Lectins are a group of proteins (or glycoproteins ) of nonimmune origin that bind specifically and reversibly to carbohydrates, particularly the sugar moiety of glycoconjugates, resulting in cell agglutination and precipitation of glycoconjugates (Goldstein et al., 1980). They are ubiquitously distributed in nature, being found in plants, fungi, viruses, bacteria, crustacea, insects, and animals (Liener et al., 1986). In the plant kingdom, lectins are particularly abundant in seeds of the family Leguminoseae (Etzler, 1986; Rudiger, 1988; Sharon and Lis, 1990).
A hemagglutinating activity in seeds of Dolichos lablab was first reported by Boyd and Reguera (1949) and then by Tobiska (1959). Dolichos lablab lectin (DLL) has been isolated and characterized by several groups. However, reports on its molecular weight and carbohydrate-binding properties have varied, the discrepancy being attributed to the different varieties of Dolichos lablab used and/or the different climates in which they were grown (Salgarkar and Sohonie, 1965; Rao et al., 1976; Guran et al., 1983; Kumar and Rao, 1986; Favero et al., 1988; Silva-Lima et al., 1988; Mo et al., 1990).
In striking contrast to the extensively studied and well documented lectin from Dolichos biflorus (Etzler, 1994), our knowledge regarding the carbohydrate-binding properties of DLL is limited to a few monosaccharides. Although its complete primary structure has been published (Gowda et al., 1994), little effort has been made to study its carbohydrate-binding specificity in more detail. The recent findings that this lectin specifically reacts with the Flk2/flt3 tyrosine kinase receptor, and activates proliferation of NIH 3T3 fibroblasts transfected with the flt3 tyrosine kinase receptor (Mo et al., 1997; Moore et al., 1997a,b) prompted us to undertake a detailed investigation of its carbohydrate-binding specificity.
In the present study, the Dolichos lablab lectin has been purified to electrophoretic homogeneity by affinity chromatography using an ovalbumin-Sepharose 4B column and its detailed carbohydrate-binding properties investigated by three different approaches, i.e., an hemagglutination inhibition assay, precipitation inhibition assay, and ELISA.
Purification of the Dolichoslablab lectin
In the present study, it was found that the hemagglutinating activity in the crude extract bound to an ovalbumin-Sepharose 4B column with great avidity and could be specifically eluted with methyl [alpha]-mannoside, resulting in a pure lectin preparation. Polyacrylamide gel electrophoresis gave a single polypeptide band under both alkaline (pH 8.9) and acidic (pH 4.3) conditions (Figure
Figure 1. Polyacrylamide (12.5%) gel electrophoresis of the purified Dolichos lablab lectin. (A) Alkaline (Tris/glycine, pH 8.3) buffer system. (B) Acidic ([beta]-alanine/acetic acid , pH 4.3) buffer system. DLL was purified from ovalbumin-Sepharose (lane 1), mannose-Sepharose (lane 2), or trehalose-Sepharose (lane 3). In each case, 20 µg of purified DLL was applied.
Figure 2. Gel filtration chromatography of the affinity-purified Dolichos lablab lectin on a BioGel P-150 column (1.45 × 120 cm, bed volume, 198 ml; void volume, 51 ml; fraction size, 2 ml/tube).
Since purification of DLL by affinity chromatography on mannose-based affinity matrices (e.g., Sepharose-mannose, etc.) has also been reported, it was necessary to compare the DLL purified on ovalbumin-Sepharose with that from mannose-based affinity absorbents. Divinylsulfone-activated Sepharose 4B was derivatized with mannose, methyl [alpha]-mannoside, or trehalose. Crude extracts of Dolichos lablab beans were applied to these affinity matrices, and DLL specifically eluted with the corresponding haptenic sugar. The lectin preparations obtained from the various affinity absorbents gave identical electrophoretic patterns (Figures
Molecular mass and molecular structure
The molecular mass of the purified DLL was estimated by gel filtration chromatography on a Bio-Gel P150 column (1.45 × 120 cm, bed volume 198 ml); it eluted as a single, symmetric peak at the same elution volume as bovine serum albumin (67 kDa), indicating that purified DLL has an apparent molecular mass of 67 kDa.
