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Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain
Cell Biology International 31 (2007) 578e585 www.elsevier.com/locate/cellbi
Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain M. Shamsul Ola*, M. Tabish, F.H. Khan, Naheed Banu Department of Biochemistry, Faculty of Life Sciences, A.M.U. Aligarh, UP, India Received 23 September 2006; revised 22 November 2006; accepted 29 November 2006
Abstract An increasing number of galectins have been found in various animal species, the most abundant of which is galectin-1. The purpose of the present study was to purify and characterize galectin-1 from buffalo brain. We purified the galectin using a combination of ammonium sulphate fractionation and affinity chromatography and the homogeneity was determined by both native polyacrylamide gel electrophoresis (PAGE) and denaturing SDS-PAGE. The molecular weight of the galectin as determined by SDS-PAGE under reducing conditions and by gel filtration column under native conditions was 13.8 and 24.5 kDa, respectively, suggesting a dimeric form of galectin. The most potent inhibitor of the galectin activity was lactose, giving complete inhibition of hemagglutination at 0.8 mM. Galectin showed higher specificity towards human blood group A. Free thiol groups were estimated at a molar ratio of 2.9. The effects of alkylating reagents (iodoacetate and iodoacetamide) on saccharide binding of the galectin were studied. Both alkylating reagents significantly inactivated the activity of the galectin within 20 min. The temperature and pH stability of the galectin were determined. Our findings based on physico-chemical properties, carbohydrate and blood group specificities of the galectin may have future implications in biological and clinical applications. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Animal lectin; Galectin; Buffalo brain galectin-1; Carbohydrate specificity; Affinity purification; Human blood types
1. Introduction Galectins are classified as a family of animal lectins that bind to b-galactoside-containing carbohydrate structures. Members in this family require fulfillment of two criteria: affinity for b-galactosides and significant sequence similarity in the carbohydrate recognition domain (Barondes et al., 1994). An increasing number of galectins have been found in various animal species, especially in mammals. Thus, the number of mammalian galectins has reached as many as 15 (galectin115), which are well characterized. The predominant forms of galectins are dimers, whereas sponge galectins have been proposed to form trimers and or tetramers. The most common and * Corresponding author. Present address: H166, Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, 500 University Drive, Hershey, PA 17033, USA. Tel.: þ1 717 531 6986; fax: þ1 717 531 7667. E-mail address: [email protected] (M.S. Ola).
abundant galectin in mammalian tissues such as muscle, heart, lung, placenta, spleen, brain and small intestine is galectin-1 which has subunit molecular weight very close to 14 kDa (Clerch et al., 1988; Ahmed et al., 1996; Ola et al., 2001; Franco-Fraguas et al., 2003). The majority of galectins have a highly conserved carbohydrate binding site with affinity for lactose and N-acetyllactosamine (Leffler and Barondes, 1986; Ahmed and Vasta, 1994). The strongest interaction is with galactose residue but interaction with glucose is also significant, making the affinity for lactose 50e150 fold higher as compared to galactose (Leffler and Barondes, 1986). Each galectin has a unique and fine specificity. For example, the affinity of galectin-1 for blood group A, tetrasaccharide, is about 100 fold lower than that galectin-3. Certain mucin derived saccharides preferentially bind to galectin-3 but do not bind to galectin-1 (Sparrow et al., 1987). Galectin-1, -3 and -5 also differ in their affinity for certain disaccharides (Leffler and Barondes, 1986). In absence of a well defined function of galectins, several biological functions have been proposed. A number of observations
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have indicated their possible involvement in a variety of cellular processes such as adhesion (Perillo et al., 1998), proliferation (Sakaguchi et al., 2006), modulation (Rabinovich et al., 2002) and signal transduction pathways (Hahn et al., 2004). The study of galectin has been an area of active research in biomedical sciences, as these molecules play an important role in a variety of biological processes. Experimental evidences support the possible use of galectins in therapy as immuno-suppressors (Kilpatrick, 2002), anti-inflammatory agents (Baum et al., 2003), anti-metastatics (Camby et al., 2006), and also as a potential therapeutic target in some neurodegenerative pathologies (Chang-Hong et al., 2005; Kadoya and Horie, 2005). Galectin-1 expression by neuronal and glial cells is closely correlated with regeneration after injury (McGraw et al., 2005) and the level of auto-antibodies to galectin-1 is significantly higher in patients with neurological disorders than in healthy controls. Galectin-1 induces astrocyte differentiation and enhances the production of brain derived neurotropic factor, which plays an important role in the survival, differentiation and synaptic plasticity of neurons (Sasaki et al., 2004). Galectin-1 also plays important roles in axonal regeneration and promotes proliferation of adult neural stem cells (Sakaguchi et al., 2006; Horie and Kadoya, 2004). It is evident from numerous studies that mammalian brain galectins participate in a number of biological processes including their potential use as a therapeutic target. However, the brain galectin-1 proteins that have been purified and characterized have been limited to human, bovine, caprine, rat and mouse brain (Caron et al., 1987; Ola et al., 2001; Bladier et al., 1989). The purpose of the present study is to contribute to the increasing knowledge of the diversity, structure, and functions of the galectins. This study serves to demonstrate the purification, partial characterization, and determination of carbohydrate and blood group specificities of the hitherto unreported new galectin from water buffalo brain extracts. 2. Materials and methods Proteins, sugars, divinyl sulfone and Bradford protein assay dye reagents were purchased from Sigma Chemical Co., Sephadex G-100 and Sepharose 4B were from Pharmacia, Sweden. Mirotiter plates (V-shaped, 96 wells) were purchased from Laxbro, India. All other chemicals used were of analytical grade. All the experimental protocols were performed according to the guideline of Animal Care and Use Committee of the University.
2.1. Preparation of bovine brain extract Total fresh brain tissue was cut into small pieces after removal of meningeal membrane and suspended (1 gm/2 ml) in ice chilled buffer A (75 mM sodium phosphate pH 7.2, containing 0.15 M NaCl, 5 mM 2-mercaptoethanol, 30 mM lactose, 10 mM EDTA and 0.02% (w/v) sodium azide). Homogenization was carried out at 4 C in a stainless steel vessel using a table homogenizer at full speed for three rounds of 1 min with intervals of 1 min. The homogenate was centrifuged in a refrigerated centrifuge at 10,000 rpm for 30 min at 4 C and supernatant collected was further sedimented at 40,000 rpm for 1 h. The supernatant was brought to 30% saturation by addition of solid ammonium sulphate and maintained under gentle agitation overnight at 4 C. The precipitated material was removed by centrifugation and the supernatant was brought to 70% saturation with solid ammonium sulphate. After agitation for 6 h at 4 C, the centrifuged precipitate (1/5 of the initial
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extraction buffer volume) was collected and dissolved in buffer B (75 mM sodium phosphate pH 7.2, containing 0.15 M NaCl, 5 mM 2-mercaptoethanol, and 0.02% (w/v) sodium azide). After extensive dialysis against buffer B, the samples were centrifuged for 15 min at 15,000 rpm at 4 C to remove any aggregate formed and the clear supernatant was subjected to affinity chromatography.
2.2. Preparation of lactosyl-Sepharose 4B column The procedure of Ersson et al. (1973) was adopted. One hundred ml of sedimented Sepharose 4B beads were suspended in one hundred ml of 0.5 M Na2CO3 and washed several times until pH 11.0 was obtained. The gel was filtered over a Buckner funnel. The drained gel was collected and resuspended in 100 ml of 0.5 M Na2CO3 and 10 ml of divinyl sulfone was slowly added. The mixture was stirred gently for 70 min at 23 C, filtered on Buchner funnel and washed several times with 0.5 M Na2CO3. The drained gel was suspended in 100 ml of a 10% lactose (w/v) solution in 0.5 M Na2CO3. The reaction was allowed to proceed for 24 h in cold with gentle mixing. The mixture was filtered and washed in water and finally with 75 mM phosphate buffer saline pH 7.2 (PBS). The column was packed and stored in PBS containing 0.02% sodium azide in cold. The column was generally repacked after 5 uses.
2.3. Purification of buffalo brain galectin by affinity chromatography The lactose free extract of buffalo brain was applied at room temperature to lactosyl-Sepharose 4B affinity column (1 6 cm), containing 5 ml of gel equilibrated with buffer B. The unbound material was eliminated by washing with buffer B until no protein was detected in the effluent. The bound material was then eluted at 4 C with 200 mM lactose in buffer B at a flow rate of 10 ml/h and 2 ml fractions were collected and assayed for hemagglutinating activity. The fractions containing hemagglutinating activity were pooled, concentrated, and protein content was determined according to the Bradford method.
