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Original Paper	Pdf file on Synergy OPEN omments
Acta Biochim Biophys Sin 2008, 40: 811-818
doi:10.1111/j.1745-7270.2008.00455.x

Thermostable mannose-binding lectin from Dendrobium findleyanum with activities dependent on sulfhydryl content

Runglawan Sudmoon¹, Nison Sattayasai¹*, Wandee Bunyatratchata², Arunrat Chaveerach³, and Suporn Nuchadomrong¹

1 Department of Biochemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

2 Department of Microbiology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

3 Department of Biology, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand

Received: April 22, 2008

Accepted: June 12, 2008

This work was supported by a grant from the Thailand Research Fund (the Royal Golden Jubilee Ph.D. Program grant No. PHD/0015/2548)

*Corresponding author: tel, 66-43-342911; fax, 66-43-342911; e-mail, [email protected]

A mannose-binding lectin was purified from Dendrobium (D.) findleyanum pseudobulb using mannan-agarose column chromatography. After heating in the presence of SDS with or without 2-mercaptoethanol on SDS-PAGE with a continuous gradient of 8%-20% acrylamide, the purified lectin showed only one protein band with a molecular mass of 14.5 kDa. Without heating, two bands were seen on the gel at the positions of 14.5 kDa and 53.7 kDa, but a higher amount of the 53.7 kDa protein was observed in the presence of 2-mercaptoethanol. Protein identification of both protein bands by liquid chromatography-tandem mass spectrometry showed three peptide fragments identical to parts of a lectin precursor from D. officinale; the lectin was named D. findleyanum agglutinin (DFA). Using various concentrations of native-PAGE and Ferguson plot, only one protein band revealed a molecular mass of 56.2 kDa, indicating four 14.5 kDa polypeptide subunits in the DFA. Isoelectric focusing revealed that the DFA had three conformational forms with an isoelectric point of 5.18, 4.87 and 4.72, whereas 2-mercaptoethanol-treated DFA showed only one band with an isoelectric point of 5.18. DFA exhibited specificity towards mannose using the solid-phase method. The binding activity, anti-fungal activity and hemagglutination activity of DFA were not affected by heat, but were increased by free sulfhydryl groups.

Keywords lectin; Orchidaceae; sulfhydryl group

Plant lectins or agglutinins are carbohydrate-binding proteins comprising at least seven families. Monocot mannose-binding lectins, one such family [1,2], differ from all other lectins in its exclusive specificity to mannose oligosaccharides [3]. It has been suggested that mannose-binding lectins play a defensive role by recognizing the high-mannose-type glycans of foreign microorganisms or plant predators [2,4]. Apart from their native functions, the mannose-binding lectins have useful applications in the analysis and isolation of mannose-containing glycoconjugates. They also have a potent inhibitory effect on human and animal retroviruses and on antiproliferative activity in some human tumor cell lines [57]. Since mannose-binding lectins are involved in many interesting activities, they have been characterized in many plants. Mannose-binding lectins have been found in several species of Orchidaceae including Listera ovata [3,8], Epipactis helleborine and Cymbidium hybrid [6,8]. In genus Dendrobium (D.), two lectin genes from D. officinale have been studied [9,10], but no native lectin was characterized.

In this study, we purified and characterized a native mannose-binding lectin from pseudo bulbs of D. findleyanum E.C. Parish & Rchb.f. [11]. The lectin showed its properties, which are somewhat different from mannose-binding lectins isolated from other species of Orchidaceae.

Materials and Methods

Plant material and crude protein extract

Mature pseudobulbs were collected from D. findleyanum. The 0.4 g plant sample was ground in 1 ml extraction buffer (200 mM Tris-HCl, 20 mM EDTA, pH 8.0, 5 mM 4-aminobenzamidine dihydrochloride, 1 mM Phenylmethylsulfonyl fluoride) with mortar and pestle. The homogenate was centrifuged for 10 min at 11,000 g. The supernatant was collected as crude protein extract.