Upon SDS-PAGE, under either reducing (Figure
Figure 3. Molecular mass determination of the subunits of the purified Dolichos lablab lectin by SDS-PAGE (reducing conditions). Lanes 1 and 3, molecular mass standard (Dalton Mark VII-L from Sigma and Benchmark Protein Ladders from Gibco BRL, respectively); lanes 2, 4, and 5, DLL purified on ovalbumin-Sepharose, mannose-Sepharose, and trehalose-Sepharose, respectively.
Taken together, these data suggest that at neutral pH, DLL exists as a heterotetramer ([alpha]2[beta]2) joined by noncovalent bonds.
Hemagglutination and hemagglutination inhibition
In agreement with the results of earlier investigators, the purified DLL agglutinates rabbit and human erythrocytes irrespective of blood group type. The minimal concentration of purified DLL required for agglutinating fresh human erythrocytes was 8 µg/ml, whereas with formaldehyde-treated rabbit and human erythrocytes (Nowak and Barondes, 1975), only 2 µg/ml of the lectin were required for hemagglutination.
The carbohydrate binding specificity of the crude extract and the purified DLL was initially investigated by hemagglutination inhibition assay. Of the monosaccharides tested, only d-mannose, N-acetyl-d-glucosamine, and d-glucose were good inhibitors. Both methyl [alpha]-d-glucoside and methyl [alpha]-d-mannoside are four to eight times better, whereas the corresponding [beta]-glycosides are inferior inhibitors compared to the parental sugars, indicating that the DLL has a strong preference for the [alpha]-anomeric configuration.
LISA
The ability of various haptenic carbohydrates to inhibit the binding of DLL to solid-phase bound IgM was investigated by an ELISA technique. The results obtained by ELISA (data not shown) are essentially in good agreement with those obtained by hemagglutination inhibition assay and quantitative precipitation inhibition assay.
Quantitative precipitation and precipitation inhibition
The ability of various glycoproteins and polysaccharides to precipitate the purified DLL was investigated by the quantitative precipitation assay. Although the hemagglutinating activity of the DLL was specifically inhibited by mannose and glucose, various structurally different yeast mannans failed to precipitate the lectin. Among those tested were highly branched mannans obtained from Saccharomyces cerevisiae and Pichia pastoris, etc., and linear mannans from Trichosporon cutaneum and H.capsulata, etc. [alpha]-d-Glucans such as glycogen, and dextran 1355 S, which has numerous [alpha]1,3-glucosidic linkages, also failed to precipitate the DLL.
Table I.
Carbohydrate | I50 (mM)a | Relative potencyb |
Mannose | 1.45 | 1 |
1,5-Anhydro-d-mannitol | 2.3 | 0.63 |
Methyl [alpha]-mannoside (M[alpha]M) | 0.4 | 3.6 |
Methyl [beta]-mannoside (M[beta]M) | 7.2 | 0.2 |
p-Nitrophenyl [alpha]-mannoside | 0.56 | 2.6 |
p-Nitrophenyl [beta]-mannoside | 0.94 | 1.5 |
N-Acetylmannosamine | N.I.(10)c | |
Glucose | 2.5-3.0 | 0.5-0.6 |
Methyl [alpha]-glucoside (G[alpha]M) | 0.8 | 1.8 |
Methyl [beta]-glucoside (G[beta]M) | 15.4 | 0.1 |
2-O-Methyl-d-glucose | 3.3 | 0.44 |
3-O-Methyl-d-glucose | N.I.(100) | |
2-Deoxy-d-glucose | 7.2 | 0.2 |
Methyl 4-O-methyl-[alpha]-glucoside | N.I.(50) | |