2.4. Hemagglutination assay Hemagglutinating activity of the galectin was determined with trypsinized rabbit erythrocytes according to the method of Lis et al. (1994). Rabbit erythrocytes were prepared from fresh blood, collected with anticoagulant and washed 4 times with 50 mM sodium phosphate buffer, pH 7.4 containing 0.15 M NaCl. A 4% erythrocyte suspension in PBS containing 1 mg/ml trypsin was incubated for 30 min at 37 C. The trypsin treated cells were washed 4 times with PBS buffer. Agglutination assays were carried out on microtiter V-shaped plates. Two fold serial dilution of 50 ml samples were made in 50 ml buffer B on microtiter V plates. To the 50 ml remaining in each well, was added 50 ml of the trypsinized erythrocytes suspension (4% v/v). The plate was incubated for 1 h at room temperature and examined for visible agglutination (Fig. 2). Agglutinating unit (titer) is defined as the reciprocal of the highest dilution giving a visible agglutination. The specific activity is defined as the ratio of titer to protein concentration. Human blood groups were also used to test hemagglutination with and without trypsin treatment using purified galectins from goat, sheep and buffalo brain.
2.5. Carbohydrate binding specificity Saccharide specificity of buffalo galectin was determined by sugar inhibition tests using 2-fold serial dilution of sugar solutions. To assess these inhibition tests, standard solutions of saccharides were prepared with the appropriate concentration in 0.15 M NaCl. The galectin dilution of 4 agglutinating units was used for these studies. The sugars tested were 200 mM each of lactose, gaclactose, methyl-b-D-galactopyranoside, p-nitrophenyl-b-Dgalactopyranoside, p-nitrophenyl-a-D-galactopyranoside, D-galactosamine, methyl-a-D-galactopyranoside, glucose, L-fucose, D-mannose, melibiose and D-fructose. To determine the minimum concentrations required for inhibition
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of hemagglutination by these different sugars, 25 ml serial diluted test sugars were added to each well containing 25 ml of brain galectin containing 4 agglutinating units. 50 ml of trypsinized erythrocytes was added into each well and contents were mixed by gentle shaking and incubated for an hour at room temperature. Highest dilution of the test sugar required for complete inhibition was noted.
NaOH buffer (pH 10.5e11.5). The hemagglutinating activity of the galectin was titrated against trypsinized rabbit erythrocytes.
3. Results 3.1. Hemagglutinating activity of brain homogenate
2.6. Polyacrylamide gel electrophoresis To test the homogeneity of the purified buffalo galectin, native polyacrylamide gel electrophoresis (PAGE) was performed. For estimation of molecular weight, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) of lectin samples was performed using 12.5% acrylamide gel according to the method of Laemmli (1970). The samples were diluted with sample buffer, 10 mM TriseHCl buffer, pH 8.0 containing 1 mM EDTA, 2% SDS, 10% glycerol and 2.5% (v/v) 2-mercaptoethanol, boiled for 5 min and loaded on the gel. Following which, electrophoresis gels were stained with Commassie Brilliant Blue R-250 dye and the relative mobility (Rm) of each band was calculated.
2.7. Gel filtration Molecular weight of native galectin in the presence of 30 mM lactose was determined by gel filtration on a column (1.6 59 cm) of Sephadex G-100, equilibrated with buffer B, using standard molecular weight proteins (b-galactosidase (116 kDa), hemoglobin (64 kDa), ovalbumin (45 kDa), soyabean trypsin inhibitor (20.1 kDa) and cytochrome C (12.4 kDa)). The lectin was eluted with buffer B at a flow rate of 15 ml/h. The molecular weight was calculated by comparing the Kav [(elution volume-void volume)/(bed volume-void volume)] of the galectin with the values obtained for calibration standards.
2.8. Determination of sulfhydryl groups The method used was essentially that described by Ellman (1959). For the determination of exposed -SH groups, an appropriate amount of protein was added in 100 mM Tris-EDTA buffer, pH 8.0 to give a total volume of 3.0 ml. To 3.0 ml of the solution, 0.1 ml DTNB solution (40 mg DTNB in 10 ml of 0.1 M TriseHCl, pH 8.0) was added and the color that developed was read at 412 nm after 15 min. A reagent blank was used to account for the absorption of the reagent at 412 nm. Cysteine was taken to obtain standard plot of SH groups. Thiol groups in m moles were calculated using the standard plot of cysteine per m mole of protein.
2.9. Alkylation of brain galectin The rates of reaction of the brain galectin with 70 mM alkylating agents (iodoacetate and iodoacetamide) were determined at room temperature in 75 mM PBS, pH 7.2 containing 1 mM 2-mercaptoethanol. After designated times, the alkylated brain galectin was titrated for hemagglutination assays.
2.10. Thermal stability The temperature stability of the galectin was determined by incubating 100 ml samples in 75 mM PBS buffer, pH 7.2 containing 5 mM 2-mercaptoethanol at various temperatures (30 to 70 C) for 30 min, cooling them on wet ice and titrating with trypsinized rabbit red blood cells using microtiter plate assay.
2.11. pH stability To determine the optimal pH and stability of brain galectin activity, purified galectin in 50 ml of normal saline containing 5 mM 2-mercaptoethanol were incubated with 50 ml of the following buffers for 24 h at 4 C: 0.1 M sodium acetate buffer (pH 3.5e5.5), 0.1 M sodium phosphate buffer (pH 6.5e7.5), 0.1 M TriseHCl buffer (pH 8.5e9.5) and 0.1 M glycine-
Buffalo brain extracts agglutinated trypsinized rabbit erythrocytes. The best solubilizing buffer was found to be 75 mM sodium phosphate pH 7.2, containing 150 mM sodium chloride, 30 mM lactose, and 5 mM 2-mercaptoethanol. Lactose was essential competing saccharides in aqueous buffer for efficient solubilization. Moreover, the presence of 2-mercaptoethanol was critical during the purification procedure. If the reducing agent was eliminated from aqueous buffer, no lectin activity was recovered. The lectin activity was unaffected by heat treatment up to 30 C for 30 min. However, the activity was considerably reduced by the addition of high concentration (over 0.5 M) of sodium chloride. The lectin-mediated hemagglutination of rabbit erythrocytes was inhibited by lactose at concentration as low as 5 mM but glucose and mannose had no effect even at concentration of 100 mM. 3.2. Purification of brain galectin The purification of brain galectin was based on the procedure reported by Avellana-Adalid et al. (1990) with some modification. Galectin from crude buffalo brain extract solubilized in buffer A was purified by a combination of ammonium sulphate fractionation and affinity chromatography. About 78% of total active protein was precipitated between 30e 70% ammonium sulphate saturation resulting in 2.3 fold purification with respect to soluble protein in the homogenate. After extensive dialysis against buffer B, the salt fractionated protein was further chromatographed on lactosyl Sepharose4B column equilibrated with the same buffer. Galectin was readily absorbed on the affinity column. No leaching of activity was observed during the washing of the column with the buffer. A 200 mM lactose solution was appropriate for the complete elution of the galectin in a single symmetrical peak. The results of purification with 100 gm brain are shown in Table 1. The yield of purified galectin was about 0.7 mg, which represented approximately 54% of total activity with a fold purification of about 1096. The pooled galectin fractions from the single peak of affinity column migrated as a single protein band in native PAGE and SDS-PAGE suggesting homogeneity of the preparation (Fig. 1A, B). 3.3. Molecular weight determination The molecular weight of buffalo brain galectin (BBG) under native condition was determined using gel filtration chromatography on Sephadex G-100 (1.659 cm) equilibrated with 75 mM PBS, pH 7.2 containing 1 mM 2-mercaptoethanol. The column was calibrated with marker proteins. The values of the ratio of elution volume to the void volume (Ve/Vo) for each
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Table 1 Purification of buffalo brain galectin Step fraction
Total protein (mg)a
Total activity (titer)b
Brain extract 30e70% ammonium sulphate fractionation Affinity purification
1413.3 19.6 469.0 17.6 0.71 0.1
Specific activity (titer/mg protein)
Purification (fold)
24447.6 232.0 19133.3 592.5
17.3 0.4 41.0 2.7
1 2.36
13217.7 617.0
18959.5 1828.3
1095.9
% yield of activity 100 78.2 54
Values are means SE of three different preparations from 100 g fresh tissue. a Determined by the method of Bradford. b The titer of the tested galectin is expressed as the reciprocal of the highest dilution showing agglutination of trypsinized rabbit erythrocytes.
marker proteins including BBG were calculated. Analysis of data indicated a linear relationship between log M and Ve/Vo. The value of Ve/Vo for the BBG was found to be 1.82, which corresponded to a molecular weight of 24.5 kDa. The subunit molecular weight of BBG was calculated using SDS-PAGE. Relative mobilities of buffalo galectin corresponded to Mr Value of 13,800 400. Stokes radius of BBG was determined from its elution volume on the Sephadex G100 column. The column was calibrated by determining the elution volume of several globular proteins with known stokes radii. The data was analyzed according to the theoretical treatment of Laurent and Killander (1964). The linear plot between stokes radii and (log Kav)1/2 was used for calculating the stokes radius of brain galectin and found to be 24.2 nM.