Affinity chromatography

Mannan-agarose (Sigma-Aldrich, St. Louis, USA) column chromatography was used for the purification of mannose-binding protein from the crude protein extract. The purification procedure was done as described by Van Damme et al [8] with some modifications. The column was equilibrated with 0.2 M NaCl. After passing the extract through the column, the column was washed with 0.2 M NaCl until the A280 was less than 0.01. Then, the bound protein was eluted with 20 mM acetic acid. The protein solution was immediately adjusted to pH 7 with 1 M Tris base. Finally, the protein was washed with a solution of 200 mM Tris-HCl and 20 mM EDTA, pH 8.0, by means of molecular filtration (Centricon YM-3; Amicon, Beverly, USA). The purity was determined by 8%-20% SDS-PAGE.

SDS-PAGE

The protein samples were mixed with an equal volume of solubilizing solution (100 mM Tris-HCl, pH 6.8, 2% SDS, 10% Glycerol, 1.4 M 2-mercaptoethanol, and 0.002% bromophenol blue) and heated in boiling water for 2 min. In some cases, 2-mercaptoethanol and/or heating were omitted. The protein mixtures were subjected to SDS-PAGE with a continuous gradient of 8%-20% acrylamide [12]. The gel was stained with 0.1% Coomassie Brilliant Blue R-250 in destaining solution (40% methanol and 10% acetic acid) and destained in the destaining solution. The molecular mass standard marker mixture (Amersham, Buckinghamshire, UK) contained phosphorylase b (97 kDa), bovine serum albumin (66 kDa), ovalbumin (45 kDa), carbonic anhydrase (30 kDa), trypsin inhibitor (20.1 kDa) and alpha-lactalbumin (14.4 kDa).

Liquid chromatography-tandem mass spectrometry (LC-MS/MS)

The protein bands with molecular mass of 53.7 kDa and 14.5 kDa were obtained from the sample prepared in the absence of 2-mercaptoethanol and without heating while a single band of 14.5 kDa was obtained from the sample prepared with 2-mercaptoethanol and heating. The bands were excised from the SDS-PAGE gel. Trypsin was used for in-gel digestion. The peptide fragments were then analyzed by LC-MS/MS (LTQ Linear Ion Trap Mass Spectrometer, ThermoFinnigan, San Jose, USA). Based on the LC-MS/MS results, a search in nr.FASTA by BioworkTM 3.1 SR1 (ThermoFinnigan) was performed to identify the protein bands. The LC-MS/MS and database search were done at the Bioservice Unit, National Science and Technology Development Agency, Bangkok, Thailand.

Native-PAGE and Ferguson plot

The purified lectin was subjected to native-PAGE with 6%, 8%, 10%, 12% and 14% acrylamide [13]. The molecular mass standard marker mixture (Serva, Heidelberg, Germany) contained ferritin horse (450 kDa), catalase bovine (240 kDa), albumin bovine (67 kDa), albumin egg (45 kDa) and myoglobin equine (17.8 kDa). The gels were stained with Coomassie brilliant blue R-250 as described above. Ferguson plot was done as previously described [14].

Isoelectric focusing (IEF)

IEF was performed on a slap gel of 5% acrylamide containing ampholytes, pH 3-10 (Fluka, Buchs, Switzerland) [15]. The gel's dimensions were 5 cm´10 cm´0.5 mm (separating distance´width´thickness). The electrode wicks were soaked in 0.02 M NaOH (cathodic wick) and 0.02 M acetic acid (anodic wick). The untreated purified lectin or purified lectin heated (2 min at 100 ºC) in the presence of 0.7 M 2-mercaptoethanol was applied to the gel surface using a slot made up of plastic sheet; isoelectric point calibration standards (Pharmacia, Piscataway, USA) were applied, as well. The gel was subjected to electrophoresis for 15 min at 100 volts, followed by 15 min at 200 volts and 30 min at 700 volts. The gel was fixed in 20% tricholoacetic acid for 10 min, followed by 3 washes of 5 min in a solution of 50% methanol and 12% acetic acid, and 2 washes of 5 min in a solution of 40% methanol and 10% acetic acid. The washed gel was stained with Coomassie brilliant blue R-250.