6-O-Methyl-d-glucose | N.I.(50) | |
1,5-Anhydro-d-glucitol | 16.0 | 0.1 |
Methyl 6-deoxy-[alpha]-glucoside | N.I.(50) | |
6-Deoxy-6-fluoroglucose | 28 | 0.05 |
6-Deoxy-6-Iodoglucose | N.I.(50) | |
N-Acetylglucosamine (GlcNAc) | 0.93 | 1.56 |
Methyl [alpha]-GlcNAcp | 0.68 | 2.1 |
Methyl [beta]-GlcNAcp | 2.4 | 0.6 |
N-Formylglucosamine | 1.52 | 0.95 |
Galactose | N.I.(100) | |
Altrose | N.I.(100) | |
Talose | N.I.(100) | |
Allose | N.I.(100) | |
d-Arabinose | N.I.(100) | |
Methyl [alpha]-d-xyloside | N.I.(50) | |
Man[alpha]1,3Man[alpha]Me | 0.83 | 1.75 |
Man[alpha]1,6Man[alpha]Me | 0.66 | 2.2 |
Man[alpha]1,3(Man[alpha]1,6)Man[alpha]Me | 0.35 | 4.1 |
Maltose (Glc[alpha]1,4Glc) | 2.9 | 0.5 |
Isomaltose (Glc[alpha]1,6Glc) | 1.65 | 0.88 |
Sucrose (Glc[alpha]1,2[beta]Fru) | 9.8 | 0.15 |
Gentiobiose (Glc[beta]1,6Glc) | 26.2 | 0.06 |
Cellobiose (Glc[beta]1,4Glc) | N.I.(50) | |
Chitobiose (GlcNAc[beta]1,4GlcNAc) | N.I.(50) | |
Kojibiose (Glc[alpha]1,2Glc) | 1.13 | 1.3 |
GlcNAc[alpha]1,6Gal | 0.75 | 1.9 |
[alpha],[alpha]-Trehalose (Glc[alpha],[alpha]Glc) | 0.4 | 3.6 |
[alpha],[beta]-Trehalose (Glc[alpha],[beta]Glc) | 2.9 | 0.5 |
[beta],[beta]-Trehalose (Glc[beta],[beta]Glc) | 42 | 0.03 |
Trehalosamine | 0.34 | 4.3 |
Mannan (S.cerevisiae) | 1.1 mg/ml | |
1355 Dextran B-S | N.I.(10 mg/ml) | |
Glycogen (rabbit liver) | N.I.(10 mg/ml) |
Although DLL was found to be retained by ovalbumin-Sepharose and ovomucoid-Separon (Guran et al., 1983), neither ovalbumin nor ovomucoid gave a detectable precipitate with the lectin. Of many native and desialylated glycoproteins examined, only murine IgM gave a pronounced precipitation reaction with DLL.
The detailed carbohydrate binding properties of the purified Dolichos lablab lectin were further elucidated by precipitation inhibition assays using murine IgM as the precipitant. The minimum concentration of each haptenic sugar required for 50% inhibition was obtained from corresponding complete inhibition curves and the results are shown in Table I. It is noteworthy that p-nitrophenyl [alpha]-mannoside was less effective as an inhibitor than methyl [alpha]-mannoside, suggesting that in contrast to concanavalin A, the Dolichos lablab lectin does not have a hydrophobic binding site adjacent to its carbohydrate combining site.Isolation of IgM on a DLL-Sepharose column
As shown in Figure
Figure 4. SDS-PAGE of IgM isolated by affinity chromatography on a DLL-Sepharose 4B column. Lane 1, crude ascitic fluids (1 µl); lane 2, IgM isolated from ascitic fluids; lane 3, molecular mass standards; lane 4, IgM isolated from spent hybridoma culture media; lane 5, spent hybridoma culture media. Arrows indicate the heavy (µ) chain (upper) and the light chain (lower) of IgM.
Awareness that some leguminous seeds contain more than one lectin (Etzler, 1986) and many lectins consist of a mixture of isolectins, led us to attempt to separate the current DLL preparation into isolectins. Methods tried include ion exchange chromatography, chromatography using affinity absorbants having more defined carbohydrate structures, and eluting the absorbed lectin with a linear gradient concentration of haptenic sugar. However, no evidence for the presence of isolectins was found.
