Bangladesh Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion-exchange chromato-grap
Trang 1Purification and characterization of three galactose specific lectins
Tanzima Yeasmin, Md Abul Kashem Tang, Abdur Razzaque and Nurul Absar
Department of Biochemistry, University of Rajshahi, Rajshahi-6205 Bangladesh
Three lectins were extracted and purified from mulberry
seeds by gel filtration of 100% ammonium sulfate saturated
crude protein extract followed by ion-exchange
chromato-graphy on DEAE and CM-cellulose The lectins were found
to be homogeneous as judged by polyacrylamide disc gel
electrophoresis The molecular masses of the lectins as
determined by gel filtration were 175 000 for MSL-1,
120 000 for MSL-2 and 89 500 for MSL-3 MSL-1 is dimer
in nature, with the two monomers held together by disulfide
bond(s), while MSL-2 and MSL-3 contain four nonidentical
subunits that are held together by nonionic hydrophobic
interactions
The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl-a-D-galactopyranoside, methyl-b-D -galactopyrano-side, lactose and raffinose The lectins MSL-1, MSL-2 and MSL-3 contained 5.7, 5.4 and 4.5% neutral sugars, respec-tively, and the sugar composition of the lectins was glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3 The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay
Keywords: mulberry seeds; galactose-specific lectins; subunit structure; hemagglutination; cytotoxicity
Mulberry (Morus alba L.) is the sole host plant of the
silkworm Bombyx mori Linn, which produces silk It
belongs to the family Moraceae, part of the genus Morus It
is a deep rooted perennial plant, widely distributed in Asia,
Europe, Africa and Latin America in a wide range of
climatic conditions varying from temperate to tropical The
silkworm can only obtain nutrients necessary for growth
from mulberry leaves
Lectin, isolated chiefly from plants, bacteria, fungi,
invertebrates and vertebrates, are nonimmunoglobulin-type
carbohydrate recognition molecules that are involved in
hemagglutination, lymphocyte transformation, inactivation
of certain types of tumor cells and precipitation of certain
polysaccharides and glycoproteins [1,2] Plant lectins
isolated from a wide variety of plants have recently attracted
great interest because of their remarkable biological
activities More recently, the lectins as the carbohydrate
binding proteins have been investigated and utilized in
various biochemical fields Some authors have described the
purification and chemical properties of lectins from many
kinds of plant seeds, such as Phaseolus vulgaris seeds [3],
Viscum album L [4], Lathyrus sativus seeds and [5] Vicia
unijuga leaves [6] Lectins are being used increasingly to
probe the structure of carbohydrates on the surfaces of
normal and malignant cells [7]
Mulberry plants are propagated either through seeds or
vegetatively Mulberry seed is oval in shape with a nearly
flat surface The seed contains about 38% carbohydrate,
32% fat and 15% protein [8] This paper describes the
purification and characterization of three galactose-specific lectins from the seeds of mulberry
M A T E R I A L S A N D M E T H O D S Mulberry seeds were collected from the experimental plot of Bangladesh Sericulture Research and Training Institute, Rajshahi Sephadex G-150, Sephadex G-75, DEAE-cellulose, CM-cellulose and Sepharose 4B were purchased from Sigma Chemical Co All the other reagents used were
of analytical grade Unless otherwise specified, all operations were performed at 4 8C
Preparation of fat free dry powder The seeds were crushed into paste using a mortar and pestle This was then mixed uniformly with precooled petroleum ether in a homogenizer at 4 8C The homogenate was filtered through a clean muslin cloth The process was repeated at least twice in order to obtain lipid-free homogenate Finally, the filtrate was clarified further by centrifugation at 8000 g,
4 8C for 10 min The precipitate obtained was collected and air-dried at room temperature
Preparation of crude protein extract The protein from fat free dry powder was extracted with five different solvents (1% CH3COOH; 10 mMTris/HCl buffer,
pH 8.4; 10 mM phosphate buffer, pH 7.2; 20 mM acetate buffer, pH 5.0 and distilled water) Water was used for preparation of crude protein extract from fat free dry powder
as the highest ratio of absorbance at 280 nm and 260 nm was found in distilled water [9] The fat free dry powder was mixed uniformly with precooled distilled water (4 mL:g21 meal) and kept overnight at 4 8C with occasional shaking The suspension was then centrifuged at 8000 g, 4 8C for
15 min The clear supernatant was collected and adjusted to
Correspondence to T Yeasmin, Department of Biochemistry,
University of Rajshahi, Rajshahi-6205 Bangladesh.
