible growth on the plate. All assays were performed in triplicate. Antifungal activity. Fungal strains were grown at 28 C in potato dextrose broth (PDB) medium. Fungal cells in PDB media were seeded to a density of 2 · 103 cells/well in a 96-well microtiter plate (100 ll per well). Test peptide solution was then added to a final concentration of 5, 30 or 100 lg/ml, and the cell suspension was incubated for 24 h at 28 C. Thereafter, 10 ll of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) solution (5 mg/ml MTT in phosphate-buffered saline (PBS), pH 7.4) was added to each well, and the plates were incubated for an additional 4 h at 37 C, after which 30 ll of 20% (w/v) SDS solution containing 0.02 M HCl was added, and the plates were incubated for 16 h at 37 C to dissolve the formazan crystals that had formed [15,16]. The turbidity of each well was then measured at 570 nm using a microtiter ELISA reader (Molecular Devices Emax, California, USA). All assays were performed in triplicate.
Antifungal activity against C. albicans also was assayed in 100 · 15 mm Petri dishes containing PDB. After a mycelial colony had developed, sterile blank paper disks (8 mm diameter) were placed 5 mm away from the leading edge of the colony. An aliquot of the test peptide in MES buffer (20 mM, pH 6.0) was added to each disk, and the plates were incubated for 72 h at 28 C. Antifungal activity was reflected by a clear zone of growth inhibition around the disk. Preparation of human red blood cells and assay of hemolytic activity. Human red blood cells (RBCs) were washed by centrifugation and resuspension three times in PBS. The hemolytic activities of potamin-G and melittin (positive control) were evaluated by measuring the release of hemoglobin from fresh RBCs. Aliquots (100 ll) of an 8% suspension of RBCs were transferred to 96-well plates, and hemolysis was determined by measuring the absorbance at 414 nm using the Emax plate reader. No hemolysis (0%) and full hemolysis (100%) were determined in PBS and 0.1% Triton X-100, respectively. Percent hemolysis was calculated using the following equation: % hemolysis = [(Abs414nm with peptide solution Abs414nm in PBS)/(Abs414nm with 0.1% Triton X-100 Abs414nm in PBS)] · 100. Amino acid sequencing of the isolated peptide. Amino acid sequencing of the purified peptide was carried out by the Sequence Centre of Korea Basic Science Institute (Seoul, Korea). Measurement of trypsin, chymotrypsin and papain inhibition. A portion of the inhibitor was incubated for 5 min at 25 C with 25 lg of trypsin, chymotrypsin or papain in 100 ll of 50 mM Tris–HCl buffer (pH 8.0) containing 200 mM CaCl2. Residual trypsin, chymotrypsin or papain activity was determined by adding 300 ll of 1% casein (w/v) and incubating at 25 C. The reaction was terminated after 15 min by adding 1 ml of cold 5% trichloroacetic acid. The reaction mixtures were then centrifuged for 20 min at 32,000g, and the absorbance of the supernatant was determined at 280 nm.