Fig. 1. Native PAGE and SDS-PAGE of buffalo brain galectin during various stages of purification. (A) Electrophoresis was performed on 10% acrylamide gel under non-reducing conditions. Lanes a, b and c contain soluble brain extract, 30e70% ammonium sulphate fraction and the purified galectin after affinity chromatography respectively. Twenty micrograms protein was applied in lane a. Lanes b and c contained 10 mg proteins. (B) Samples were electrophoressed on 12.5% SDS-PAGE under reducing conditions. Lanes a, b and c contain soluble extract, 30e70% ammonium sulphate fraction and the purified galectin on lactosyl-Sepharose 4B respectively. Thirty microgram protein was applied in lanes a and b. Lanes c and d contained 10 mg protein. Lane d shows the molecular weight markers; 68 kDa (bovine serum albumin), 43 kDa (ovalbumin), 29 kDa (carbonic anhydrase), 20 kDa (soyabean trypsin inhibitor), 14.3 kDa (Lysozyme).
3.4. Carbohydrate specificity The inhibitory effect of saccharides on agglutination of trypsinized rabbit erythrocytes by purified galectin was tested using a constant hemagglutinating unit of 4. Increasing concentration of a specific saccharide caused substantial decrease in hemagglutination. The most potent inhibitor of buffalo brain galectin was lactose giving complete inhibition of hemagglutination at concentration 0.8 mM followed by p-nitrophenylb-D-galactopyranoside at concentration 50 mM. 3.5. Blood group specificity The ability of the affinity purified galectins from goat, sheep and buffalo brain were scanned using trypsinized, as well as untrypsinized preparation of the blood cells to agglutinate human erythrocytes (Blood group A, B and O). The galectins were titrated against erythrocytes, showing positive agglutination using microtiter plate assay. The results are shown in Table 2. The concentration of brain galectins required for agglutination varied markedly with the type of cells. The rabbit erythrocytes treated with trypsin were most sensitive requiring only 1e1.5 mg/ml of galectin to agglutinate, while the concentration required for trypsinized human erythrocytes was rather high (8e16 mg/ml). The goat galectin did not agglutinate any type of untreated human erythrocytes, but it could cause agglutination of trypsinized blood cells. Moreover, goat galectin exhibited preference for type A erythrocytes rather than type O or B. Sheep brain galectin also did not agglutinate any type of native human erythrocytes, but agglutinated all the three groups of trypsinized blood cells in the order of preference A > O > B. In contrast to goat and sheep galectin, the buffalo galectin agglutinated native human erythrocytes with marked preference for the blood group A. Trypsinized human erythrocytes were found to be more sensitive towards buffalo galectin in the order of blood group A ¼ O > B. 3.6. Free thiol group Thiol groups were estimated in the purified brain galectins using DTNB as described by Ellman (1959). The results are shown in Table 3. Thiol group analysis of buffalo galectin indicated a molar ratio of 2.9, suggesting the presence of three moles of sulfhydral group per mole of protein.
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Goat galectin Untreated
Trypsin-treated
Untreated
Sheep galectin Trypsin-treated
Untreated
Buffalo galectin Trypsin-treated
A B O
e e e
16 8 8
e e e
16 4 8
8 4 8
32 16 32
Brain galectins of titer 256 against trypsinized rabbit erythrocytes were taken for human blood typing. Titer, the reciprocal of the highest dilution giving a visible hemagglutination.
3.7. Effect of thiol blocking reagents Several galectins require thiol-protecting reagents for the activity (Levi and Teichberg, 1985; Whitney et al., 1986). Like other galectins, BBG lost hemagglutinating activity in the absence of 2-mercaptoethanol. To examine the role of thiol groups in the saccharide bindings, these groups were modified by iodoacetate and iodoacetamide. The effect of these alkylating reagents on the hemagglutinating activity of BBG was studied by measuring the time course of inactivation by 70 mM each of iodoacetate and iodoacetamide in 75 mM sodium phosphate buffer, pH 7.2 containing 0.15 M NaCl and 1 mM 2-mercaptoethanol. Iodoacetamide inactivated more than 90% activity of the galectin within 20 min, but no further significant loss of activity was observed. Treatment of galectin with iodoacetate also led to a significant loss of activity within the first 20 min and a further loss of activity with more time. 3.8. Thermal and pH stabilities Temperature stability of the brain galectin was determined by the method discussed in the method section. The buffalo galectin retained full hemagglutinating activity when maintained at temperatures up to 45 C. A sudden drop of activity was observed when protein was heated between 45 to 65 C. The activity was completely abolished when the protein was heated at 70 C for 30 min. The BBG showed maximum hemagglutinating activity at pH 7.5. Brain galectin was quite stable in the pH range 6.5e8.5. 4. Discussion A growing number of lectins with specificity for b-galactosides have been characterized in brain and cerebellum as well as other tissues. The present study demonstrated that the brain Table 3 Summary of physico-chemical properties of buffalo brain galectin Subunit molecular weight (SDS-PAGE) Molecular weight (native condition) Subunit structure Stokes radius Most potent inhibitor of galectin activity pH stability Thermal stability Number of free thiol group
13.8 0.4 kDa 24.5 kDa dimer 24.2 nm lactose pH (6.5e9.0) 45e60 C 3 moles/mole of galectin
tissue extracts from buffalo contains soluble protein that was able to agglutinate trypsinized rabbit erythrocytes. The agglutination activities were specifically inhibited by lactose. The soluble protein having lectin activity (galectin) was purified using a combination of ammonium sulphate fractionation and affinity chromatography on lactosyl-Sepharose 4B. The ammonium sulphate fractionation prior to affinity chromatography resulted in a partial purification and enrichment of specific activity of galectin. Affinity chromatography was effective in eliminating nearly all of the contaminating proteins and the bound preparation was eluted in a single peak using 0.2 M lactose, resulting in a significant enhancement in specific activities as evident from Table 1. The yield of the purified galectin obtained by this method was comparable to that obtained by others (Caron et al., 1987; Bladier et al., 1989). Affinity purified brain galectin moved as a single protein band in native PAGE suggesting homogeneity of the galectin with respect to charge (Fig. 1A). The brain galectin was also found to be electrophoretically homogeneous in SDS-PAGE under reducing conditions (Fig. 1B). The subunit molecular weight of the buffalo galectin was found to be slightly lower than brain galectins isolated from bovine, rat and human (Caron et al., 1987; Avellana-Adalid et al., 1990). The molecular weight of the galectin under native condition was elucidated using gel filtration chromatography. Fetal calf skeletal muscle lectin was found to be retained on Sepahex G-100 and G-75 columns, but in presence of 25 mM lactose the lectin-gel interaction was blocked (Montelione et al., 1981). The binding site of galectins also contains a conserved tryptophan residue and the tryptophan containing peptides are known to interact with Sephadex (Hirabayashi, 1997; Hirabayashi and Kasai, 1991). Therefore in order to determine the molecular of the brain galectin under native condition using Sephadex gel filtration chromatography, 30 mM lactose was added into the protein and loaded onto the column to inhibit the galectin and gel interaction. The molecular weight was found to be 24.5 kDa. Thus the existence of non-covalent association between the subunits of the buffalo galectin is suggested. Hence, there is dimerization of the BBG like reported for most of the other galectins isolated from different mammalian tissues including human, bovine and rat brain (Cerra et al., 1985; Hirabayashi et al., 1987; Caron et al., 1987; Bladier et al., 1989). For most galectin the dimeric form provides them the potential to bind to glycoconjugates. It is reported that galectin-1 causes biphasic modulation of cell growth. The positive and negative
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effects of galectin-1 on cell growth might be influenced by both dose and the relative distribution of monomeric versus dimeric forms (Camby et al., 2006). Most galectins bind lactose and N-acetyllactosamine (LacNAc) but there are subtle differences in their carbohydrate specificities (Leffler and Barondes, 1986; Ahmed and Vasta, 1994). The BBG showed high carbohydrate specificity towards lactose and was found to be less specific for carbohydrates other than lactose (Fig. 2), suggesting that each galectin differs in its unique and fine specificity. The results of inhibition studies using a number of saccharides lead to the conclusion that brain galectin is specific for saccharides bearing non-reducing galactose linked in a b-configuration. This was supported by the evidence that the methyl-a-D-galactopyranoside and p-nitro-a-D-galactopyranoside were weak inhibitors compared to methyl-b-D-galactopyranoside and p-nitrophenyl-b-D-galactopyranoside. The nitrophenylated galactose was a more effective inhibitor of buffalo galectin activity than galactose or methyl-b-D-galactopyranoside, suggesting the involvement of hydrophobic interaction in the saccharide binding (Ali and Salahuddin, 1989). The hemagglutinating activity was unaffected by the presence of sugars like mannose, glucose, fucose and fructose. The fact that lactose is far more inhibitory for hemagglutinating activity of the galectin suggests that the carbohydrate binding site of the galectin could have extended geometry which is only partially occupied by the lactose molecule. X-ray crystallographic studies using small disaccharides predicted that the 4-OH and 6-OH groups of the galactose and the 3-OH group of the glucose (in lactose) are critically important for high affinity binding (Liao et al., 1994). As indicated in the Table 2, unlike goat and sheep brain galectin, BBG agglutinates all types of native human erythrocytes apart from agglutination of trypsin treated human blood cells. Goat and sheep brain galectins exhibited agglutination with preference for type A trypsin treated erythrocytes, rather than O and B. Thus it is possible that galectins may bind to blood group A
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determinants having GalNAc instead of Gal and GlcNAc (Sparrow et al., 1987). BBG agglutinated equally well to both blood group A and O erythrocytes, probably mediated by the polylactosaminoglycans found on the surface of human erythrocytes (Fukuda, 1985). Most of the known galectins require reducing agents such as 2-mercaptoethanol for the hemagglutinating activity and stability (Cerra et al., 1985; Southan et al., 1987) while some do not require them at all (Tracey et al., 1992; Kasai and Hirabayashi, 1996). Buffalo brain galectin strictly requires reducing agents such as 2-mercaptoethanol to show activity for long periods of time. Cerra et al. (1985) observed that for initial purification of the galectin, the reducing agent is necessary, but the reducing environment is not required for its continued activity. Moreover, it is observed that lactose maintains the brain galectin in the active form even in the absence of a reducing agent, possibly by preventing oxidative inactivation due to formation of intradisulfide bonds (Tracey et al., 1992; Cho and Cummings, 1995). The number of thiol groups titrated in the BBG was 2.9 (Table 3). In order to investigate whether free thiol groups were necessary to maintain galectins in their active form, the native brain galectin was alkylated by iodoacetate and iodoacetamide. This led to a wide range in rates at which the thiol groups were alkylated and resulted in inactivation of hemagglutination activity (Fig. 3). The inactivation of hemagglutinating activity indicates that the reduced form of cysteine is however, required for the maintenance of the galectin in the active form. The slow rate of galectin inactivation suggests that the cysteine molecule could be partially buried inside the protein and possibly not involved directly in carbohydrate binding but present at a relatively distant site (Whitney et al., 1986). It is important to point out that most galectins are not inactivated by iodoacetamide treatment (Hirabayashi et al., 1987; Ali and Salahuddin, 1989), rather some modified galectins show higher activity (Whitney et al., 1986; Clerch et al., 1988). Surprisingly, buffalo galectin was readily inactivated.