Determination of sulfhydryl groups

Sulfhydryl groups in untreated purified D. findleyanum agglutinin (DFA) and 2-mercaptoethanol treated DFA were determined using DTNB (Ellman's reagent) in 0.1 M PBS, pH 8.5 [7,16]. The 2-mercaptoethanol-treated DFA was washed extensively with PBS by means of molecular filtration before reacting with DTNB.

Solid-phase method

Binding activity of the lectin was determined by binding horseradish peroxidase (HRP), a mannose-rich glycoprotein [17], to the purified lectin using the solid-phase method. Wells of F96 Maxisorp Immuno plate (Nunc, New York, USA) were incubated with 50 ml purified lectin (corresponding to 1 mg protein) overnight at 4 ºC followed by incubation at 37 ºC for 30 min. After 5 washes with PBS (137 mM NaCl, 2.68 mM KCl, 10 mM Na2HPO4 and 1.7 mM KH2PO4, pH 7.4), the wells were incubated with 100 ml 5% bovine serum albumin in PBS for 60 min at 37 ºC. Followed by 5 washes with PBS and incubation with 50 ml 0.02% HRP (Sigma-Aldrich) in 0.1 M sodium phosphate buffer, pH 6.0, for 2 h at 37 ºC. For binding competition, mannose (10, 20, 30, 40 or 50 mM), glucose, galactose, arabinose, ribose or xylose (30, 60, 90, 120 or 150 mM) was present in the enzyme solution. The wells were washed twice with PBS containing 0.05% Tween and five times with PBS. Enzymatic activity was determined using the ABTS substrate [0.1 M citrate buffer, pH 4.2, containing 0.5 mg/ml 2,2'-azino-bis(3-ethylbenzo-thiazoline-6-sulfonic acid) and 0.03% hydrogen peroxide]. The absorbance was measured at 415 nm on a microplate reader. The percentage of binding activity was calculated and measured against the control (without competition with sugar), the absorbance of which was designated as 100% activity. To determine the effect of heat, the lectin was incubated for 10 min at 85 ºC followed by 30 min at 37 ºC.

To determine the effect of the sulfhydryl content on binding activity, the same method was repeated, though the incubation procedure was changed. One microgram protein with 2-mercaptoethanol (0.14, 0.35, 0.56 or 0.7 M) or iodoacetamide (0.14 or 0.35 M) was incubated in wells for 10 min at 85 ºC followed by 30 min at 37 ºC. Blank wells were incubated with only 2-mercaptoethanol or iodoacetamide, without protein. Control wells were incubated with only the lectin, without 2-mercaptoethanol or iodoacetamide. The percentage of binding activity was calculated and measured against the control, the absorbance of which was designated as 100% activity.

Hemagglutination assay

Hemagglutination activity of the purified lectin was determined with trypsinized chicken erythrocytes according to the procedures previously described [18] with some modifications. Chicken erythrocytes were prepared from fresh blood collected with anticoagulant. After washing four times with PBS (50 mM sodium phosphate buffer, pH 7.4, containing 150 mM NaCl), 4% erythrocyte suspension in PBS containing 1 mg/ml trypsin (Sigma-Aldrich) was incubated for 30 min at 37 ºC. The trypsin-treated erythrocytes were washed four times with PBS and made into 4% erythrocyte suspension. Two-fold serial dilution of 50 ml purified protein was made in 50 ml PBS on a microplate. To the 50 ml remaining in each well, 50 ml 4% erythrocyte suspension was added. The plate was incubated for 1 h at room temperature and examined for visible agglutination. Under some conditions, the protein was heated (5 min at 100 ºC) in the presence or absence of 0.7 M 2-mercaptoethanol before being used.