Results and discussion An antimicrobial peptide obtained from potato tubers was purified using ultrafiltration and DEAE-cellulose and C18-reverse-phase HPLC. In the first isolation step, ultrafiltration through a 10-kDa molecular weight cut-off membrane yielded two components: one with molecular masses >10 kDa and one with molecular masses <10 kDa. In the second isolation step, <10-kDa samples were fractionated on DEAE-cellulose into an unadsorbed fraction 1 with antimicrobial activity and an adsorbed fraction 2 without activity (data not shown). Fraction 1 was subjected to further fractionation by C18-reverse-phase HPLC (Fig. 1A). The major single peak (arrow), which we refer to as potide-G, was then further purified through two additional steps of C18-reverse-phase HPLC (Fig. 1B). The protein yields at the various chromatographic steps are shown in Table 1. Upon tricine gel electrophoresis, the isolated potide-G appeared as a single band with a molecular weight of about 6 kDa (Fig. 2). Consistent with that finding, the relative molecular weight calculated based on the protein sequence of potide-G was 5578.9 Da, which was in close agreement with the molecular weight of 5578.9 Da directly determined by MALDI-MS (Fig. 3). We next examined the antimicrobial activity of potide-G against several human and plant pathogenic microbial strains (Table 1). The microbial cells were treated with potide-G and then spread on agar plates (Figs. 4 and 5). Potide-G exerted an antibacterial action against the
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Fig. 1. Elution profile of potide-G. (A) The fraction containing the major peak was separated by reversed-phase HPLC using a C18 column. (B) The purified antimicrobial peptide was then again subjected to reversed-phase HPLC using a C18 column. In (A) and (B) the peptides were eluted with a linear gradient (2% increase/min) of acetonitrile (10–95%) containing 0.1% trifluoroacetic acid at a flow rate of 1 ml/min. The effluent was monitored at 214 nm.
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Fig. 2. Tricine gel electrophoresis of the purified potide-G. Lane 1: molecular size markers (in kDa) (26.6, triosephosphate isomerase; 17 kDa, myoglobin; 14.2, a-lactalbumin; 6.5, aprotinin; 3.5, insulin chain B; 1, bradykinin). Lane 2: potide-G.
Table 1 Steps in the purification of potide-G from potato tubers Fraction
Yield (mg)
Potato tuber Protein extraction buffer Cut-off under 10 kDa DEAE-cellulose C18-HPLC
200,000 83.72 9.8 2.83 0.04
pathogenic bacterial strains S. aureus, L. monocytogenes and C. michiganense subsp. michiganinse; in each case, the MIC was <30 lg/ml (Table 2 and Fig. 4). Potide-G also showed potent antifungal activity against the human and plant pathogenic fungi C. albicans and Saccharomyces cerevisiae, with MICs of <30 lg/ml (Table 2 and Fig. 5). PotideG was somewhat less effective against Escherichia coli and R. solani, with MICs of >30 and 100 lg/ml, respectively. We also evaluated the cytotoxicity of potide-G against mammalian cells by assessing its hemolytic activity using human RBCs. Potide-G showed no hemolytic activity, whereas melittin, which served as a positive control, was strongly hemolytic (Table 3). It thus appears that potide-G possesses remarkable antimicrobial activity against a variety of microbial cells, with no hemolytic activity. When we used the N-terminal sequence of potideG (NH2-Gln-Ile-Cys-Thr-Asn-Cys-Cys-Ala-Gly-Arg-Lys-)
Fig. 3. MALDI-MS (matrix-assisted laser desorption/ionization mass spectrometry) determination of potide-G molecular mass.
for a homology search, we found that it was 40% identical to potato serine proteinase inhibitor, a member of Kunitz superfamily [17]. Notably, the 11 N-terminal amino acids of potide-G were identical the corresponding amino acids in proteinase inhibitor (Fig. 6), suggesting potide-G from Solanum tuberosum L. cv. Golden Valley also is a member of the Kunitz superfamily. Consistent with that idea, potide-G dose-dependently inhibited the activities of trypsin, chymotrypsin and papain, suggesting that this potently antimicrobial peptide is indeed a serine protease inhibitor (Fig. 7).
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Fig. 4. Antibacterial assays. Potide-G exhibited antibacterial activity against S. aureus, L. monocytogenes, C. michiganense subsp. michiganinse and E. coli: (A) untreated S. aureus; (B) S. aureus + potide-G (30 lg/ml); (C) untreated L. monocytogenes; (D) L. monocytogenes + potide-G (30 lg/ml); (E) untreated C. michiganense subsp. michiganinse; (F) C. michiganense subsp. michiganinse + potide-G (30 lg/ml); (G) untreated E. coli; (H) E. coli + potide-G (30 lg/ ml).