The molecular weight of the Dolichos lablab lectin reported in the literature varies from 60 kDa to 110 kDa. In the present study, the affinity purified DLL was eluted as a single symmetric peak on a Bio-Gel P150 column, with an apparent molecular weight of 67 kDa, which is in good agreement with the results obtained by Kumar and Rao (1986) and Gowda et al. (1994). However, the subunit pattern of the present preparation is quite different from those reported previously (Guran et al., 1983; Gowda et al., 1994). Upon SDS-PAGE under both reducing and nonreducing condition, the purified lectin separated into five bands with apparent subunit masses ranging from 10 kDa to 22 kDa, of which band 3 and band 4 appear as doublets or triplets of closely migrating bands (Figure
The majority of lectins characterized thus far fall into two categories, namely, one-chain lectins (homopolymeric lectins) and two-chain lectins (i.e., [alpha][beta] or [alpha]2[beta]2 etc.).The subunits can originate from different genes, by proteolytic splitting of a single gene product, or by differential posttranslational modification (Etzler, 1994). The Dolichos lablab lectin has a very heterogeneous [alpha] chain, which has rarely been reported in the literature.
In the present study, the carbohydrate binding specificity of DLL was elucidated by three different approaches, i.e., hemagglutination inhibition assay, quantitative precipitation inhibition assay (in solution binding assay), and ELISA (solid-phase binding assay), in order to determine whether different approaches and binding kinetics would give different results. The relative inhibitory potencies of a given saccharide obtained by these three different methods were generally in good agreement with one another within the limits of experimental error.
The carbohydrate binding properties of the purified Dolichos lablab lectin were investigated in detail by precipitation inhibition assays. Of the monosaccharides tested, only mannose, glucose and N-acetylglucosamine were good inhibitors, whereas neither the C-4 epimers of d-glucose (i.e., d-galactose), d-mannose (i.e., d-talose) nor the C-3 epimers of d-mannose (i.e., d-altrose), d-glucose (i.e., d-allose) were inhibitory up to 100 mM. Methyl 6-deoxy-[alpha]-d-glucoside and the pentoses d-arabinose and methyl [alpha]-d-xyloside were also noninhibitory up to 100 mM. These results indicate that the C-3, C-4 equatorial hydroxyl groups and the C-5 equatorial hydroxymethyl group of the d-pyranose ring are crucial for DLL binding. This conclusion was further confirmed by the observation that except for 6-deoxy-6-fluoro-d-glucose, none of the sugars substituted in these positions (e.g., methyl 4-O-methyl [alpha]-glucoside, 6-O-methyl glucose, methyl 6-deoxy-[alpha]-glucoside, 6-deoxy-6-iodo-d-glucose, or 3-O-methyl glucose) was inhibitory up to 50 mM, indicating that unsubstituted, free hydroxyl groups at C-3, C-4 and C-6 position of the pyranose ring were essential for DLL binding, most likely serving as donors and/or acceptors of hydrogen bonds (Elgavish and Shaanan, 1997). Both mannose and methyl [alpha]-mannoside were two times more effective as inhibitors than glucose and methyl [alpha]-glucoside respectively, implying a positive contribution of a C-2 axial hydroxyl group of the pyranose ring to the lectin binding. The fact that both N-formyl-d-glucosamine and N-acetyl-d-glucosamine were more potent than glucose as inhibitors, and 2-O-methyl glucose also was inhibitory, whereas N-acetyl-d-mannosamine was noninhibitory implies that the C-2 axial hydroxyl group of mannose must remain unsubstituted, perhaps due to steric hindrance, whereas the C-2 equatorial hydroxyl group of glucose tolerates some kinds of substitution, e.g., 2-O-methylation, without abolishing DLL binding. Moreover, some modifications as in the cases of N-formyl-d-glucosamine and N-acetyl-d-glucosamine even favor DLL binding, most likely because the N-formyl or N-acetyl group might be involved in additional hydrogen bonding with lectin. Furthermore, the methyl group might also make an extra positive contribution to DLL binding through a hydrophobic interaction with the carbohydrate combining site.
