J Biol Chem, Vol. 274, Issue 47, 33300-33305, November 19, 1999

Xanthosoma sagittifolium Tubers Contain a Lectin with Two Different Types of Carbohydrate-binding Sites^*

Hanqing Mo, Kevin G. Rice^§, David L. Evers^§, Harry C. Winter, Willy J. Peumans^¶, Els J. M. Van Damme^¶**, and Irwin J. Goldstein

From the  Department of Biological Chemistry, University of Michigan, Medical School, Ann Arbor, Michigan 48109-0606, the ^§ Department of Medicinal Chemistry, College of Pharmacy, University of Michigan, Ann Arbor, Michigan 48109-1065, and the ^¶ Laboratorium voor Fytopathologie en Plantenbescherming, Katholieke Universiteit Leuven, Willem de Croylaan 42, B-3001 Leuven (Heverlee), Belgium

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

An unusual lectin possessing two distinctly different types of carbohydrate-combining sites was purified from tubers of Xanthosoma sagittifolium L. by consecutive passage through two affinity columns, i.e. asialofetuin-Sepharose and invertase-Sepharose. SDS-polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and gel filtration chromatography of the purified lectin showed that the X. sagittifolium lectin is a heterotetrameric protein composed of four 12-kDa subunits (₂₂) linked by noncovalent bonds. The results obtained by quantitative precipitation and hapten inhibition assays revealed that the lectin has two different types of carbohydrate-combining sites: one type for oligomannoses, which preferentially binds to a cluster of nonreducing terminal 1,3-linked mannosyl residues, and the other type for complex N-linked carbohydrates, which best accommodates a non-sialylated, triantennary oligosaccharide with N-acetyllactosamine (i.e. Gal1,4GlcNAc-) or lacto-N-biose (i.e. Gal1,3GlcNAc-) groups at its three nonreducing termini.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Lectins are (glyco)proteins of nonimmune origin that agglutinate cells and/or precipitate complex carbohydrates (1). It is their unique ability to recognize and bind reversibly to specific carbohydrate ligands without chemically modifying them that distinguishes lectins from other carbohydrate-binding proteins and enzymes and that makes lectins invaluable tools in biomedical and glycoconjugate research.

Lectins are ubiquitous in the biosphere. In the plant kingdom, they are traditionally found in the dicotyledons, especially in seeds (2-4). However, during the last decade, lectins from monocotyledonous families such as Alliaceae (5-7), Amaryllidaceae (8-10), Araceae (11-14), Liliaceae (15, 16), and Orchidaceae (17) have been isolated and characterized. Interestingly, they all belong to a single monocot mannose-binding lectin superfamily with respect to their molecular structures, sequence homologies, and exclusive specificity for D-mannose (18).

Recently, Van Damme et al. (11) described the isolation of lectins from several Araceae species, viz. Arum maculatum, Colocasia esculenta, Xanthosoma sagittifolium, and Dieffenbachia sequina. Based on their molecular structure, taxonomic relationship, and high sequence homology to the aforementioned mannose-binding lectins, they clearly belong to the monocot mannose-binding lectin superfamily. However, compared with the other members of the superfamily, these lectins exhibit either very weak or no affinity for D-mannose, whereas they bind with great avidity to asialoglycoproteins. Therefore, a detailed investigation of their carbohydrate-binding specificities has been undertaken.

The results presented in this study reveal that one of these lectins, i.e. the lectin from X. sagittifolium (XSL),¹ possesses two distinctly different types of binding sites: one type for oligomannoses and the other for complex carbohydrates, which best accommodates an asialo triantennary N-linked glycan. The knowledge obtained from this study might also shed light on the nature of the binding sites of other lectins with "complex" carbohydrate-binding specificity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Tubers of X. sagittifolium L. Schott were purchased from a local store in Leuven, Belgium. Yeast mannan from Saccharomyces cerevisiae, glycogens (from both rabbit liver and oyster), invertase (from bakers' yeast), mucins, and various glycoproteins such as fetuin, ₁-acid glycoprotein, human transferrin, and thyroglobulin (bovine) were obtained from Sigma. The glycoproteins were desialylated by heating in 0.1 N hydrochloric acid at 80 °C for 1 h, followed by dialysis and lyophilization; removal of sialic acid was confirmed by the thiobarbituric acid assay (19). All monosaccharides and their methyl or p-nitrophenyl glycosides were purchased from Sigma. Man1,3Man--OMe, Man1,6Man--OMe, and Man1,6(Man1,3)Man--OMe were also available from Sigma; Man1,3Man, Man1,6Man, Man1,2Man, Man1,4GlcNAc, Man1,6(Man1,3)Man, and branched mannopentaose were obtained from Dextra Laboratories Ltd. (Reading, United Kingdom). The mannan of S. cerevisiae mutant 1B4 and Kloeckera brevis mannan were generous gifts of Dr. Akira Misaki (Konan Women's University, Kobe, Japan).