Fig. 2. Determination of minimum inhibitory concentration (MIC) of various carbohydrates required for the complete inhibition of 4 agglutinating units of buffalo brain galectin. Each well contains 25 ml of a serial diluted test sugar, 25 ml of galectin solution, and 50 ml of 4% suspension of trypsinized rabbit erythrocytes. The minimum inhibitory concentration is obtained by dividing the starting concentration of the carbohydrate (which was 200 mM for all sugars tested) by reciprocal of the highest inhibitory dilution, taking in account that due to the addition of the galectin, the dilution of the sugar is 1:2 already in the first well. NI, non-inhibitory.
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Fig. 3. Effect of iodoacetate and iodoacetamide on hemagglutinating activity of buffalo brain galectin. The galectin was diluted to get hemagglutinating units of 256 and incubated with 70 mM of either iodoacetate (,) or iodoacetamide (:) at room temperature for different time intervals (0e60 min) and residual activity was measured by microtiter plate assay. The initial activity refers to hemagglutination activity of the unincubated galectin.
The BBG was irreversibly inactivated by exposure to high temperature during a relatively short period of time. The galectin was completely inactivated at 70 C. Bovine galectin has been found to be more resistant to thermal inactivation compared to BBG as it retained 6% of total activity even at 100 C for 30 min (Ahmed et al., 1996). The BBG was quite stable between pH 6.5e9.5 and overlapped with that of its natural physiological environment as reported earlier for human spleen galectin (Ahmed et al., 1990). Galectin-1 shows the characteristics of typical cytoplasmic proteins as well as an acetylated N-terminus and a lack of glycosylation (Clerch et al., 1988). These findings, based on unique physico-chemical properties, carbohydrate and blood group specificities of the BBG may have further theoretical, biological and clinical applications. Acknowledgements Thanks are due to the University Grant Commission, New Delhi, for financial support to the author (MSO) and also to Aligarh Muslim University for providing the facilities. References Ahmed H, Vasta GR. Galectins: conservation of functionally and structurally relevant amino acid residues defines two types of carbohydrate recognition domains. Glycobiology 1994;4:545e8. Ahmed H, Allen HJ, Sharma A, Matta KL. Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site. Biochemistry 1990;29:5315e9. Ahmed H, Pohl J, Fink NE, Strobel F, Vasta GR. The primary structure and carbohydrate specificity of a beta-galactosyl-binding lectin from toad (Bufo arenarum Hensel) ovary reveal closer similarities to the mammalian galectin-1 than to the galectin from the clawed frog Xenopus laevis. J Biol Chem 1996;271:33083e94.
Ali N, Salahuddin A. Isolation and characterization of soluble beta-galactoside-binding lectins from mammalian liver. Biochim Biophys Acta 1989; 992:30e4. Avellana-Adalid V, Joubert R, Bladier D, Caron M. Biotinylated derivative of a human brain lectin: synthesis and use in affinoblotting for endogenous ligand studies. Anal Biochem 1990;190:26e31. Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, et al. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994;76:597e8. Baum LG, Blackall DP, Arias-Magallano S, Nanigian D, Uh SY, Browne JM, et al. Amelioration of graft versus host disease by galectin-1. Clin Immunol 2003;109:295e307. Bladier D, Joubert R, Avellana-Adalid V, Kemeny JL, Doinel C, Amouroux J, et al. Purification and characterization of a galactoside-binding lectin from human brain. Arch Biochem Biophys 1989;269:433e9. Camby I, Mercier ML, Lefranc F, Kiss R. Galectin-1: a small protein with major functions. Glycobiology 2006. PMID: 16840800. Caron M, Joubert R, Bladier D. Purification and characterization of a betagalactoside-binding soluble lectin from rat and bovine brain. Biochim Biophys Acta 1987;925:290e6. Cerra RF, Gitt MA, Barondes SH. Three soluble rat beta-galactoside-binding lectins. J Biol Chem 1985;260:10474e7. Chang-Hong R, Wada M, Koyama S, Kimura H, Arawaka S, Kawanami T, et al. Neuroprotective effect of oxidized galectin-1 in a transgenic mouse model of amyotrophic lateral sclerosis. Exp Neurol 2005;194(1):203e11. Cho M, Cummings RD. Galectin-1, a beta-galactoside-binding lectin in Chinese hamster ovary cells. I. Physical and chemical characterization. J Biol Chem 1995;270:5198e206. Clerch LB, Whitney P, Hass M, Brew K, Miller T, Werner R, et al. Sequence of a full-length cDNA for rat lung beta-galactoside-binding protein: primary and secondary structure of the lectin. Biochemistry 1988;27:692e9. Ellman GL. Tissue sulfhydryl groups. Arch Biochem Biophys 1959;82:70e7. Ersson B, Aspberg K, Porath J. The phytohemagglutinin from sunn hemp seeds (Crotalaria juncea). Purification by biospecific affinity chromatography. Biochim Biophys Acta 1973;310:446e52. Franco-Fraguas L, Batista-Viera F, Carlsson J. Isolation of a beta-galactosidebinding lectin from cat liver. Braz J Med Biol Res 2003;36:447e57. Fukuda M. Cell surface glycoconjugates as onco-differentiation markers in hematopoietic cells. Biochim Biophys Acta 1985;780:119e50. Hahn HP, Pang M, He J, Hernandez JD, Yang RY, Li LY, et al. Galectin-1 induces nuclear translocation of endonuclease G in caspase- and cytochrome c-independent T cell death. Cell Death Differ 2004;12:1277e86. Hirabayashi J. Application of site-directed mutagenesis to structure-function studies of carbohydrate-binding proteins. In: Gabius H-J, Gabius S, editors. Glycosciences status and perspectives. Weinheim: Chapman & Hall; 1997. p. 355e68. Hirabayashi J, Kasai K. Effect of amino acid substitution by sited-directed mutagenesis on the carbohydrate recognition and stability of human 14-kDa beta-galactoside-binding lectin. J Biol Chem 1991;266:23648e53. Hirabayashi J, Kawasaki H, Suzuki K, Kasai K. Complete amino acid sequence of 14 kDa beta-galactoside-binding lectin of chick embryo. J Biochem (Tokyo) 1987;101:775e87. Horie H, Kadoya T. Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1. Glycoconjugate J 2004;19:479e89. Kadoya T, Horie H. Structural and functional studies of galectin-1: a novel axonal regeneration-promoting activity for oxidized galectin-1. Curr Drug Targets 2005;6:375e83. Kasai K, Hirabayashi J. Galectins: a family of animal lectins that decipher glycocodes. J Biochem (Tokyo) 1996;119:1e8. Kilpatrick DC. Animal lectins: a historical introduction and overview. Biochim Biophys Acta 2002;1572:187e97. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227:680e5. Laurent TC, Killander J. A theory of gel filtration and its experimental verification. J Chromatogr 1964;14:317. Leffler H, Barondes SH. Specificity of binding of three soluble rat lung lectins to substituted and unsubstituted mammalian beta-galactosides. J Biol Chem 1986;261:10119e26.