The fact that both methyl [alpha]-d-mannoside and methyl [alpha]-d-glucoside were four times more potent, whereas the corresponding [beta]-glycosides were five times less effective inhibitors than the parent monosaccharides suggested that DLL had a strong preference for the [alpha]-anomeric configuration. This [alpha]-anomeric preference was also observed in the disaccharides tested, of which [alpha]-glycosidic linked disaccharides such as [alpha],[alpha]-trehalose was an excellent inhibitor; Man[alpha]1,6Man[alpha]Me, Man[alpha]1,3Man[alpha]Me, GlcNAc[alpha]1,6Gal, and kojibiose were good inhibitors; and maltose, isomaltose, and [alpha],[beta]-trehalose were moderate inhibitors. On the other hand, of the [beta]-glycosidically linked disaccharides tested, neither cellobiose nor chitobiose was inhibitory up to 50 mM; gentiobiose and [beta],[beta]-trehalose were very poor inhibitors. The Pauling-Corey-Koltun space-filling models of these disaccharides revealed that the reducing d-glucosyl residues of these [beta]-glycosidic linked disaccharides perhaps sterically hinder the binding of the nonreducing terminal glucosyl residues to the DLL carbohydrate combining sites. In the case of gentiobiose and [beta],[beta]-trehalose, more flexibility around a [beta]1,6 and a [beta],[beta]-linkage than around a [beta]1,4 linkage (in the case of cellobiose and chitobiose), causes less hindrance; this may explain why gentiobiose and [beta],[beta]-trehalose are still inhibitors, although very poor ones.
It is particularly noteworthy that among the oligosaccharides tested, with the exception of trehalose and trehalosamine, the branched trimannoside, i.e., [alpha]-d-Man-(1-3)-[[alpha]-d-Man-(1-6)][alpha]-d-ManOMe is the best ligand for DLL binding. However, this branched trimannoside is only slightly more potent than methyl [alpha]-d-mannoside. All the disaccharides examined are less inhibitory than methyl [alpha]-d-mannoside, implying that the Dolichos lablab lectin probably does not have an extended carbohydrate combining site (or subsites; Elgavish and Shaanan, 1997).
The branched trimannosyl unit, Man[alpha]1,3(Man[alpha]1,6)Man, exists in the core region of all N-glycan-bearing glycoproteins. However, in most glycoproteins, especially those of complex and hybrid types, this trimannosyl unit is masked by peripheral sugars, which abrogate the DLL binding. On the other hand, ovalbumin contains both hybrid and high mannose type glycans with nonreducing terminal branching trimannosyl units (Kuo et al., 1996), and the heavy chain of murine IgM bears five N-linked glycans, of which the glycan at asparagine 563 is of high mannose type with unmasked branched trimannosyl core (Anderson et al., 1985). This may explain why, except for IgM and ovalbumin, many native and desialylated glycoproteins tested in the present study fail to react with the Dolichos lablab lectin.
A surprise of the present study was the finding that trehalose and trehalosamine were among the best ligands for DLL binding. Except for a report that [alpha],[alpha]-trehalose was a potent inhibitor of ConA-dextran interaction in 1960s (So and Goldstein, 1967a), few plant lectins (Wang et al., 1974) were reported to recognize trehalose. [alpha],[alpha]-Trehalose is a disaccharide formed by two d-glucose residues joined through their reducing carbon atoms. The absence of direct internal hydrogen bonds renders great flexibility around the glycoside oxygen bond, enabling this disaccharide to conform to the carbohydrate combining site of the Dolichos lablab lectin, while additional, water-mediated hydrogen bonds can be formed between DLL and the two associated water molecules in trehalose dihydrate (Panek, 1995; Elgavish and Shaanan, 1997), enhancing significantly the binding affinity.