Fig. 5. Antifungal activity of potide-G against C. albicans (a) and S. cerevisiae (b). The white disks were left untreated (A) or were treated with 5 lg of potide-G (B) or 30 lg of potide-G (C). Table 2 Antimicrobial activities of purified potide-G Microorganisms
MIC (lg/ml)
S. aureus L. monocytogenes C. michiganense subsp. michiganinse E. coli
<30 <30 <30
Human pathogenic fungi
C. albicans S. cerevisiae
<30 <30
Plant pathogenic fungi
R. solani
100
Pathogenic bacteria
>30
Table 3 Hemolytic activity of potide-G Peptide (lM)
Potide-G Melittin
% Hemolysis 40
20
10
5
2.5
1.25
0.62
0.31
0 100
0 100
0 100
0 95
0 73
0 36
0 19
0 0
Protease inhibitors in plant tubers and plant seeds are generally thought to serve as storage proteins and to act in the defense against insects and microorganisms [18]. For instance, Conconi et al. [19] showed that protease inhibitors are involved in the wound-induced defense responses of plants against herbivores and pathogens, while Terras et al. [20] suggested that a 2S-albumin-like trypsin inhibitor from barley seeds had some antifungal activity and acted synergistically with thionins to permeabilize fungal membranes [21]. In addition, Chen et al. [22] isolated a 14-kDa trypsin inhibitor from corn that retarded the growth of Aspergillus flavus and was present at high concentrations in Aspergillus-resistant genotypes, but was present at low levels or not at all in Aspergillus-susceptible genotypes. Chen et al. [23] also showed that the resistance of certain corn genotypes to fungal infection is likely related to the action of trypsin inhibitor, which lowers the production and activity of fungal a-amylase, thereby reducing the availability of simple sugars for fungal
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Fig. 6. N-terminal amino acid sequence of potide-G. Note that the 11 N-terminal amino acids of potide-G are identical to the corresponding sequence in proteinase inhibitor from Solanum melongena.
Fig. 7. Inhibition of trypsin, chymotrypsin and papain by potide-G. Fluorescently labeled casein was incubated for 60 min at room temperature with 25 lg of the indicated enzyme, with or without the indicated concentration of potide-G, after which the fluorescences were read.
growth. A trypsin inhibitor from wheat kernel also elicits a potent antifungal effect [24]. What’s more, another Kunitztype proteinase inhibitor from Prosopis juliflora with activity against papain, trypsin and chymotrypsin exerts insecticide effects against C. maculatus larvae by blocking the affected enzymes in the insect’s digestive system [25]. Finally, protease inhibitors also have recently attracted attention because of their potent anticarcinogenic effect in various in vivo and in vitro systems [26]. Several nontoxic protease inhibitors, mostly of bacterial or plant origin (e.g., from barley seeds, cabbage leaves and Streptomyces), have been purified and are now commercially available for use in preventing protease-induced perianal dermatitis [27]. Potato tuber proteins also have been shown to efficiently inhibit human fecal proteases, and could be useful in the treatment of perianal dermatitis [27]. We found that potide-G exerts an antifungal effect against C. albicans, which is the most common cause of oral, esophageal, vaginal and urinary candidiasis [28], as well as against R. solani, a rice pathogen [29]; C. michiganense subsp. michiganense, a gram-positive coryneform bacterium that is a pathogen in a variety of agriculturally important plants, including tomato, potato and maize [30]; and L. monocytogenes, a Shiga toxin-producing, foodborne pathogen [31]. Thus, like other members of this class of protease inhibitor, potide-G may have a number of beneficial and therapeutic uses. It is also noteworthy that potide-G is heat-stable, which would be expected to enhance the therapeutic utility of this low molecular weight protein. Acknowledgments This work was supported by grant from the Ministry of Science and Technology, Korea and the Korea Science and Engineering Foundation through the Research Center for Proteineous Materials and this study was supported (in
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