M.S. Ola et al. / Cell Biology International 31 (2007) 578e585 Levi G, Teichberg VI. Isolation and characterization of chicken thymic electrolectin. Biochem J 1985;226:379e84. Liao DI, Kapadia G, Ahmed H, Vasta GR, Herzberg O. Structure of S-lectin, a developmentally regulated vertebrate beta-galactoside-binding protein. Proc Natl Acad Sci USA 1994;91:1428e32. Lis H, Belenky D, Rabinkov A, Sharon N. Purification of lectins and determination of their carbohydrate specificity. In: Celis JE, editor. Cell biology: a laboratory handbook, vol. 3. Academic Press, Inc.; 1994. p. 3332e8. McGraw J, Gaudet AD, Oschipok LW, Kadoya T, Horie H, Steeves JD, et al. Regulation of neuronal and glial galectin-1 expression by peripheral and central axotomy of rat primary afferent neurons. Exp Neurol 2005;195: 103e14. Montelione GT, Callahan S, Podleski TR. Physical and chemical characterization of the major lactose-blockable lectin activity from fetal calf skeletal muscle. Biochim Biophys Acta 1981;670:110e23. Ola MS, Shahwan M, Banu N. Galectin from caprine brain: Subunit structure, carbohydrate specificity and its immunological relationship. J Biochem Mol Biol Biophys 2001;5:473e80. Perillo NL, Marcus ME, Baum LG. Galectins: versatile modulators of cell adhesion, cell proliferation, and cell death. J Mol Med 1998;76:402e12.
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Rabinovich GA, Rubinstein N, Toscano MA. Role of galectins in inflammatory and immunomodulatory processes. Biochim Biophys Acta 2002;1572: 274e84. Sakaguchi M, Shingo T, Shimazaki T, Okano HJ, Shiwa M, Ishibashi S, et al. A carbohydrate-binding protein, Galectin-1, promotes proliferation of adult neural stem cells. Proc Natl Acad Sci USA 2006;103:7112e7. Sasaki T, Hirabayashi J, Manya H, Kasai K, Endo T. Galectin-1 induces astrocyte differentiation, which leads to production of brain derived neurotrophic factor. Glycobiology 2004;14:357e63. Southan C, Aitken A, Childs RA, Abbott WM, Feizi T. Amino acid sequence of beta-galactoside-binding bovine heart lectin. Member of a novel class of vertebrate proteins. FEBS Lett 1987;214:301e4. Sparrow CP, Leffler H, Barondes SH. Multiple soluble beta-galactoside-binding lectins from human lung. J Biol Chem 1987;262:7383e90. Tracey BM, Feizi T, Abbott WM, Carruthers RA, Green BN, Lawson AM. Subunit molecular mass assignment of 14,654 Da to the soluble betagalactoside-binding lectin from bovine heart muscle and demonstration of intramolecular disulfide bonding associated with oxidative inactivation. J Biol Chem 1992;267:10342e7. Whitney PL, Powell JT, Sanford GL. Oxidation and chemical modification of lung beta-galactoside-specific lectin. Biochem J 1986;238:683e9.
Purification and some properties of galectin-1 derived from water buffalo (Bubalus bubalis) brain M. Shamsul Ola*, M. Tabish, F.H. Khan, Naheed Banu Department of Biochemistry, Faculty of Life Sciences, A.M.U. Aligarh, UP, India Received 23 September 2006; revised 22 November 2006; accepted 29 November 2006
Abstract An increasing number of galectins have been found in various animal species, the most abundant of which is galectin-1. The purpose of the present study was to purify and characterize galectin-1 from buffalo brain. We purified the galectin using a combination of ammonium sulphate fractionation and affinity chromatography and the homogeneity was determined by both native polyacrylamide gel electrophoresis (PAGE) and denaturing SDS-PAGE. The molecular weight of the galectin as determined by SDS-PAGE under reducing conditions and by gel filtration column under native conditions was 13.8 and 24.5 kDa, respectively, suggesting a dimeric form of galectin. The most potent inhibitor of the galectin activity was lactose, giving complete inhibition of hemagglutination at 0.8 mM. Galectin showed higher specificity towards human blood group A. Free thiol groups were estimated at a molar ratio of 2.9. The effects of alkylating reagents (iodoacetate and iodoacetamide) on saccharide binding of the galectin were studied. Both alkylating reagents significantly inactivated the activity of the galectin within 20 min. The temperature and pH stability of the galectin were determined. Our findings based on physico-chemical properties, carbohydrate and blood group specificities of the galectin may have future implications in biological and clinical applications. Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. Keywords: Animal lectin; Galectin; Buffalo brain galectin-1; Carbohydrate specificity; Affinity purification; Human blood types
1. Introduction Galectins are classified as a family of animal lectins that bind to b-galactoside-containing carbohydrate structures. Members in this family require fulfillment of two criteria: affinity for b-galactosides and significant sequence similarity in the carbohydrate recognition domain (Barondes et al., 1994). An increasing number of galectins have been found in various animal species, especially in mammals. Thus, the number of mammalian galectins has reached as many as 15 (galectin115), which are well characterized. The predominant forms of galectins are dimers, whereas sponge galectins have been proposed to form trimers and or tetramers. The most common and * Corresponding author. Present address: H166, Department of Cellular and Molecular Physiology, College of Medicine, Pennsylvania State University, 500 University Drive, Hershey, PA 17033, USA. Tel.: þ1 717 531 6986; fax: þ1 717 531 7667. E-mail address: [email protected] (M.S. Ola).
abundant galectin in mammalian tissues such as muscle, heart, lung, placenta, spleen, brain and small intestine is galectin-1 which has subunit molecular weight very close to 14 kDa (Clerch et al., 1988; Ahmed et al., 1996; Ola et al., 2001; Franco-Fraguas et al., 2003). The majority of galectins have a highly conserved carbohydrate binding site with affinity for lactose and N-acetyllactosamine (Leffler and Barondes, 1986; Ahmed and Vasta, 1994). The strongest interaction is with galactose residue but interaction with glucose is also significant, making the affinity for lactose 50e150 fold higher as compared to galactose (Leffler and Barondes, 1986). Each galectin has a unique and fine specificity. For example, the affinity of galectin-1 for blood group A, tetrasaccharide, is about 100 fold lower than that galectin-3. Certain mucin derived saccharides preferentially bind to galectin-3 but do not bind to galectin-1 (Sparrow et al., 1987). Galectin-1, -3 and -5 also differ in their affinity for certain disaccharides (Leffler and Barondes, 1986). In absence of a well defined function of galectins, several biological functions have been proposed. A number of observations
1065-6995/$ - see front matter Ó 2006 International Federation for Cell Biology. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.cellbi.2006.11.032
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have indicated their possible involvement in a variety of cellular processes such as adhesion (Perillo et al., 1998), proliferation (Sakaguchi et al., 2006), modulation (Rabinovich et al., 2002) and signal transduction pathways (Hahn et al., 2004). The study of galectin has been an area of active research in biomedical sciences, as these molecules play an important role in a variety of biological processes. Experimental evidences support the possible use of galectins in therapy as immuno-suppressors (Kilpatrick, 2002), anti-inflammatory agents (Baum et al., 2003), anti-metastatics (Camby et al., 2006), and also as a potential therapeutic target in some neurodegenerative pathologies (Chang-Hong et al., 2005; Kadoya and Horie, 2005). Galectin-1 expression by neuronal and glial cells is closely correlated with regeneration after injury (McGraw et al., 2005) and the level of auto-antibodies to galectin-1 is significantly higher in patients with neurological disorders than in healthy controls. Galectin-1 induces astrocyte differentiation and enhances the production of brain derived neurotropic factor, which plays an important role in the survival, differentiation and synaptic plasticity of neurons (Sasaki et al., 2004). Galectin-1 also plays important roles in axonal regeneration and promotes proliferation of adult neural stem cells (Sakaguchi et al., 2006; Horie and Kadoya, 2004). It is evident from numerous studies that mammalian brain galectins participate in a number of biological processes including their potential use as a therapeutic target. However, the brain galectin-1 proteins that have been purified and characterized have been limited to human, bovine, caprine, rat and mouse brain (Caron et al., 1987; Ola et al., 2001; Bladier et al., 1989). The purpose of the present study is to contribute to the increasing knowledge of the diversity, structure, and functions of the galectins. This study serves to demonstrate the purification, partial characterization, and determination of carbohydrate and blood group specificities of the hitherto unreported new galectin from water buffalo brain extracts. 2. Materials and methods Proteins, sugars, divinyl sulfone and Bradford protein assay dye reagents were purchased from Sigma Chemical Co., Sephadex G-100 and Sepharose 4B were from Pharmacia, Sweden. Mirotiter plates (V-shaped, 96 wells) were purchased from Laxbro, India. All other chemicals used were of analytical grade. All the experimental protocols were performed according to the guideline of Animal Care and Use Committee of the University.