Fax: 1 880 721 750064, Tel.: 1 880 880 721 750294,
E-mail: rajucc@citechco.net or makashem72@yahoo.com
(Received 8 May 2001, accepted 21 August 2001)
Trang 2100% saturation by adding solid ammonium sulfate The
precipitate was again collected by centrifugation, dissolved
in the minimum volume of water and dialyzed against 5 mM
phosphate buffer, pH 7.6, for 24 h at 4 8C After
centrifugation the clear supernatant was used as crude
protein extract
Purification of lectin
Gel Filtration Gel filtration of crude protein extract was
performed on Sephadex G-75 using 5 mMphosphate buffer,
pH 7.6 at 4 8C
DEAE-cellulose chromatography The active protein
frac-tion obtained after gel filtrafrac-tion was dialyzed against
distilled water for 12 h and against 10 mMTris/HCl buffer
pH 8.4 overnight, and then loaded onto the DEAE-cellulose
column at 4 8C The protein was eluted from the column by
buffer containing different concentrations of NaCl (0.06,
0.18 and 0.3M)
CM-cellulose chromatography The above protein fractions
needed for further purification after DEAE-cellulose
chromatography was dialyzed 12 h against distilled water
and overnight against 5 mM phosphate buffer, pH 6.5 and
then loaded onto the column The protein was eluted from
the column stepwisely using the same buffer containing
0.2MNaCl
Polyacrylamide disc gel electrophoresis Polyacrylamide
disc gel electrophoresis was conducted at room temperature,
pH 8.4 on 7.5% gel as described by Ornstein [10] and 1%
amido black was used as staining reagent
Characterization of lectin
Molecular mass determination: gel filtration The molecular
masses of the lectins were determined by gel filtration on
Sephadex G-150 (0.75 100 cm) using lysozyme, trypsin
inhibitor, a-amylase, BSA and b-amylase as reference
proteins
Molecular mass determination: SDS/PAGE SDS/PAGE was
conducted on a 10% acrylamide gel according to Weber &
Osborn [11] and the marker proteins used were same as
those used for the gel filtration Dissociation and reduction
of proteins were performed by heating for 5 min at 100 8C
in 0.1% SDS with 0.1% 2-mercaptoethanol and the proteins
were stained with Coomassie Brilliant Blue R-250
Hemagglutination studies
Hemagglutinating activity was assayed by the serial dilution
technique using 2% albino rat red blood cells as described
by Lin et al [12] Protein solution (0.2 mL) in 5 mM
phosphate buffer saline, pH 7.2, was mixed with 0.2 mL of
2% rat red blood cell and incubated at 37 8C for 1 h The
degree of hemagglutination was observed under a
microscope
The agglutinating activity was expressed as titre (the
reciprocal of the greatest dilution at which visible
agglutination could be detected) The specific activity was
expressed as the titre per mg protein The hemagglutination
inhibition test was performed in the presence of different saccharides following the same procedure as described above
Affinity chromatography The pure proteins obtained after dialysis against 5 mM
NaCl/Pi, pH 7.2, were applied to a Sepharose 4B column previously equilibrated with the same buffer at 4 8C The adsorbed protein was eluted from the column with the buffer containing 0.2Mgalactose
Protein and carbohydrate analysis The concentration of protein was measured by the method of Lowry et al [13] using BSA as the standard The presence of sugar in the protein was detected by periodic acid Schiff’s method [14] and the total neutral carbohydrate contents of the proteins were estimated by phenol/sulfuric acid method
of Dubois et al [15] withD-glucose as the standard For identification of sugars, the lectins were hydrolyzed with 1M HCl for 4 h at 100 8C under vacuum The sugar component was determined by the one-dimensional TLC method described by Joseph & Murrell [16] using different standard sugars The chromatogram was developed with the solvent: Isopropanol, acetic acid and water (3 : 1 : 1, v/v/v) and the spots were identified by spraying with aniline/ phthalate solution
Toxicity study Cytotoxicity was studied using the eggs of the brine shrimp nauplii (Artemia salina L.) Eggs were placed in one side of
a small tank divided by a net containing 3.8% NaCl solution for hatching In the other side of the tank, a light source was placed in order to attract the nauplii Two days were allowed for the hatching of all the eggs and sufficient maturation of the nauplii for the experiment described by Meyer et al [17]