Fungus growth assay by drop plate method

The inhibition effect of purified lectin on fungus growth was determined using Alternaria alternata. The fungus was grown on potato dextrose agar plate for 7 d at 30 ºC. When the diameter of the mycelia was approximately 4 cm, wells of 0.5 cm in diameter were made 1 cm from the rim. Twenty-five micrograms purified lectin in 80 ml dissolving solution (200 mM Tris-HCl, 20 mM EDTA, pH 8.0) with 4 different treatments were added to the wells. The treatments were as followed: untreated, treated with 0.14 M 2-mercaptoethanol, heated at 100 ºC for 5 min, treated with 2-mercaptoethanol and heat. The dissolving solution and the dissolving solution containing 0.14 M 2-mercaptoethanol were used as the controls. After incubation for another 4 d, growth inhibition zones were observed.

Results

Purification of mannose-binding protein

The SDS-PAGE results indicated that the crude protein extract contained many protein bands, whereas the protein eluted from the mannan-agarose column contained only one band at 14.5 kDa (Fig. 1). The flow-through also showed many bands, but the intense 14.5 kDa protein band disappeared. However, when the sample preparations were done without heating, in either the presence or absence of 2-mercaptoethanol, the purified protein showed two bands at 14.5 kDa and 53.7 kDa on SDS-PAGE. A greater amount of the 53.7 kDa protein band was seen in the sample prepared in the presence of 2-mercaptoethanol (Fig. 2).

Protein identification by LC-MS/MS

The purified protein at 53.7 kDa and 14.5 kDa gave the same sequence tags by LC-MS/MS (Table 1). Using the database search, the tags were identified as parts of a mannose-binding lectin precursor from D. officinale called D. officinale agglutinin (DOA) [9]. The protein, therefore, was named D. findleyanum agglutinin (DFA).

Native form of DFA and its sulfhydryl content

Relative migrations of standard proteins and DFA on native-PAGE with 6%, 8%, 10%, 12% and 14% acrylamide were used to determine DFA molecular mass by Ferguson plots (Figs. 3 and 4). The protein contained one band at 56.2 kDa. However, when IEF was performed, there were three bands of native DFA with pI of 5.18, 4.87, and 4.72. Interestingly, 2-mercaptoethanol-treated DFA showed only one band with pI of 5.18 (Fig. 5).

Native DFA had approximately 1.6 sulfhydryl groups per molecule, whereas the 2-mercaptoethanol-treated DFA contained approximately 6.9 sulfhydryl groups per molecule, by using Ellman's reagent.

Binding specificity of DFA

Binding activity of DFA in the presence of sugar was calculated and measured against the control (Fig. 6). Mannose showed 50% inhibition at about 15 mM, whereas galactose showed only 20% inhibition at 150 mM. No inhibition was seen in the presence of glucose, arabinose, ribose or xylose. Therefore, DFA exhibited greater specificity towards mannose than towards other sugars.

Effect of heat and 2-mercaptoethanol on lectin activity

Binding activity was not affected by heat, but it was increased by the addition of 2-mercaptoethanol (Figs. 7 and 8). The highest activity, approximately three to four times that of the control, occurred after the addition of 0.7 M 2-mercaptoethanol. In contrast, the activity was largely inhibited by the addition of iodoacetamide. The remaining activity was only 27% when 0.35 M iodoacetamide was added.

The purified native DFA inhibited mycelial growth of Alternaria alternata and inhibitory effect was not changed by heat treatment (Fig. 9). The lectin showed only a slightly better inhibitory effect when it was treated with 2-mercaptoethanol without heat. However, heat treatment showed a synergistic effect of the inhibitory activity of DFA treated with 2-mercaptoethanol.

For hemagglutination activity, 0.18 mg DFA and 0.18 mg heated DFA was the lowest amount that agglutinated chicken erythrocytes, whereas 0.09 mg DFA heated in the presence of 2-mercaptoethanol could precipitate the erythrocytes (data not shown).