Purification of X. sagittifolium Lectin-- Asialofetuin and invertase were coupled to cyanogen bromide-activated Sepharose 4B according to the procedure described by March et al. (20). Affinity absorbents containing 3.1 mg of asialofetuin/ml and 5.2 mg of invertase/ml of settled gel were obtained.

The Xanthosoma lectin was isolated from tuber extracts by affinity chromatography on an asialofetuin-Sepharose 4B column essentially as described previously (11). The final purification of the lectin was accomplished by a second affinity chromatography step on an invertase-Sepharose 4B column. The affinity-adsorbed lectin was desorbed with 0.1 M acetic acid (pH 3.0), collected, dialyzed immediately against distilled water, and lyophilized.

Preparation of Complex Oligosaccharides-- The N-tert-butoxycarbonyl-L-tyrosine-derivatized oligosaccharides Bi 2, Bi 4, Tri 2, Tri 3, Tri 4, and Tri 5 (Fig. 1) were prepared from bovine fetuin as described previously (21, 22). Man₉GlcNAc₂-N-tert-butoxycarbonyl-L-tyrosine was prepared from soybean agglutinin (23), Bis 2 from ovotransferrin (24), and Fuc-Tri and Tetra from human orosomucoid (25). The N-linked bi-, tri-, and tetraantennary oligosaccharides were isolated by peptide N-glycosidase (EC 3.2.2.18) treatment of the tryptic glycopeptides. The reducing ends were converted into glycosylamines and then derivatized with N-tert-butoxycarbonyl-L-tyrosine N-hydroxysuccinimide ester (Sigma). The tyrosinylated oligosaccharides were purified by RP-HPLC, and the structures were characterized by ¹H NMR and fast atom bombardment mass spectrometry (21-27).

View larger version (31K): [in this window] [in a new window]   Fig. 1.   Structures and trivial names of the oligosaccharides used in this study. N-Linked bi-, tri-, and tetraantennary oligosaccharides were isolated from various sources as described under "Experimental Procedures" by peptide N-glycosidase treatment of the tryptic glycopeptides. The reducing ends were converted into glycosylamines and then derivatized with N-tert-butoxycarbonyl-L-tyrosine N-hydroxysuccinimide ester. The tyrosinylated oligosaccharides were purified by RP-HPLC and characterized by proton NMR and fast atom bombardment mass spectrometry (21-27).

Molecular Mass Estimation-- The molecular mass of the purified lectin was estimated by gel filtration chromatography on an Amersham Pharmacia Biotech Superose 12 column at room temperature in PBS (10 mM phosphate-buffered saline (pH 7.2) containing 0.15 M NaCl), both in the presence and absence of 0.2 M methyl--D-mannoside. The column was precalibrated with blue dextran, bovine serum albumin (66 kDa), ovalbumin (44 kDa), chymotrypsinogen (25 kDa), and cytochrome c (12.5 kDa) as well as with the previously characterized mannose-binding lectins from Galanthus nivalis (50 kDa) and Allium sativum (25 kDa).

Electrophoresis-- Native gel electrophoresis using a 12.5% slab gel was conducted in Tris/glycine running buffer (pH 8.3) according to the method of Davis (28). SDS-PAGE using a 12.5% acrylamide slab gel was carried out in Tris/Tricine running buffer (pH 8.3) as described by Schagger and von Jagow (29).

N-terminal Amino Acid Sequence Analysis-- After SDS-PAGE, the protein bands were transferred from the slab gel to a polyvinylidene difluoride membrane and visualized with Coomassie Brilliant Blue. Bands were excised and directly subjected to amino acid sequence analysis essentially as described previously (11) using an Applied Biosystems Model 477A protein sequencer interfaced with an Applied Biosystems Model 120A on-line analyzer.