From the stock solution of the lectins (0.9 mg:mL21), 10,
20, 40, 80 and 160 mL were placed in different vials and NaCl solution was added to each vial make the volume up to
5 mL; the final concentration of the sample in the vials became 1.8, 3.6, 7.2, 14.4 and 28.8 mg:mL21, respectively One-hundred brine shrimp nauplii were then placed in each vial Three experiments were carried out for the same concentration and a control experiment was performed containing 100 nauplii in 5 mL of seawater After 24 h of incubation, the vials were observed using a magnifying glass and the number of survivors in each vial were counted and noted From this data, the mean percentage of mortality of the nauplii was calculated for each concentration
R E S U L T S Purification of mulberry seed lectins The 100% ammonium sulfate saturated crude protein extract after dialysis against 5 mM phosphate buffer, pH 7.6, was applied to a Sephadex G-75 column at 4 8C previously equilibrated with the same buffer As shown in Fig 1, the proteins were eluted as one main broad peak, i.e fraction F-1 and another small peak, i.e fraction F-2 The active
Trang 3fraction, F-1, as indicated by the solid line was pooled,
precipitated with 100% saturation by ammonium sulfate,
and purified further by ion-exchange chromatography The
fraction F-2 was not used for further study as it contained
mainly colored materials and small amounts of low
molecular mass proteins
The precipitate was dissolved in a minimum volume of
distilled water and dialyzed against 10 mMTris/HCl buffer
pH 8.4 at 4 8C for 24 h After removal of the insoluble
material, the clear supernatant was applied to a
DEAE-cellulose column at 4 8C, previously equilibrated with the
same buffer, and the protein was eluted by a linear gradient
of NaCl from 0.0 to 0.3Min the buffer The components of
F-1 were eluted as a single, but broad, peak indicating the
presence of more than one component (data not shown) In
order to separate the components, the elution was carried out
in a stepwise fashion with an increasing concentration of
NaCl in the same buffer Fig 2, shows that the components
of F-1 fraction were separated into three different fractions, F-1a, F-1b and F-1c, which were eluted with the buffer containing 0.06, 0.18 and 0.3M NaCl, respectively The fractions indicated by the solid bars were pooled separately and their homogeneity was checked by polyacrylamide disc gel electrophoresis It is evident from Fig 2 (inset) that the fractions F-1b and F-1c contained pure protein as they gave single bands while F-1a gave more than one band on the gel The fraction F-1a was further purified by CM-cellulose chromatography (see below) All three fractions displayed lectin activity
The fraction F-1a obtained after DEAE-cellulose chromatography was dialyzed overnight against 5 mM
phosphate buffer, pH 6.5, and then applied to a CM-cellulose column at 4 8C Fig 3 shows that fraction F-1a was separated into two fractions, F-1a0 and F-1a00 The F-1a0 fraction was eluted by the buffer only, while F-1a00 was eluted by the buffer containing 0.2M NaCl Of these two fractions, only F-1a0 displayed lectin activity The fraction F-1a0might contain pure protein as it gave a single band on a polyacrylamide gel (Fig 3, inset) Table 1 summarizes the data for the purification of mulberry seed lectins The fraction F-1b showed maximum hemagglutinating activity with a purification of 15.28-fold while F-1a0 and F-1c showed 12.48- and 10.69-fold increases in hemagglutinating activity, respectively Although the yield of these proteins was found to be decreased by the purification steps and over 96% of protein was lost, the purification of the proteins was increased after each subsequent purification step This low yield may be due to denaturation of the protein during the lengthy purification procedure
Molecular masses of the lectins and their subunits The molecular masses of the lectins, as determined by gel filtration, were estimated to be 175 000, 120 000 and
89 500 Da for F-1a (mulberry seed lectin-1 i.e MSL-1),
Fig 1 Gel filtration of crude protein extract on Sephadex G-75.