Discussion

This work is the first study on the purification and characterization of native mannose-binding lectin in genus Dendrobium. SDS-PAGE indicated that only one protein, with a molecular mass of 14.5 kDa, bound to the mannan-agarose column (Fig. 1). Protein identification was performed using LC-MS/MS and a database search. Three sequence tags were identified as parts of DOA, a mannose-binding lectin from D. officinale [9], and the protein was named D. findleyanum agglutinin (DFA). The binding specificity of DFA (GenBank accession no. ABU62812) was then determined using the solid-phase method. Binding of HRP to DFA was strongly competed by mannose, especially compared to galactose, glucose, arabinose, ribose and xylose. The results indicated DFA has significantly binding specificity towards mannose (Fig. 6).

DFA appears to be an important substance for D. findleyanum's defense functions, as it inhibited the growth of Alternaria alternata and was the largest band in the crude extract (Fig. 1). The gradient SDS-PAGE with the unheated sample showed an additional band at 53.7 kDa (Fig. 2). Identification using the proteomic method yielded the same result in the upper band as in the 14.5 kDa band (Table 1). This band, therefore, was the native complex of DFA. The native-PAGE, with varying concentrations of acrylamide and Ferguson plot, confirmed the conclusion. One protein band was seen on the gels; it had molecular mass of 56.2 kDa by Ferguson plot (Figs. 3 and 4), similar in size to the upper band on the SDS-PAGE. Therefore, the native complex of DFA is a homotetramer that differs from the other orchid mannose-binding lectins [8-10]. The monomers are not linked by disulfide bond since heated DFA in the absence of 2-mercaptoethanol yielded one band on the gradient SDS-PAGE with a molecular mass of 14.5 kDa (Fig. 2). However, the association force between the monomers appears to be strong since SDS without heat could not destroy all of the complexes in the lectin samples.

Free sulfhydryl groups and 2-mercaptoethanol were found to be important for stability and the activity of Aspidistra elatior Blume lectin and some galectins [7,18,19]; this information was applied to DFA. Since a greater amount of the 53.7 kDa protein was seen on the SDS-PAGE when 2-mercaptoethanol was added to the sample without heating (Fig. 2), we assumed that the increased sulfhydryl content strengthened the association force among the monomers and stabilized the conformation of DFA. This made the DFA more resistant to SDS, a strong dissociating substance. However, there was still a question as to whether the higher sulfhydryl content increased DFA activities. Binding activity, antifungal activity and hemagglutination activity were determined in order to address the question. The binding activity to HRP increased after treatment with 2-mercaptoethanol but decreased after treatment with iodoacetamide. Antifungal activity of DFA also increased after treatment with 2-mercaptoethanol, and similar results were obtained from the hemagglutination test. To find more information on DFA, IEF was performed to find its pI. Interestingly, there were three bands of native DFA that had different pI, 5.18, 4.87 and 4.72 (Fig. 5), indicating that native DFA has three isoforms. Since DFA treated with 2-mercaptoethanol showed only one band at pI of 5.18, the various isoforms may be caused by different numbers of the sulfhydryl groups. In addition, the 2-mercaptoethanol-treated DFA showed higher activities than untreated DFA; the isoform with pI of 5.18 appeared to be the most active form and contained the largest number of thiol groups. However, the treated DFA's higher pI is not the result of ionization of the free sulfhydryl group, because this functional group is an acidic group. Therefore, the varying pI of DFA isoforms is the result of different conformations and the cysteine residues located in the area important for DFA conformation. Similar to the previous report on cysteine residues in galectin-1 [18], the slow rate of DFA inactivation by iodoacetamide suggested that the location of cysteine residues was partially inside the protein molecule and may not be a part of the active site (Fig. 8). As all activities tested were not decreased by heating up to 100 ºC, DFA should be a thermostable protein. This property is different from DOA2 whose antifungal activity was destroyed by heat [10], and was never found in mannose-binding lectins from the other orchids.

Our work is the first report on the native mannose-binding lectin from Orchidaceae, a homotetramer containing more than one conformational form. It shows higher thermostability than most of the other reported mannose-binding lectins. Its conformations and activities are clearly affected by the sulfhydryl content of the molecule. Additional investigations are required to identify useful applications for the lectin.

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