2.1. Preparation of bovine brain extract Total fresh brain tissue was cut into small pieces after removal of meningeal membrane and suspended (1 gm/2 ml) in ice chilled buffer A (75 mM sodium phosphate pH 7.2, containing 0.15 M NaCl, 5 mM 2-mercaptoethanol, 30 mM lactose, 10 mM EDTA and 0.02% (w/v) sodium azide). Homogenization was carried out at 4 C in a stainless steel vessel using a table homogenizer at full speed for three rounds of 1 min with intervals of 1 min. The homogenate was centrifuged in a refrigerated centrifuge at 10,000 rpm for 30 min at 4 C and supernatant collected was further sedimented at 40,000 rpm for 1 h. The supernatant was brought to 30% saturation by addition of solid ammonium sulphate and maintained under gentle agitation overnight at 4 C. The precipitated material was removed by centrifugation and the supernatant was brought to 70% saturation with solid ammonium sulphate. After agitation for 6 h at 4 C, the centrifuged precipitate (1/5 of the initial
579
extraction buffer volume) was collected and dissolved in buffer B (75 mM sodium phosphate pH 7.2, containing 0.15 M NaCl, 5 mM 2-mercaptoethanol, and 0.02% (w/v) sodium azide). After extensive dialysis against buffer B, the samples were centrifuged for 15 min at 15,000 rpm at 4 C to remove any aggregate formed and the clear supernatant was subjected to affinity chromatography.
2.2. Preparation of lactosyl-Sepharose 4B column The procedure of Ersson et al. (1973) was adopted. One hundred ml of sedimented Sepharose 4B beads were suspended in one hundred ml of 0.5 M Na2CO3 and washed several times until pH 11.0 was obtained. The gel was filtered over a Buckner funnel. The drained gel was collected and resuspended in 100 ml of 0.5 M Na2CO3 and 10 ml of divinyl sulfone was slowly added. The mixture was stirred gently for 70 min at 23 C, filtered on Buchner funnel and washed several times with 0.5 M Na2CO3. The drained gel was suspended in 100 ml of a 10% lactose (w/v) solution in 0.5 M Na2CO3. The reaction was allowed to proceed for 24 h in cold with gentle mixing. The mixture was filtered and washed in water and finally with 75 mM phosphate buffer saline pH 7.2 (PBS). The column was packed and stored in PBS containing 0.02% sodium azide in cold. The column was generally repacked after 5 uses.
2.3. Purification of buffalo brain galectin by affinity chromatography The lactose free extract of buffalo brain was applied at room temperature to lactosyl-Sepharose 4B affinity column (1 6 cm), containing 5 ml of gel equilibrated with buffer B. The unbound material was eliminated by washing with buffer B until no protein was detected in the effluent. The bound material was then eluted at 4 C with 200 mM lactose in buffer B at a flow rate of 10 ml/h and 2 ml fractions were collected and assayed for hemagglutinating activity. The fractions containing hemagglutinating activity were pooled, concentrated, and protein content was determined according to the Bradford method.
2.4. Hemagglutination assay Hemagglutinating activity of the galectin was determined with trypsinized rabbit erythrocytes according to the method of Lis et al. (1994). Rabbit erythrocytes were prepared from fresh blood, collected with anticoagulant and washed 4 times with 50 mM sodium phosphate buffer, pH 7.4 containing 0.15 M NaCl. A 4% erythrocyte suspension in PBS containing 1 mg/ml trypsin was incubated for 30 min at 37 C. The trypsin treated cells were washed 4 times with PBS buffer. Agglutination assays were carried out on microtiter V-shaped plates. Two fold serial dilution of 50 ml samples were made in 50 ml buffer B on microtiter V plates. To the 50 ml remaining in each well, was added 50 ml of the trypsinized erythrocytes suspension (4% v/v). The plate was incubated for 1 h at room temperature and examined for visible agglutination (Fig. 2). Agglutinating unit (titer) is defined as the reciprocal of the highest dilution giving a visible agglutination. The specific activity is defined as the ratio of titer to protein concentration. Human blood groups were also used to test hemagglutination with and without trypsin treatment using purified galectins from goat, sheep and buffalo brain.
2.5. Carbohydrate binding specificity Saccharide specificity of buffalo galectin was determined by sugar inhibition tests using 2-fold serial dilution of sugar solutions. To assess these inhibition tests, standard solutions of saccharides were prepared with the appropriate concentration in 0.15 M NaCl. The galectin dilution of 4 agglutinating units was used for these studies. The sugars tested were 200 mM each of lactose, gaclactose, methyl-b-D-galactopyranoside, p-nitrophenyl-b-Dgalactopyranoside, p-nitrophenyl-a-D-galactopyranoside, D-galactosamine, methyl-a-D-galactopyranoside, glucose, L-fucose, D-mannose, melibiose and D-fructose. To determine the minimum concentrations required for inhibition
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of hemagglutination by these different sugars, 25 ml serial diluted test sugars were added to each well containing 25 ml of brain galectin containing 4 agglutinating units. 50 ml of trypsinized erythrocytes was added into each well and contents were mixed by gentle shaking and incubated for an hour at room temperature. Highest dilution of the test sugar required for complete inhibition was noted.
NaOH buffer (pH 10.5e11.5). The hemagglutinating activity of the galectin was titrated against trypsinized rabbit erythrocytes.
3. Results 3.1. Hemagglutinating activity of brain homogenate
2.6. Polyacrylamide gel electrophoresis To test the homogeneity of the purified buffalo galectin, native polyacrylamide gel electrophoresis (PAGE) was performed. For estimation of molecular weight, sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE) of lectin samples was performed using 12.5% acrylamide gel according to the method of Laemmli (1970). The samples were diluted with sample buffer, 10 mM TriseHCl buffer, pH 8.0 containing 1 mM EDTA, 2% SDS, 10% glycerol and 2.5% (v/v) 2-mercaptoethanol, boiled for 5 min and loaded on the gel. Following which, electrophoresis gels were stained with Commassie Brilliant Blue R-250 dye and the relative mobility (Rm) of each band was calculated.
2.7. Gel filtration Molecular weight of native galectin in the presence of 30 mM lactose was determined by gel filtration on a column (1.6 59 cm) of Sephadex G-100, equilibrated with buffer B, using standard molecular weight proteins (b-galactosidase (116 kDa), hemoglobin (64 kDa), ovalbumin (45 kDa), soyabean trypsin inhibitor (20.1 kDa) and cytochrome C (12.4 kDa)). The lectin was eluted with buffer B at a flow rate of 15 ml/h. The molecular weight was calculated by comparing the Kav [(elution volume-void volume)/(bed volume-void volume)] of the galectin with the values obtained for calibration standards.
2.8. Determination of sulfhydryl groups The method used was essentially that described by Ellman (1959). For the determination of exposed -SH groups, an appropriate amount of protein was added in 100 mM Tris-EDTA buffer, pH 8.0 to give a total volume of 3.0 ml. To 3.0 ml of the solution, 0.1 ml DTNB solution (40 mg DTNB in 10 ml of 0.1 M TriseHCl, pH 8.0) was added and the color that developed was read at 412 nm after 15 min. A reagent blank was used to account for the absorption of the reagent at 412 nm. Cysteine was taken to obtain standard plot of SH groups. Thiol groups in m moles were calculated using the standard plot of cysteine per m mole of protein.
2.9. Alkylation of brain galectin The rates of reaction of the brain galectin with 70 mM alkylating agents (iodoacetate and iodoacetamide) were determined at room temperature in 75 mM PBS, pH 7.2 containing 1 mM 2-mercaptoethanol. After designated times, the alkylated brain galectin was titrated for hemagglutination assays.
2.10. Thermal stability The temperature stability of the galectin was determined by incubating 100 ml samples in 75 mM PBS buffer, pH 7.2 containing 5 mM 2-mercaptoethanol at various temperatures (30 to 70 C) for 30 min, cooling them on wet ice and titrating with trypsinized rabbit red blood cells using microtiter plate assay.