The seeds of Dolichos lablab (Hyacinth bean) were obtained from Stokes Seeds Inc. (Buffalo, NY). Various glycoproteins including ovalbumin, fetuin, transferrin, glycophorin, and thyroglobulin were purchased from Sigma (St. Louis, MO). The desialylated glycoproteins were prepared by heating the parent glycoproteins in 0.1 M hydrochloric acid at 80°C for 1 h, followed by dialysis and lyophilization, the removal of sialic acid was ascertained by the thiobarbituric acid assay (Warren, 1959). Sepharose 4B was a product of Pharmacia Fine Chemicals (Uppsala, Sweden); EZ-Link Sulfo-NHS-LC-Biotin (Sulfosuccinimidyl-6-[biotinamido] Hexanoate) was a product of Pierce; alkaline phosphatase-streptavidin was obtained from Zymed Laboratories Inc. (San Francisco, CA), and p-nitrophenyl phosphate (di[cyclohexylammonium] salt) was from Sigma. Murine IgM, IgA, and IgG were the generous gifts of Dr. J.L.Claflin of the University of Michigan. Methyl 3-O-[alpha]-d-mannopyranosyl-[alpha]-d-mannopyranoside, methyl 6-O-[alpha]-d-mannopyranosyl-[alpha]-d-mannopyranoside, and methyl 3,6-di-O-([alpha]-d-mannopyranosyl)-[alpha]-d-mannopyranoside were purchased from Toronto Research Chemicals Inc. (Ontario, Canada).
d-Mannose, d-glucose, d-galactose, d-altrose, d-allose, d-talose, methyl [alpha]-d-mannopyranoside, methyl [alpha]-d-glucopyranoside, methyl [beta]-d-glucopyranoside, 2-deoxy-d-glucose, 1,5-anhydro-d-mannitol, [beta]-gentiobiose (6-O-[beta]-d-glucopyranosyl-[beta]-d-glucose), N-acetyl-d-glucosamine, maltose, isomaltose, mannan (from Saccharomyces cerevisiae), dextran (produced by leuconostoc mesenteroides strain N0. B1355), glycogen (from rabbit liver), p-nitrophenyl [alpha]-d-mannopyranoside, p-nitrophenyl [beta]-d-mannopyranoside, 3-O-methyl-d-glucopyranose, methyl 6-deoxy-[alpha]-d-glucopyranoside, methyl [alpha]-d-xylopyranoside, cellobiose ([beta]-d-Glc-[1-4]-d-Glc), arabinose, N-acetyl-mannosamine, [alpha],[alpha]-trehalose ([alpha]-d-glucopyranosyl-[alpha]-d-glucopyranoside), [beta],[beta]-trehalose, and [alpha],[beta]-trehalose were purchased from Sigma. Methyl 2-acetamido-2-deoxy-[alpha]-d-glucopyranoside, methyl 2-acetamido-2-deoxy-[beta]-d-glucopyranoside, 2-O-methyl-d-glucopyranose, 6-O-methyl-d-glucopyranose, and 6-O-(2-acetamido-2-deoxy-[alpha]-d-glucopyranosyl)-d-galactopyranose were synthesized in this laboratory. Methyl [beta]-d-mannopyranoside isopropylate, kojibiose (2-O-[alpha]-d-glucopyranosyl-d-glucose), N,N'-diacetylchitobiose, methyl 6-deoxy-6-fluoro-d-glucopyranoside, trehalosamine, methyl 6-deoxy-6-iodo-d-glucopyranoside, 1,5-anhydroglucitol, N-formyl-d-glucosamine, mannans obtained from Pichia pastoris, Trichosporon cutaneum, and H.capsulata etc. were from previous studies.
Molecular weight standards used in SDS-PAGE, Dalton Mark VII-L were from Sigma; Benchmark Protein Ladders was from Gibco BRL, Bio-Gel P-150 (50-100 mesh); and the Ready-Gels (Tris-Tricine Gel, 10-20% gradient gel) were from Bio-Rad. Divinyl sulfone was obtained from Aldrich Chemical Company Inc. (Milwaukee, MI).All other chemicals used were of the highest purity available. Preparation of various affinity adsorbents:
Methyl [alpha]-d-mannoside-Sepharose 4B, mannose-Sepharose 4B, and trehalose-Sepharose 4B were prepared as described by Fornstedt and Porath (1975), via divinyl sulfone activation of Sepharose 4B.
