Original Paper
|
|
|
file on Synergy OPEN |
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 ?C) 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 ?C followed by incubation at 37 ?C 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 ?C. 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 ?C. 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 ?C followed by 30 min at 37 ?C.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 ?C
followed by 30 min at 37 ?C. 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 ?C. 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 ?C) 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 ?C. 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 ?C 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 ?C, 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.
References
1 Van Damme EJM, Peumans
WJ, Barre A, Rouge P. Plant lectins: a composite of several distinct families
of structurally and evolutionary related proteins with diverse biological role.
Crit Rev Plant Sci 1998, 17: 575692
2 Jiang JF, Han Y, Xing LJ,
Xu YY, Xu ZH, Chong K. Cloning and expression of a novel cDNA encoding a
mannose-specific jacalin-related lectin from Oryza sativa. Toxicon 2006,
47: 133139
3 Van Damme EJM, Allen AK,
Peumans WJ. Leaves of the orchid twayblade (Listera ovata) contain a
mannose-specific lectin. Plant Physiol 1987, 85: 566569
4 Barre A, Bourne Y, Van
Damme EJM, Peumans WJ, Rouge P. Mannose-binding plant lectins: different
structural scaffolds for a common sugar-recognition process. Biochimie 2001,
83: 645651
5 Balzarini J, Schols D,
Neyts J, Van Damme E, Peumans W, De Clercq E. ?-(1-3)- and
?-(1-6)-D-mannose-specific plant lectins are markedly inhibitory to human
immunodeficiency virus and cytomegalovirus infections in vitro.
Antimicrob Agents Chemother 1991, 35: 410416
6 Balzarini J, Neyts J,
Schols D, Hosoya M, Van Damme EJM, Peumans WJ, De Clercq E. The
mannose-specific plant lectins from Cymbidium hybrid and Epipactis
helleborine and the (N-acetylglucosamine)-specific plant lectin from Urtica
dioica are potent and selective inhibitors of human immunodeficiency virus
and cytomegalovirus replication in vitro. Antiviral Res 1992, 18:
191207
7 Xu X, Wu C, Liu C, Luo Y,
Li J, Zhao X, Van Damme E et al. Purification and characterization of a
mannose-binding lectin from the rhizomes of Aspidistra elation Blume
with antiproliferative activity. Acta Biochim Biophys Sin 2007, 39: 507519
8 Van Damme EJM, Smeets K,
Torrekens S, Van Leuven F, Peumans WJ. Characterization and molecular cloning
of mannose-binding lectins from the Orchidaceae species Listera ovata, Epipactis
helleborine and Cymbidium hybrid. Eur J Biochem 1994, 221: 769777
9 Chen Z, Sun X, Tang K.
Molecular cloning and characterization of mannose-binding lectin gene from Dendrobium
officinale. J Plant Biochem Biotech 2005, 14: 3336
10 Chen Z, Sun X, Tang K. Cloning
and expression of a novel cDNA encoding a mannose-binding lectin from Dendrobium
officinale. Toxicon 2005, 45: 535540
11 Holttum RE. A Revised Flora of
Malaya: An Illustrated Systematic Account of the Malayan Flora Including
Commonly Cultivated Plants Vol. I Orchid of Malaya. Singapore: The Government
Printing Office, 1964
12 Laemmli UK. Cleavage of
structural proteins during the assembly of the head of bacteriophage T4. Nature
1970, 227: 680685
13 Davis BJ. Disc electrophoresis
II: Method and application to human serum protein. Ann N Y Acad Sci 1964, 121:
404427
14 Kong Y, Kin WB, Kang SY, Cho
SY. Molecular weight of major component proteins in crude saline extract of
adult Paragonimus westermani. Korean J Parasitol 1991, 29: 113120
15 Van den Berg BM. Isoelectric
focusing in the vegetable seed industry. Electrophoresis 1998, 19: 17801787
16 Ellman GL. Tissue sulfhydryl
groups. Arch Biochem Biophys 1959, 82: 7077
17 Heth CA, Bernstein MH.
Mannose-sensitive HRP endocytosis by the retinal pigment epithelium. Exp Eye
Res 1991, 52: 7582
18 Ola MS, Tabish M, Khan FH, Banu
N. Purification and some properties of galectin-1 derived from water buffalo (Bubalus
bubalis) brain. Cell Biol Int 2007, 31: 578585
19 Cerra RF, Gitt MA, Barondes SH. Three
soluble rat b-galactoside-binding lectins. J Biol Chem 1985, 260: 1047410477