Quantitative Precipitation and Hapten Inhibition Assays-- Quantitative microprecipitation assays were performed as described previously (30). Briefly, varying amounts of glycoproteins (or polysaccharides), ranging from 0 to 100 µg, were mixed with 17 µg of purified lectin in a total volume of 120 µl of PBS (pH 7.2). After standing at 37 °C for 1 h, the reaction mixtures were kept at 4 °C for 48 h. The precipitates formed were centrifuged, washed three times with ice-cold PBS, dissolved in 0.05 N NaOH, and assayed for protein content by the method of Lowry et al. (31) using bovine serum albumin as a standard. For hapten inhibition assays, increasing amounts of haptenic saccharides were added to the reaction mixture consisting of 17 µg of the lectin and 40 µg of asialoorosomucoid (or 10 µg of yeast mannan) in a final volume of 120 µl of PBS (pH 7.2). The precipitated protein was determined, and the percentage of inhibition was calculated.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Purification of the Xanthosoma Lectin-- After two successive affinity chromatography steps, first on an asialofetuin-Sepharose 4B column and then on an immobilized invertase column, an electrophoretically homogeneous Xanthosoma lectin preparation was obtained with a yield of 4 mg/g (wet weight). The purified lectin gave a single protein band upon native PAGE (Fig. 2A) and a single symmetric protein peak on gel filtration chromatography (data not shown).

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Gel electrophoresis of the purified Xanthosoma lectin. A, native polyacrylamide gel electrophoresis of the purified Xanthosoma lectin on a 12.5% gel at pH 8.3 in Tris/glycine running buffer; B, SDS-PAGE of the purified Xanthosoma lectin on a 12.5% gel at pH 8.3 in the Tris/Tricine running buffer of Schagger and von Jagow (29) . First lane, purified Xanthosoma lectin; second lane, molecular mass standards (BenchMark protein ladder, Life Technologies, Inc.).

Molecular Mass and Molecular Structure-- The molecular mass of the purified Xanthosoma lectin was estimated by gel filtration chromatography on a precalibrated Superose 12 column. The lectin eluted as a single symmetric peak in the presence or absence of methyl--D-mannoside, at an elution volume corresponding to an apparent molecular mass of 50 kDa (data not shown). Upon SDS-PAGE by the method of Schagger and von Jagow (29), both in the presence and absence of -mercaptoethanol, the lectin gave a single band with an apparent molecular mass of 12 kDa (Fig. 2B). However, N-terminal sequencing of the purified Xanthosoma lectin (Table I) gave two amino acids in each Edman degradation cycle in equimolar amounts, revealing that this 12-kDa band was an equimolar mixture of two different types of 12-kDa subunits. Taken together, these data suggest that at neutral pH, the native Xanthosoma lectin exists as a heterotetramer composed of four 12-kDa subunits (₂₂) joined by noncovalent bonds.

View this table: [in this window] [in a new window]   Table I N-terminal amino acid sequences of the two subunits of the purified Xanthosoma lectin The 12-kDa band from SDS-PAGE containing - and -subunits was blotted onto polyvinylidene difluoride membrane and sequenced by automated Edman degradation. The sequences of the individual subunits were deduced from the double sequence using the method previously described (11).

Precipitation Assays-- Yeast mannan (S. cerevisiae), which contains clusters of 1,3-linked mannosyl residues attached to an 1,6-linked mannose backbone (32), precipitated the Xanthosoma lectin at an appropriate stoichiometric ratio (Fig. 3A), whereas the mannan obtained from S. cerevisiae mutant 1B4 and the K. brevis mannan (32), both of which contain 1,2- and 1,6-linked mannosyl residues, but lack 1,3-linked mannosyl residues, failed to precipitate the lectin. Neither glycogens nor dextrans interacted with the Xanthosoma lectin. In this regard, the Xanthosoma lectin not only exhibits exclusive specificity for mannose, but more definitively has a linkage preference for terminal 1,3-linked mannosyl residues.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Quantitative precipitation of the Xanthosoma lectin by yeast mannan (S. cerevisiae) and glycoproteins. Varying amounts of polysaccharides or glycoproteins, ranging from 0 to 100 µg, were incubated with 17 µg of lectin in a total volume of 120 µl of PBS (pH 7.2). After 48 h at 4 °C, the amounts of protein precipitated were quantified. A, quantitative precipitation of the Xanthosoma lectin by yeast mannan (S. cerevisiae) () and invertase (); B, quantitative precipitation of the purified Xanthosoma lectin by several asialoglycoproteins: asialothyroglobulin (), asialofetuin (), asialoorosomucoid (), and asialotransferrin ().