2.11. pH stability To determine the optimal pH and stability of brain galectin activity, purified galectin in 50 ml of normal saline containing 5 mM 2-mercaptoethanol were incubated with 50 ml of the following buffers for 24 h at 4 C: 0.1 M sodium acetate buffer (pH 3.5e5.5), 0.1 M sodium phosphate buffer (pH 6.5e7.5), 0.1 M TriseHCl buffer (pH 8.5e9.5) and 0.1 M glycine-
Buffalo brain extracts agglutinated trypsinized rabbit erythrocytes. The best solubilizing buffer was found to be 75 mM sodium phosphate pH 7.2, containing 150 mM sodium chloride, 30 mM lactose, and 5 mM 2-mercaptoethanol. Lactose was essential competing saccharides in aqueous buffer for efficient solubilization. Moreover, the presence of 2-mercaptoethanol was critical during the purification procedure. If the reducing agent was eliminated from aqueous buffer, no lectin activity was recovered. The lectin activity was unaffected by heat treatment up to 30 C for 30 min. However, the activity was considerably reduced by the addition of high concentration (over 0.5 M) of sodium chloride. The lectin-mediated hemagglutination of rabbit erythrocytes was inhibited by lactose at concentration as low as 5 mM but glucose and mannose had no effect even at concentration of 100 mM. 3.2. Purification of brain galectin The purification of brain galectin was based on the procedure reported by Avellana-Adalid et al. (1990) with some modification. Galectin from crude buffalo brain extract solubilized in buffer A was purified by a combination of ammonium sulphate fractionation and affinity chromatography. About 78% of total active protein was precipitated between 30e 70% ammonium sulphate saturation resulting in 2.3 fold purification with respect to soluble protein in the homogenate. After extensive dialysis against buffer B, the salt fractionated protein was further chromatographed on lactosyl Sepharose4B column equilibrated with the same buffer. Galectin was readily absorbed on the affinity column. No leaching of activity was observed during the washing of the column with the buffer. A 200 mM lactose solution was appropriate for the complete elution of the galectin in a single symmetrical peak. The results of purification with 100 gm brain are shown in Table 1. The yield of purified galectin was about 0.7 mg, which represented approximately 54% of total activity with a fold purification of about 1096. The pooled galectin fractions from the single peak of affinity column migrated as a single protein band in native PAGE and SDS-PAGE suggesting homogeneity of the preparation (Fig. 1A, B). 3.3. Molecular weight determination The molecular weight of buffalo brain galectin (BBG) under native condition was determined using gel filtration chromatography on Sephadex G-100 (1.659 cm) equilibrated with 75 mM PBS, pH 7.2 containing 1 mM 2-mercaptoethanol. The column was calibrated with marker proteins. The values of the ratio of elution volume to the void volume (Ve/Vo) for each
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Table 1 Purification of buffalo brain galectin Step fraction
Total protein (mg)a
Total activity (titer)b
Brain extract 30e70% ammonium sulphate fractionation Affinity purification
1413.3 19.6 469.0 17.6 0.71 0.1
Specific activity (titer/mg protein)
Purification (fold)
24447.6 232.0 19133.3 592.5
17.3 0.4 41.0 2.7
1 2.36
13217.7 617.0
18959.5 1828.3
1095.9
% yield of activity 100 78.2 54
Values are means SE of three different preparations from 100 g fresh tissue. a Determined by the method of Bradford. b The titer of the tested galectin is expressed as the reciprocal of the highest dilution showing agglutination of trypsinized rabbit erythrocytes.
marker proteins including BBG were calculated. Analysis of data indicated a linear relationship between log M and Ve/Vo. The value of Ve/Vo for the BBG was found to be 1.82, which corresponded to a molecular weight of 24.5 kDa. The subunit molecular weight of BBG was calculated using SDS-PAGE. Relative mobilities of buffalo galectin corresponded to Mr Value of 13,800 400. Stokes radius of BBG was determined from its elution volume on the Sephadex G100 column. The column was calibrated by determining the elution volume of several globular proteins with known stokes radii. The data was analyzed according to the theoretical treatment of Laurent and Killander (1964). The linear plot between stokes radii and (log Kav)1/2 was used for calculating the stokes radius of brain galectin and found to be 24.2 nM.
Fig. 1. Native PAGE and SDS-PAGE of buffalo brain galectin during various stages of purification. (A) Electrophoresis was performed on 10% acrylamide gel under non-reducing conditions. Lanes a, b and c contain soluble brain extract, 30e70% ammonium sulphate fraction and the purified galectin after affinity chromatography respectively. Twenty micrograms protein was applied in lane a. Lanes b and c contained 10 mg proteins. (B) Samples were electrophoressed on 12.5% SDS-PAGE under reducing conditions. Lanes a, b and c contain soluble extract, 30e70% ammonium sulphate fraction and the purified galectin on lactosyl-Sepharose 4B respectively. Thirty microgram protein was applied in lanes a and b. Lanes c and d contained 10 mg protein. Lane d shows the molecular weight markers; 68 kDa (bovine serum albumin), 43 kDa (ovalbumin), 29 kDa (carbonic anhydrase), 20 kDa (soyabean trypsin inhibitor), 14.3 kDa (Lysozyme).
3.4. Carbohydrate specificity The inhibitory effect of saccharides on agglutination of trypsinized rabbit erythrocytes by purified galectin was tested using a constant hemagglutinating unit of 4. Increasing concentration of a specific saccharide caused substantial decrease in hemagglutination. The most potent inhibitor of buffalo brain galectin was lactose giving complete inhibition of hemagglutination at concentration 0.8 mM followed by p-nitrophenylb-D-galactopyranoside at concentration 50 mM. 3.5. Blood group specificity The ability of the affinity purified galectins from goat, sheep and buffalo brain were scanned using trypsinized, as well as untrypsinized preparation of the blood cells to agglutinate human erythrocytes (Blood group A, B and O). The galectins were titrated against erythrocytes, showing positive agglutination using microtiter plate assay. The results are shown in Table 2. The concentration of brain galectins required for agglutination varied markedly with the type of cells. The rabbit erythrocytes treated with trypsin were most sensitive requiring only 1e1.5 mg/ml of galectin to agglutinate, while the concentration required for trypsinized human erythrocytes was rather high (8e16 mg/ml). The goat galectin did not agglutinate any type of untreated human erythrocytes, but it could cause agglutination of trypsinized blood cells. Moreover, goat galectin exhibited preference for type A erythrocytes rather than type O or B. Sheep brain galectin also did not agglutinate any type of native human erythrocytes, but agglutinated all the three groups of trypsinized blood cells in the order of preference A > O > B. In contrast to goat and sheep galectin, the buffalo galectin agglutinated native human erythrocytes with marked preference for the blood group A. Trypsinized human erythrocytes were found to be more sensitive towards buffalo galectin in the order of blood group A ¼ O > B. 3.6. Free thiol group Thiol groups were estimated in the purified brain galectins using DTNB as described by Ellman (1959). The results are shown in Table 3. Thiol group analysis of buffalo galectin indicated a molar ratio of 2.9, suggesting the presence of three moles of sulfhydral group per mole of protein.
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582 Table 2 Human blood type specificity of brain galectins Titer Human blood type
Goat galectin Untreated
Trypsin-treated
Untreated
Sheep galectin Trypsin-treated
Untreated
Buffalo galectin Trypsin-treated
A B O
e e e
16 8 8
e e e
16 4 8
8 4 8
32 16 32
Brain galectins of titer 256 against trypsinized rabbit erythrocytes were taken for human blood typing. Titer, the reciprocal of the highest dilution giving a visible hemagglutination.
