Purification and characterization of three galactose specific lectins from Mulberry seeds ( Morus sp.) 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. MATERIALS AND METHODS 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% CH 3 COOH; 10 mM Tris/HCl buffer, pH 8.4; 10 m M 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 : g 21 meal) and kept overnight at 4 8C with occasional shaking. The suspension was then centrifuged at 8000 g,48C 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) Eur. J. Biochem. 268, 6005–6010 (2001) q FEBS 2001 100% saturation by adding solid ammonium sulfate. The precipitate was again collected by centrifugation, dissolved in the minimum volume of water and dialyzed against 5 m M 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 m M phosphate buffer, pH 7.6 at 4 8C. DEAE-cellulose chromatography. The active protein frac- tion obtained after gel filtration was dialyzed against distilled water for 12 h and against 10 m M Tris/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 0.3 M). 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 m M phosphate buffer, pH 6.5 and then loaded onto the column. The protein was eluted from the column stepwisely using the same buffer containing 0.2 M NaCl. 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 Â 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 m M 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 m M NaCl/P i , 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 0.2 M galactose. 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] with D-glucose as the standard. For identification of sugars, the lectins were hydrolyzed with 1 M 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 : mL 21 ), 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 : mL 21 , 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. RESULTS Purification of mulberry seed lectins The 100% ammonium sulfate saturated crude protein extract after dialysis against 5 m M 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 6006 T. Yeasmin et al. (Eur. J. Biochem. 268) q FEBS 2001 fraction, 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 m M Tris/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 0.3 M in 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 0.3 M 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 m M 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-1a 0 and F-1a 00 . The F-1a 0 fraction was eluted by the buffer only, while F-1a 00 was eluted by the buffer containing 0.2 M NaCl. Of these two fractions, only F-1a 0 displayed lectin activity. The fraction F-1a 0 might 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-1a 0 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 Â 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 Â 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). q FEBS 2001 Lectins from mulberry seeds (Morus sp.) (Eur. J. Biochem. 268) 6007 F-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 m M phosphate buffer, pH 7.2, containing 0.2 M galactose (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 : mL 21 , 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 and D-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 LC 50 (concentration at which 50% mortality of the napulii occurs) as estimated by extrapolation was 10.96 mg : mL 21 for MSL-1, 17.37 mg : mL 21 for MSL-2, and 6.30 mg : mL 21 for 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 Â 15 cm), pre-equilibrated with 5m 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-1a 0 on 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 : mg 21 ) Yield (%) Purification (fold) Crude extract 600 4230 7.05 100 1.00 100% (NH 4 ) 2 SO 4 220 3344 15.20 79.06 2.16 Saturated After gel filtration 80 2842 35.53 67.06 5.04 DEAE-cellulose fraction F- 1a 12.2 650 53.29 15.37 12.48 F-1b 7.6 818 107.72 19.35 7.56 F-1c 4.8 362 75.38 8.55 15.28 CM-cellulose F-1a 0 7.3 640 87.98 15.13 10.69 6008 T. Yeasmin et al. (Eur. J. Biochem. 268) q FEBS 2001 Table 2. Hemagglutination inhibition assay of mulberry seed lectins. NI, No inhibition; I, Inhibition. Proteins Sugar Concentration (m M) InhibitionMaximum Minimum MSL-1 D-Glucose 110 – NI D-Mannose 110 – NI D-Galactose – 20 I N-Acetyl D-glucosamine 110 – NI Methyl-a- D-galactopyranoside – 28 I Methyl-b- D-galactopyranoside – 15 I N-acetyl-galactosamine 110 – NI D-glucosamine-HCl 110 – NI Lactose – 20 I D-Raffinose – 25 I MSL-2 D-Glucose 110 – NI D-Mannose 110 – NI D-Galactose – 22 I N-Acetyl D-glucosamine 110 – NI Methyl-a- D-galactopyranoside – 30 I Methyl-b- D-galactopyranoside – 20 I N-Acetyl-galactosamine 110 – NI D-Glucosamine-HCl 110 – NI Lactose – 20 I D-Raffinose – 30 I D-Glucose 110 – NI D-Mannose 110 – NI D-Galactose – 25 I N-Acetyl D-glucosamine 110 – NI Methyl-a- D-galactopyranoside – 30 I Methyl-b- D-galactopyranoside – 20 I MSL-3 N-Acetyl-galactosamine 110 – NI D-glucosamine-HCl 110 – NI Lactose – 20 I D-Raffinose – 35 I 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-1a 0 (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. q FEBS 2001 Lectins from mulberry seeds (Morus sp.) (Eur. J. Biochem. 268) 6009 DISCUSSION 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. ACKNOWLEDGEMENTS 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. REFERENCES 1. Lis, H. & Sharon, N. (1986) Lectins as molecules and as tools. Annu. Rev. Biochem. 55, 35–67. 2. Goldstein, I.J. & Hayes, C.E. 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(1984) Purification and characterization of Abrus precatorius Agglutinin. J. Fac. Agr. Kyushu University Japan. 29, 103 – 115. 21. Olsnes, S., Saltvedt, E. & Phil, A. (1974) Isolation and comparison of galactose-binding lectins from Abrus precatorius and Ricinus communis. J. Biol. Chem. 249, 803– 810. 6010 T. Yeasmin et al. (Eur. J. Biochem. 268) q FEBS 2001 . Purification and characterization of three galactose specific lectins from Mulberry seeds ( Morus sp. ) Tanzima Yeasmin, Md Abul Kashem. lethality bioassay. Keywords: mulberry seeds; galactose- specific lectins; subunit structure; hemagglutination; cytotoxicity. Mulberry (Morus alba L .) is the