Among various glycoproteins and mucins tested, the Xanthosoma lectin readily formed precipitins with various asialoglycoproteins such as asialo-₁-acid glycoprotein (i.e. asialoorosomucoid), asialofetuin, asialotransferrin, and asialothyroglobulin (Fig. 3B), whereas the corresponding sialylated glycoproteins were inert. On the other hand, neither the native nor desialylated mucins tested precipitated the lectin. Taken together, it is apparent that the Xanthosoma lectin binds specifically to N-linked glycans, and the presence of terminal sialic acid groups abolishes the binding.

Inhibition of Precipitation by Haptenic Carbohydrates-- To investigate the carbohydrate-binding properties of the Xanthosoma lectin, detailed precipitation inhibition assays were conducted using either yeast mannan (S. cerevisiae) or asialoorosomucoid as the precipitants. The results are shown in Fig. 4 and Table II and in Fig. 5 and Table III, respectively.

View larger version (12K): [in this window] [in a new window]   Fig. 4.   Hapten inhibition of XSL/yeast mannan precipitation. The inhibition assays were conducted under maximum precipitating conditions (i.e. 17 µg of lectin and 10 µg of mannan). Varying amounts of haptenic saccharides were added to the reaction mixture containing 17 µg of lectin and 10 µg of S. cerevisiae mannan in a total volume of 120 µl of PBS. After 48 h, the precipitated protein was determined, and the percentage of inhibition was calculated. , branched mannopentaose; , branched mannotriose; , 1,3-mannobiose; , 1,2- and 1,6-mannobioses; , methyl--D-mannopyranoside.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Inhibition of the XSL/asialoorosomucoid precipitation reaction by various complex oligosaccharides. For structures, see Fig. 1. Varying amounts of oligosaccharides were added to the reaction mixture containing 17 µg of lectin and 40 µg of asialoorosomucoid in a final volume of 120 µl of PBS. After 48 h at 4 °C, the precipitated protein was measured, and the percentage of inhibition was calculated. ×, Fuc-Tri; , Tri 4; , Tri 2; , Bi 2; , Tri 3; , Bi 4; , Tetra; , Bis 2; , branched mannopentaose.

For the XSL/yeast mannan precipitation system, only D-mannose, of the monosaccharides tested, had a very weak inhibitory activity (5% inhibition at 200 mM); methyl--D-mannopyranoside was slightly more active than mannose (14% inhibition at 200 mM), whereas methyl--D-mannopyranoside and epimers of D-mannose, i.e. D-glucose (C-2 epimer), D-altrose (C-3 epimer), and D-talose (C-4 epimer), were inactive up to 200 mM. Of the mannobioses tested, 1,3-mannobiose was the only inhibitor; 1,2- and 1,6-mannobioses and Man1,4GlcNAc all failed to show inhibitory activity at the highest concentration tested (20 mM). The branched mannopentaose, i.e. Man1,6(Man1,3)Man1,6(Man1,3)Man, was by far the best inhibitor, followed by the branched mannotriose, i.e. Man1,6(Man1,3)Man, being 10- and 2-fold more active, respectively, than 1,3-mannobiose.