Ovalbumin (200 mg) was coupled to cyanogen bromide-activated Sepharose 4B (24 ml) according to the procedure described by March et al. (1974). Based on the amount of unbound ovalbumin remaining in the coupling buffer, it was estimated that ~58% of ovalbumin was coupled to Sepharose 4B, yielding an affinity absorbant containing 4.8 mg of ovalbumin per milliliter of settled gel.
Purified Dolichos lablab lectin was also coupled to cyanogen bromide-activated Sepharose 4B in the same manner, giving a product containing ~4 mg protein/ml gel. Purification of Dolichos lablab lectin
After careful manual removal of the seed coat and embryo, the Dolichos lablab beans were finely ground using an IKA laboratory mill (Janke & Kunkel GMBH & CO. KG., Germany). Twenty grams of the seed meal were delipidated with 300 ml of a mixture of methylene chloride/methanol (2:1, v/v) by gently stirring at room temperature for 2 h. The suspension was filtered on a buchner funnel and the bean meal was air-dried at room temperature.
The dried, defatted bean flour was extracted at 4° C for 4 h with 200 ml of 0.01 M phosphate-buffered saline (PBS, pH 7.4) containing 1mM phenylmethylsulfonyl fluoride and 10 mM thiourea. After centrifugation (15,000 r.p.m. × 30 min), the residue was reextracted once with 100 ml of the same PBS solution for an additional 2 h and centrifuged (15,000 r.p.m. × 30 min).
The two supernatant solutions were combined and subjected to ammonium sulfate fractionation. The precipitate formed between 20% and 60% (NH4)2SO4 saturation was collected and dialyzed extensively against 0.01 M PBS, pH 7.4, containing 1 mM CaCl2. After removing the insoluble materials by centrifugation, the dialysate was applied onto an ovalbumin-Sepharose 4B column (1.0 × 17 cm, bed volume 15 ml), preequilibrated with PBS, at a flow rate of 15 ml/h; the eluate was monitored by absorbance at 280 nm. After washing the column with PBS until the absorbance of the effluent had become negligible, the affinity-adsorbed lectin was displaced with 0.2 M methyl [alpha]-d-mannoside in PBS, collected, dialyzed against distilled water and lyophilized. The yield of the lectin was ~30-40 mg from 20 g of seed meal. Protein estimation
Protein concentration was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard. Polyacrylamide gel electrophoresis (PAGE) and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE)
Native gel electrophoresis using a 12.5% slab gel was conducted in both alkaline (Tris/glycine, pH 8.3) (Davis, 1964) and acidic ([beta]-alanine/acetic acid, pH 4.3; Reisfeld et al., 1962) buffer systems. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was carried out using a Bio-Rad 10-20% gradient Ready Gel (Tris-Tricine Gels, 10-20%T gradient gels) running in Tris/tricine buffer system as instructed by the manufacturer. Protein bands were visualized by Coomassie brilliant blue R-250 staining. Preparation of biotin-Dolichos lablab lectin conjugate
Biotinylation of the purified Dolichos lablab lectin was performed using EZ-Link sulfo-NHS-LC-Biotin as described by the manufacturer in the presence of 0.2 M methyl [alpha]-d-mannopyranoside to protect the sugar-binding sites. After coupling, the lectin activity was ascertained by hemagglutination assay. Hemagglutination and inhibition assays
The hemagglutinating activity of the lectin against rabbit and human cells and the inhibitory potency of various carbohydrates were determined using 96-well V-shaped microtiter plates by a 2-fold serial dilution procedure as described previously (Crowley and Goldstein,1982). LISA
The plates were coated with 100 µl/well of a 10 µg/ml IgM solution (i.e., 1 µg/well) in 0.1 M Na2CO3 buffer, pH 9.6, and incubated at 37°C for 3 h or 4°C overnight. After washing three times with 150 µl/well of buffer A (0.01M PBS, pH 7.4, containing 0.1% Tween 20), the plates were blocked with 150 µl/well of buffer B (0.01M PBS, pH 7.4, containing 1% bovine serum albumin and 0.1% NaN3) and stored at 37°C overnight. After washing, the wells were incubated with a serial dilution of various haptenic saccharides in the presence of 5 µg/ml (0.5 µg/well) of biotinylated Dolichos lablab lectin at 37°C for 3 h. Following washing four times (5 min/each) with buffer A, 100 µl of alkaline phosphatase-streptavidin diluted appropriately with PBS containing 0.5% BSA and 0.1% Tween 20 was added to each well and the reaction was allowed to proceed at 37°C for 1 h. The wells were washed four times with 150 µl/well of buffer A and once with 150 µl/well of Na2CO3 buffer (0.1 M, pH 9.8) containing 1 mM MgCl2. One hundred microliters of substrate solution (1 mg p-nitrophenyl phosphate/ml of 0.1 M Na2CO3 buffer, pH 9.8, containing 1 mM MgCl2) was added to each well, and the reaction was allowed to proceed at room temperature for 10-20 min, before quenching the reaction with 100 µl of 1 M NaOH. The adsorbance at 410 nm was determined using a microplate reader. Quantitative precipitation and hapten inhibition assay
Quantitative precipitation assays were performed by a microprecipitation technique as described by So and Goldstein (So and Goldstein, 1967b). Briefly, varying amounts of glycoproteins or polysaccharides, ranging from 0 to 100 µg, were added to 20 µl (20 µg) of purified Dolichos lablab lectin in a total volume of 120 µl of PBS, pH 7.2. After incubation at 37°C for 1 h, the reaction mixtures were stored at 4°C for 48 h. The precipitins formed were centrifuged, washed three times with 150 µl of cold PBS, dissolved in 0.05 M NaOH and determined for protein content by Lowry's method using bovine serum albumin as standard.
For hapten inhibition assays, increasing amounts of various haptenic saccharides were added to the reaction mixture consisting of 20 µl (16 µg) of DLL and 25 µl (10 µg) of IgM in a final volume of 120 µl of PBS, pH 7.2. After incubation at 37°C for 1 h and 4° C for 48 h, the precipitated protein was determined, the percentage of inhibition calculated, and inhibition curves constructed. Molecular mass and molecular structure
The molecular mass and molecular structure of the purified Dolichos lablab lectin was determined by gel filtration and SDS-PAGE performed in the presence and absence of 2-mercaptoethanol (Laemmli, 1970). The subunit masses were calculated from their electrophoretic mobilities in comparison with those of the molecular weight standards.
Gel filtration chromatography was carried out on a Bio-Gel P-150 column (1.45 × 120 cm, bed volume 198 ml) operating at room temperature in PBS, pH 7.2. The column was calibrated with the following standard proteins: thyroglobulin (670 kDa), gamma globulin (158 kDa), bovine serum albumin (67 kDa), ovalbumin (45 kDa), carbonic anhydrase (29 kDa), and chymotrypsinogen A (25 kDa). Isolation of IgM on the DLL-Sepharose column
Crude IgM preparations, either from spent hybridoma culture media or from peritoneal fluid, were applied to a DLL-Sepharose 4B column (0.7 × 18 cm, bed volume 7 ml) preequilibrated with PBS, pH 7.4. The affinity-bound IgM was eluted with 0.2 M trehalose in PBS.
We thank Dr. J. L. Claflin (University of Michigan) for providing us with various IgM preparations and advice, and Dr. Harry Winter for helpful discussions and assistance in preparing the manuscript. This investigation was supported by the National Institutes of Health Grant GM 29470 (to I.J.G.).
BSA, bovine serum albumin; DLL, Dolichos lablab lectin; ELISA, Enzyme linked immunosorbent assay; PAGE, polyacrylamide gel electrophoresis; PBS, 10 mM phosphate-buffered saline, pH7.2, containing 0.15 M NaCl; PMSF, phenylmethylsulfonyl fluoride; SDS, sodium dodecyl sulfate.
3To whom correspondence should be addressed at: Department of Biological Chemistry, The University of Michigan, 1301 Catherine Road, Ann Arbor, MI 48109-0606, USA
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
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