The crude extract (85 mg) was applied to the column (2.5 100 cm),
pre-equilibrated with 5 m M phosphate buffer, pH 7.6, at 4 8C and
developed with the same buffer.
Fig 2 Ion-exchange chromatography of
fraction F-1 on DEAE-cellulose F-1 (27 mg),
obtained from gel filtration was applied to the
column (2.1 24 cm) which was pre-equilibrated
with 10 m M Tris/HCl buffer, pH 8.4 at 4 8C and
eluted by stepwise increases of NaCl concentration
in the same buffer Insets, polyacrylamide disc gel
electrophoresis of different fractions at room
temperature on 7.5% gel (staining reagent: 1%
amido black).
Trang 4F-1b (MSL-2) and F-1c (MSL-3), respectively It was found
that in the presence of 0.1% SDS, MSL-1 gave a single
band, while MSL-2 and MSL-3 gave four distinct bands on
SDS/PAGE (Fig 4) In the presence of 0.1% SDS and 0.1%
2-mercaptoethanol, MSL-1 gave a strong band
correspond-ing to a molecular mass of 110 000 Da and a weak band
corresponding to molecular mass of 70 000 Da, while the
MSL-2 and MSL-3 gave four bands with molecular masses
of 42 000, 35 000, 25 000 and 19 000, and 35 000, 22 500,
17 000 and 14 950 Da, respectively
Affinity for Sepharose 4B
All the three purified lectins, MSL-1; MSL-2 and MSL-3
bound very tightly to Sepharose 4B even at room
temperature; the bound lectins were eluted by 5 mM
phosphate buffer, pH 7.2, containing 0.2Mgalactose (data not shown)
Hemagglutinating properties The lectins MSL-1, MSL-2 and MSL-3 agglutinated specifi-cally the albino rat red blood cells; a minimum protein concentration of 4.8, 6.7 and 10.5 mg:mL21, respectively, was needed for visible agglutination The results of the hemagglutination inhibition test of mulberry seed lectins with haptenic sugars are presented in Table 2 It is evident from the results that galactose, methyl-a-D -galactopyrano-side, methyl-b-D-galactopyranoside, lactose andD-raffinose are the most potent inhibitors for all three lectins; the b-anomers were found to be slightly more potent inhibitors than the a-anomer
Lectin concentration and carbohydrate composition Purified mulberry seed lectins in aqueous solution gave maximal absorption < 276 – 280 nm and minimal absorp-tion < 246 – 248 nm The absorbance of 1.0 at 280 nm for MSL-1, MSL-2 and MSL-3 corresponded to 0.98, 0.94 and 0.84 mg of protein, respectively, as determined by drying the proteins under vacuum Similar results were obtained when the concentration of the proteins were measured by the Lowry method
The neutral sugar contents of the lectins, MSL-1, MSL-2, and MSL-3 were found to be 5.7, 5.4 and 4.5%, respectively The sugar composition of the lectins as identified by TLC was found to be glucose and mannose for MSL-1 and galactose for both MSL-2 and MSL-3
Cytotoxic effects All three lectins were found to be toxic and the mortality rate of brine shrimp nauplii were found to be increased with concentration of the lectins As shown in the Fig 5, the LC50 (concentration at which 50% mortality of the napulii occurs)
as estimated by extrapolation was 10.96 mg:mL21 for MSL-1, 17.37 mg:mL21for MSL-2, and 6.30 mg:mL21for MSL-3
Fig 3 Ion-exchange chromatography of fraction F-1a on
CM-cellulose F-1a (12 mg), obtained from DEAE-cellulose
chromato-graphy was applied to the column (0.5 15 cm), pre-equilibrated with
5 m M sodium phosphate buffer, pH 6.5 at 4 8C and eluted by the buffer
containing NaCl Inset, polyacrylamide disc gel electrophoresis of
fraction F-1a0on 7.5% gel at room temperature (staining reagent: 1%
amido black).