On the other hand, the precipitation reaction between the Xanthosoma lectin and asialoorosomucoid was not inhibited by any mono-, di-, or trisaccharide tested. Of the various oligosaccharides examined (Fig. 1), Tri 4 (a triantennary complex-type oligosaccharide with two LacNAc- residues and one Gal1,3GlcNAc- group at its three nonreducing termini) and Fuc-Tri (with three LacNAc- residues at its three nonreducing ends and an additional fucosyl residue substituted at the penultimate GlcNAc residue of the Gal1,4GlcNAc1,4- branch) were the best inhibitors. Tri 5 (same as Tri 4, except that the penultimate GlcNAc residue of the Gal1,3GlcNAc1,4- branch was substituted with an 2,6-linked sialic acid residue) was non-inhibitory, suggesting that this complex carbohydrate-combining site can tolerate a neutral sugar substituted at the 3-OH of the GlcNAc1,4- branch, but not a negatively charged sialic acid in the same vicinity. The facts that both the biantennary oligosaccharide (Bi 2) and the tetraantennary oligosaccharide (Tetra) were inferior to their triantennary counterparts (Tri 2 and Tri 4) and that the longer chain analogs (Tri 2, Tri 4, and Bi 2, all with LacNAc at their nonreducing termini) were superior to the shorter chain analogs (Tri 3 and Bi 4, in which the nonreducing terminal galactoses were absent) indicated that this binding site best accommodated triantennary complex carbohydrates with LacNAc or lacto-N-biose residues at their nonreducing termini. A bisecting GlcNAc1,4- at the 1,4-linked mannosyl residue abolished the binding (as evident by the fact that Bis 2 was non-inhibitory).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As noted above, during the last decade, an increasing number of lectins have been isolated from various monocotyledonous families such as Alliaceae, Amaryllidaceae, Araceae, Liliaceae, and Orchidaceae. Data accumulated from physicochemical characterization and molecular cloning of these lectins reveal that, with the major exception of the Gramineae family, they all belong to a superfamily of monocot mannose-binding lectins with respect to their sequence homology, conserved domains, tissue occurrence, and exclusive specificity for mannose. However, recently, we isolated lectins from the tubers of several Araceae species, viz. A. maculatum, C. esculenta, X. sagittifolium, and D. sequina, and observed that in contrast to our previous findings, these monocot lectins are barely inhibited by mannose, whereas they strongly react with asialoglycoproteins (11). Simultaneously, Shangary et al. (13) reported the isolation of four monocot lectins from the family Araceae, viz. Arisaema consanguineum, Arisaema curvatum, Sauromatum guttatum, and Gonatanthus pumilus; they made the same observations. This discrepancy with other monocot lectins prompted us to make a detailed investigation into the carbohydrate-binding properties of these lectins. X. sagittifolium (also known as tannia, taro, cocoyam, and yautia), an edible member of the Araceae family, was chosen to pursue a detailed investigation.

Inasmuch as the hemagglutination activity of the tuber extracts of X. sagittifolium was inhibited by asialofetuin, affinity chromatography of the crude extract was performed on an asialofetuin-Sepharose column. Preliminary experiments showed that this lectin preparation gave a single low molecular mass band migrating close to the bromphenol blue dye front upon conventional SDS-PAGE (33), which had insufficient resolution in the low molecular mass range. Therefore, the Tricine/SDS-PAGE system of Schagger and von Jagow (29), which allowed an excellent separation of small proteins, was used instead, revealing a minor impurity in this lectin preparation (data not shown). By introducing a second affinity chromatography step on immobilized invertase, taking advantage of its nine N-linked high mannose-type glycans (34), a final purification of the lectin was achieved.

The data presented in this paper indicate that the Xanthosoma lectin possesses two distinctly different types of carbohydrate-combining sites: one type for oligomannoses and the other for N-linked complex carbohydrates. Since the lectin is precipitated by both mannan and asialoglycoproteins, it presumably contains at least two binding sites of each type per molecule.

The oligomannose-binding site not only is exclusively specific for mannose, but also has very strict linkage specificity for terminal 1,3-linked mannosyl residues. As revealed by precipitation and inhibition assays, it reacts only with mannans and oligomannoses bearing nonreducing terminal 1,3-mannosyl groups, such as yeast mannan (S. cerevisiae) and branched tri- and pentamannoses, but not with glucans (e.g. glycogens, dextrans, etc.) and other types of mannans (such as the mutant 1B4 mannan and K. brevis mannan, both of which lack terminal 1,3-linked mannosyl residues). The oligomannose-binding site does not react with Man₉GlcNAc₂, which has internal 1,3-linked mannosyl residues substituted by a number of 1,2-linked mannoses. The branched mannopentaose is 5-fold more potent as an inhibitor than the branched mannotriose, suggesting there is a clustering effect, which is quite common for lectin/carbohydrate interactions.