3.7. Effect of thiol blocking reagents Several galectins require thiol-protecting reagents for the activity (Levi and Teichberg, 1985; Whitney et al., 1986). Like other galectins, BBG lost hemagglutinating activity in the absence of 2-mercaptoethanol. To examine the role of thiol groups in the saccharide bindings, these groups were modified by iodoacetate and iodoacetamide. The effect of these alkylating reagents on the hemagglutinating activity of BBG was studied by measuring the time course of inactivation by 70 mM each of iodoacetate and iodoacetamide in 75 mM sodium phosphate buffer, pH 7.2 containing 0.15 M NaCl and 1 mM 2-mercaptoethanol. Iodoacetamide inactivated more than 90% activity of the galectin within 20 min, but no further significant loss of activity was observed. Treatment of galectin with iodoacetate also led to a significant loss of activity within the first 20 min and a further loss of activity with more time. 3.8. Thermal and pH stabilities Temperature stability of the brain galectin was determined by the method discussed in the method section. The buffalo galectin retained full hemagglutinating activity when maintained at temperatures up to 45 C. A sudden drop of activity was observed when protein was heated between 45 to 65 C. The activity was completely abolished when the protein was heated at 70 C for 30 min. The BBG showed maximum hemagglutinating activity at pH 7.5. Brain galectin was quite stable in the pH range 6.5e8.5. 4. Discussion A growing number of lectins with specificity for b-galactosides have been characterized in brain and cerebellum as well as other tissues. The present study demonstrated that the brain Table 3 Summary of physico-chemical properties of buffalo brain galectin Subunit molecular weight (SDS-PAGE) Molecular weight (native condition) Subunit structure Stokes radius Most potent inhibitor of galectin activity pH stability Thermal stability Number of free thiol group
13.8 0.4 kDa 24.5 kDa dimer 24.2 nm lactose pH (6.5e9.0) 45e60 C 3 moles/mole of galectin
tissue extracts from buffalo contains soluble protein that was able to agglutinate trypsinized rabbit erythrocytes. The agglutination activities were specifically inhibited by lactose. The soluble protein having lectin activity (galectin) was purified using a combination of ammonium sulphate fractionation and affinity chromatography on lactosyl-Sepharose 4B. The ammonium sulphate fractionation prior to affinity chromatography resulted in a partial purification and enrichment of specific activity of galectin. Affinity chromatography was effective in eliminating nearly all of the contaminating proteins and the bound preparation was eluted in a single peak using 0.2 M lactose, resulting in a significant enhancement in specific activities as evident from Table 1. The yield of the purified galectin obtained by this method was comparable to that obtained by others (Caron et al., 1987; Bladier et al., 1989). Affinity purified brain galectin moved as a single protein band in native PAGE suggesting homogeneity of the galectin with respect to charge (Fig. 1A). The brain galectin was also found to be electrophoretically homogeneous in SDS-PAGE under reducing conditions (Fig. 1B). The subunit molecular weight of the buffalo galectin was found to be slightly lower than brain galectins isolated from bovine, rat and human (Caron et al., 1987; Avellana-Adalid et al., 1990). The molecular weight of the galectin under native condition was elucidated using gel filtration chromatography. Fetal calf skeletal muscle lectin was found to be retained on Sepahex G-100 and G-75 columns, but in presence of 25 mM lactose the lectin-gel interaction was blocked (Montelione et al., 1981). The binding site of galectins also contains a conserved tryptophan residue and the tryptophan containing peptides are known to interact with Sephadex (Hirabayashi, 1997; Hirabayashi and Kasai, 1991). Therefore in order to determine the molecular of the brain galectin under native condition using Sephadex gel filtration chromatography, 30 mM lactose was added into the protein and loaded onto the column to inhibit the galectin and gel interaction. The molecular weight was found to be 24.5 kDa. Thus the existence of non-covalent association between the subunits of the buffalo galectin is suggested. Hence, there is dimerization of the BBG like reported for most of the other galectins isolated from different mammalian tissues including human, bovine and rat brain (Cerra et al., 1985; Hirabayashi et al., 1987; Caron et al., 1987; Bladier et al., 1989). For most galectin the dimeric form provides them the potential to bind to glycoconjugates. It is reported that galectin-1 causes biphasic modulation of cell growth. The positive and negative
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effects of galectin-1 on cell growth might be influenced by both dose and the relative distribution of monomeric versus dimeric forms (Camby et al., 2006). Most galectins bind lactose and N-acetyllactosamine (LacNAc) but there are subtle differences in their carbohydrate specificities (Leffler and Barondes, 1986; Ahmed and Vasta, 1994). The BBG showed high carbohydrate specificity towards lactose and was found to be less specific for carbohydrates other than lactose (Fig. 2), suggesting that each galectin differs in its unique and fine specificity. The results of inhibition studies using a number of saccharides lead to the conclusion that brain galectin is specific for saccharides bearing non-reducing galactose linked in a b-configuration. This was supported by the evidence that the methyl-a-D-galactopyranoside and p-nitro-a-D-galactopyranoside were weak inhibitors compared to methyl-b-D-galactopyranoside and p-nitrophenyl-b-D-galactopyranoside. The nitrophenylated galactose was a more effective inhibitor of buffalo galectin activity than galactose or methyl-b-D-galactopyranoside, suggesting the involvement of hydrophobic interaction in the saccharide binding (Ali and Salahuddin, 1989). The hemagglutinating activity was unaffected by the presence of sugars like mannose, glucose, fucose and fructose. The fact that lactose is far more inhibitory for hemagglutinating activity of the galectin suggests that the carbohydrate binding site of the galectin could have extended geometry which is only partially occupied by the lactose molecule. X-ray crystallographic studies using small disaccharides predicted that the 4-OH and 6-OH groups of the galactose and the 3-OH group of the glucose (in lactose) are critically important for high affinity binding (Liao et al., 1994). As indicated in the Table 2, unlike goat and sheep brain galectin, BBG agglutinates all types of native human erythrocytes apart from agglutination of trypsin treated human blood cells. Goat and sheep brain galectins exhibited agglutination with preference for type A trypsin treated erythrocytes, rather than O and B. Thus it is possible that galectins may bind to blood group A
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determinants having GalNAc instead of Gal and GlcNAc (Sparrow et al., 1987). BBG agglutinated equally well to both blood group A and O erythrocytes, probably mediated by the polylactosaminoglycans found on the surface of human erythrocytes (Fukuda, 1985). Most of the known galectins require reducing agents such as 2-mercaptoethanol for the hemagglutinating activity and stability (Cerra et al., 1985; Southan et al., 1987) while some do not require them at all (Tracey et al., 1992; Kasai and Hirabayashi, 1996). Buffalo brain galectin strictly requires reducing agents such as 2-mercaptoethanol to show activity for long periods of time. Cerra et al. (1985) observed that for initial purification of the galectin, the reducing agent is necessary, but the reducing environment is not required for its continued activity. Moreover, it is observed that lactose maintains the brain galectin in the active form even in the absence of a reducing agent, possibly by preventing oxidative inactivation due to formation of intradisulfide bonds (Tracey et al., 1992; Cho and Cummings, 1995). The number of thiol groups titrated in the BBG was 2.9 (Table 3). In order to investigate whether free thiol groups were necessary to maintain galectins in their active form, the native brain galectin was alkylated by iodoacetate and iodoacetamide. This led to a wide range in rates at which the thiol groups were alkylated and resulted in inactivation of hemagglutination activity (Fig. 3). The inactivation of hemagglutinating activity indicates that the reduced form of cysteine is however, required for the maintenance of the galectin in the active form. The slow rate of galectin inactivation suggests that the cysteine molecule could be partially buried inside the protein and possibly not involved directly in carbohydrate binding but present at a relatively distant site (Whitney et al., 1986). It is important to point out that most galectins are not inactivated by iodoacetamide treatment (Hirabayashi et al., 1987; Ali and Salahuddin, 1989), rather some modified galectins show higher activity (Whitney et al., 1986; Clerch et al., 1988). Surprisingly, buffalo galectin was readily inactivated.
Fig. 2. Determination of minimum inhibitory concentration (MIC) of various carbohydrates required for the complete inhibition of 4 agglutinating units of buffalo brain galectin. Each well contains 25 ml of a serial diluted test sugar, 25 ml of galectin solution, and 50 ml of 4% suspension of trypsinized rabbit erythrocytes. The minimum inhibitory concentration is obtained by dividing the starting concentration of the carbohydrate (which was 200 mM for all sugars tested) by reciprocal of the highest inhibitory dilution, taking in account that due to the addition of the galectin, the dilution of the sugar is 1:2 already in the first well. NI, non-inhibitory.
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Fig. 3. Effect of iodoacetate and iodoacetamide on hemagglutinating activity of buffalo brain galectin. The galectin was diluted to get hemagglutinating units of 256 and incubated with 70 mM of either iodoacetate (,) or iodoacetamide (:) at room temperature for different time intervals (0e60 min) and residual activity was measured by microtiter plate assay. The initial activity refers to hemagglutination activity of the unincubated galectin.
The BBG was irreversibly inactivated by exposure to high temperature during a relatively short period of time. The galectin was completely inactivated at 70 C. Bovine galectin has been found to be more resistant to thermal inactivation compared to BBG as it retained 6% of total activity even at 100 C for 30 min (Ahmed et al., 1996). The BBG was quite stable between pH 6.5e9.5 and overlapped with that of its natural physiological environment as reported earlier for human spleen galectin (Ahmed et al., 1990). Galectin-1 shows the characteristics of typical cytoplasmic proteins as well as an acetylated N-terminus and a lack of glycosylation (Clerch et al., 1988). These findings, based on unique physico-chemical properties, carbohydrate and blood group specificities of the BBG may have further theoretical, biological and clinical applications. Acknowledgements Thanks are due to the University Grant Commission, New Delhi, for financial support to the author (MSO) and also to Aligarh Muslim University for providing the facilities. References Ahmed H, Vasta GR. Galectins: conservation of functionally and structurally relevant amino acid residues defines two types of carbohydrate recognition domains. Glycobiology 1994;4:545e8. Ahmed H, Allen HJ, Sharma A, Matta KL. Human splenic galaptin: carbohydrate-binding specificity and characterization of the combining site. Biochemistry 1990;29:5315e9. Ahmed H, Pohl J, Fink NE, Strobel F, Vasta GR. The primary structure and carbohydrate specificity of a beta-galactosyl-binding lectin from toad (Bufo arenarum Hensel) ovary reveal closer similarities to the mammalian galectin-1 than to the galectin from the clawed frog Xenopus laevis. J Biol Chem 1996;271:33083e94.
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