Table 1 Purification of mulberry seed lectins.
Fraction
Total protein (mg)
Total hemagglutination activity (titre)
Specific activity (titre:mg21)
Yield (%)
Purification (fold)
Saturated
DEAE-cellulose fraction
CM-cellulose
Trang 5Table 2 Hemagglutination inhibition assay of mulberry seed lectins NI, No inhibition; I, Inhibition.
Proteins Sugar
Concentration (m M )
Inhibition Maximum Minimum
Fig 4 SDS/PAGE patterns of the proteins on 10% gel at room
temperature (staining reagent: Coomassie Brilliant Blue) A, F-1a 0
(MSL-) in the presence of SDS; B, F-1a0(MSL-) in the presence of SDS
and 2-mercaptoethanol; C, F-1b (MSL-2) in presence of SDS; D, F-1c
(MSL-3) in the presence of SDS.
Fig 5 Determination of LC 50 of mulberry seed lectins (W) for MSL-1 (*) for MSL-2 and (K) for MSL-3.
Trang 6D I S C U S S I O N
Three lectins have been isolated and purified from a crude
extract of mulberry seeds; the lectins are glycoproteins as
they gave an orange-yellow color in the presence of phenol/
sulfuric acid The presence of sugar in the lectins was further
confirmed by the findings that they produced a pinkish-red
band on a polyacrylamide gel when the gels are stained with
periodic acid Schiff’s staining reagent after electrophoresis
(data not shown)
The agglutination of rat red blood cells by the lectins
was inhibited specifically in the presence of galactose,
methyl-a-D-galactopyranoside, methyl-b-D
-galactopyrano-side, lactose and raffinose This finding was further
supported because all three lectins showed binding affinity
to Sepharose 4B It is concluded from the above findings that
mulberry seeds contained at least three lectins that are
specific for D-galactose Although the purified mulberry
seed lectins were galactose-specific, the crude protein
extract of mulberry seeds did not bind to Sepharose 4B
column at room temperature or at 4 8C This may be due to
nonexposure of the saccharide binding sites of lectins in the
crude state
Although the lectins purified from mulberry seeds are
similar in their sugar specificity, they are found to be quite
different from each other in respect to molecular mass,
subunit structure, neutral sugar content and sugar
com-position The three lectins were each found to migrate as a
single band with slightly different mobilities on
polyacryl-amide gels In the presence of SDS, MSL-1 moved as a
single band while MSL-2 and MSL-3 were transformed into
four subunits of different molecular mass Further, in the
presence of SDS and 2-mercaptoethanol, MSL-1 was
trans-formed into two subunits From these results, it was
con-cluded that MSL-1 is dimer, with the two monomers held
together by disulfide bond(s), while MSL-2 and MSL-3 are
heterotetrameric, with the monomers held together by
nonionic hydrophobic interaction Although the lectins
purified from mulberry seeds are quite different from most
of the lectins purified from other sources, the subunit
structure of mulberry seed lectins are very similar to those of
ant egg lectins [18] The lectins purified from plant sources
contained mostly four subunits of two identical pairs, e.g
Indian bean (Dolichos lablab L.) [19], Arbus precatorius
[20], and Ricinus comminis agglutinin [21] and very few
contained four subunits of identical molecular mass, e.g
Phaseolus vulgaris [3]
The mulberry seed lectins, like those from Abrus
precatorus, Ricinus communis and mistletoe, are toxic in
nature However it has yet to be determined whether all the
subunits of mulberry seed lectins possess toxicity in addition
to their hemagglutinating properties
In conclusion, the purified lectins, MSL-1, MSL-2 and
MSL-3, besides being specific for rat red blood cell
agglutination, are members of the galactose-binding lectins
A C K N O W L E D G E M E N T S
The authors thank Dr Abdul Aziz Sarkar, Senior Scientific Officer,
Bangladesh Sericulture Research and Training Institute, Rajshahi,
Bangladesh for supplying mulberry seeds during the period of the
research.
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