On the other hand, of a variety of native and desialylated glycoproteins and mucins tested, all those that precipitate the Xanthosoma lectin bear asialo N-linked glycans, and the precipitation reactions are not inhibited by any mono-, di-, or trimannoses, suggesting that the lectin might possess yet another type of binding sites for complex N-glycans. In an effort to elucidate the structural requirements of this type of binding site, a group of oligosaccharides that represent the major components of the asparagine-linked (N-linked) glycans was prepared. By studying the effects of these complex oligosaccharides on both XSL/yeast mannan and XSL/asialoorosomucoid precipitation reactions, the following was revealed.

1) The Xanthosoma lectin does possess a distinct type of extended binding site for complex N-glycans, which best accommodates a non-sialylated, triantennary carbohydrate with LacNAc (i.e. Gal1,4GlcNAc-, such as Tri 2) or lacto-N-biose (i.e. Gal1,3GlcNAc-, such as Tri 4) groups at its three nonreducing termini. This binding site can tolerate a substitution at the Gal1,4GlcNAc1,4- branch by an L-fucose group 1,3-linked to the penultimate GlcNAc residue (as in Fuc-Tri), but not substitution by a sialic acid group (as in Tri 5) or a bisecting GlcNAc (as in Bis 2).

2) The oligomannose-binding sites and the complex carbohydrate-combining sites are most likely non-overlapping, i.e. the oligomannose-binding site is not part of the complex carbohydrate-combining site. This was indicated by the fact that none of the complex carbohydrates tested is inhibitory in the XSL/yeast mannan precipitation system. On the other hand, the branched mannopentaose, which is by far the best inhibitor of the XSL/mannan precipitation system, is an extremely weak inhibitor of XSL/asialoorosomucoid precipitation, being 3 orders of magnitude less effective compared with the complex carbohydrates; it is also an order of magnitude less effective than it is against mannan precipitation.

Allen (12) characterized the lectin from A. maculatum as being inhibited (hemagglutination activity) primarily by N-acetyllactosamine, but not by lacto-N-biose. Our data (Table III) clearly indicate that in contrast to the A. maculatum lectin, glycoprotein precipitation by the Xanthosoma lectin is inhibited by N-acetyllactosamine and lacto-N-biose only when they occur at the nonreducing termini of complex N-linked glycans, but not by the free disaccharides (up to 100 mM).

Although the gene(s) for the Xanthosoma lectin has not yet been cloned, the conclusion that the lectin is a heterotetramer of two different subunits of nearly identical size, but with distinct N-terminal amino acid sequences, is supported by cloning and sequencing of the genes of closely related species, especially C. esculenta (11). The sequence contains an open reading frame comprising a 24-amino acid leader sequence followed by an 11-amino acid sequence identical to the X. sagittifolium lectin -subunit N-terminal sequence; 140 residues from the initiation site is found a sequence identical to the N-terminal sequence of the -subunit. Two clones from A. maculatum show highly homologous, but not identical sequences similarly placed. Thus, these aroid lectins are most likely synthesized as a single 27-kDa peptide that undergoes post-translational cleavage of the 24-residue leader sequence and a second cleavage near the center into - and -subunits of nearly identical size, which probably remain associated and assemble into the ()₂-heterotetramer before or after the cleavage.

We have observed that other aroid lectins, including those from A. maculatum and C. esculenta, are also precipitated by both asialoglycoproteins and yeast mannan,² suggesting that the presence of two distinct types of binding sites might be a general characteristic of the Araceae lectins. It is likely that the two types of sites reside specifically in the - or -subunits (or their juxtaposition) of the heterotetramer, but the relationship between the different subunits and the distinct binding sites has yet to be resolved.

	FOOTNOTES

^* This work was supported by National Institutes of Health Grant GM 29470 (to I. J. G.), by National Institutes of Health Training Grant GM 07767 (to D. L. E.), and by grants from the Katholieke Universiteit Leuven and the Fund for Scientific Research Flanders.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Research Director of the Fund for Scientific Research Flanders.

^** Postdoctoral Fellow of the Fund for Scientific Research Flanders.

To whom correspondence should be addressed. Tel.: 734-763-3511; Fax: 734-763-4581; E-mail: igoldste@umich.edu.

² H. Mo and I. J. Goldstein, unpublished data.

	ABBREVIATIONS

The abbreviations used are: XSL, X. sagittifolium lectin; -OMe, -methoxy; RP-HPLC, reverse-phase high performance liquid chromatography; PAGE, polyacrylamide gel electrophoresis; Tricine, N- [2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; LacNAc, N-acetyl- lactosamine.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES