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Glycobiology Advance Access originally published online on January 26, 2007
Glycobiology 2007 17(5):479-491; doi:10.1093/glycob/cwm007
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© The Author 2007. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

Strict specificity for high-mannose type N-glycans and primary structure of a red alga Eucheuma serra lectin

Kanji Hori1,2, Yuichiro Sato2,4, Kaori Ito2,5, Yoshifumi Fujiwara2,6, Yasumasa Iwamoto2,7, Hiroyuki Makino3,6 and Akihiro Kawakubo3,6

2 Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-4-4, Higashi-Hiroshima 739-8528, Japan
3 Marine Greens Laboratory Co., Mori, Iyo 799-3125, Japan
4 National Research Institute of Brewing, Kagamiyama 3-7-1, Higashi-Hiroshima 739-0046, Japan
5 Sumika Chemical Analysis service, Ltd., Konohanaku 3-1-1353, Osaka 554-0022, Japan
6 Yamaki Co., Kominato, Iyo 799-3194, Japan
7 Otsuka Food Co., Chiyodaku, Tokyo 101-0053, Japan

1 Author to whom correspondence should be addresserd; Tel: +81-82-424-7931; Fax: +81-82-424-7916; e-mail: kanhori{at}hiroshima-u.ac.jp

Received on September 12, 2006; revised on January 14, 2007; accepted on January 16, 2007


   Abstract
Top
 Abstract
Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
We have elucidated the carbohydrate-binding profile of a non-monosaccharide-binding lectin named Eucheuma serra lectin (ESA)-2 from the red alga Eucheuma serra using a lectin-immobilized column and a centrifugal ultrafiltration-high performance liquid chromatography method with a variety of fluorescence-labeled oligosaccharides. In both methods, ESA-2 exclusively bound with high-mannose type (HM) N-glycans, but not with any of other N-glycans including complex type, hybrid type and core pentasaccharides, and oligosaccharides from glycolipids. These findings indicate that ESA-2 recognizes the branched oligomannosides of the N-glycans. However, ESA-2 did not bind with any of the free oligomannoses examined that are constituents of the branched oligomannosides implying that the portion of the core N-acetyl-D-glucosamine (GlcNAc) residue(s) of the N-glycans is also essential for binding. Thus, the algal lectin was strictly specific for HM N-glycans and recognized the extended carbohydrate structure with a minimum size of the pentasaccharide, Man({alpha}1-3)Man({alpha}1-6)Man(ß1-4)GlcNAc(ß1-4) GlcNAc. Kinetic analysis of binding with a HM heptasaccharide (M5) showed that ESA-2 has four carbohydrate-binding sites per polypeptide with a high association constant of 1.6 x 108 M–1. Sequence analysis, by a combination of Edman degradation and mass analyses of the intact protein and of peptides produced by its enzymic digestions, showed that ESA-2 is composed of 268 amino acids (molecular weight 27950) with four tandemly repeated domains of 67 amino acids. The number of repeats coincided with the number of carbohydrate-binding sites in the monomeric molecule. Surprisingly, the marine algal lectin was homologous to hemagglutinin from the soil bacterium Myxococcus xanthus.

Key words: algal lectin / amino acid sequence / Eucheuma / high-mannose N-glycan specificity / Myxococcus xanthus hemagglutinin


   Introduction
Top
 Abstract
 Introduction
Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Lectins, carbohydrate-binding proteins widely distributed in organisms ranging from viruses to humans, play important roles as recognition molecules in cell–cell or cell–matrix interactions. The proteins are also convenient tools in the field of glycomics, because they can discriminate differences in carbohydrate structures. Although the molecular structures of several families of lectins from plant, animal, and bacteria is known (Sharon and Lis 2003Go), lectins from aquatic plant have only recently been characterized. Lectins have been isolated and partially characterized from more than 45 species of marine algae. These studies revealed that many of the algal lectins share some common characteristics of low molecular weight (MW), monomeric, thermostable, and metal-independent hemagglutination, and that they have affinity for glycoproteins but not monosaccharides (Hori et al. 2000Go). These properties suggest that these lectins possess molecular structures and carbohydrate-binding specificities distinct from known lectins from other sources.

On the basis of their binding preference for N-linked glycoproteins, marine algal lectins seem to be grouped into three types: those specific for high-mannose (Man), complex, or both N-glycans (Hori et al. 1990Go, 2000). However, the detailed carbohydrate (oligosaccharide)-binding specificities of the algal lectins have not yet been investigated. Additionally, the molecular basis of strong hemagglutination by the monomeric algal proteins is unknown. The features of algal lectins do not exclude their participation in various biological activities including lymphocyte-mitogenic activity and inhibition of tumor cell growth. Furthermore, novel lectins showing strong anti-human immunodeficiency virus (anti-HIV) activity have recently been discovered from a red alga Griffithsia sp. (Mori et al. 2005Go) and cyanobacteria (blue-green algae) such as Nostoc ellipsosporum (Boyd et al. 1997Go; Dey et al. 2000Go), Scytonema varium (Bokesch et al. 2003Go), and Microcystis viridis (Williams et al. 2005Go). The HIV-inhibiting lectins from algae commonly showed the high-Man-binding nature that was critical for their antiviral activities (Botos and Wlodawer 2005Go). Thus, marine algae are a new source of novel lectin molecules for basic research and application.

In our continuing studies on algal lectins, we found a new family of lectins in lower organisms such as marine red algae belonging to the genera Solieria, Eucheuma, and Gracilaria and a freshwater cyanobacterium (blue-green alga) (Sato et al. 2000Go). This family of lectins shared similar N-terminal amino acid sequences and hemagglutination profiles that are inhibited by high-Man N-linked glycoproteins, in addition to other intrinsic characteristics common to many macroalgal lectins. Additionally, their N-terminal sequences resembled that of a hemagglutinin (MBHA) from a soil myxobacterium Myxococcus xanthus (Romeo et al. 1986Go), suggesting that lectins belonging to this family may be widely distributed in lower organisms. However, the complete amino acid sequence of a lectin from this family has not yet been elucidated, except that of MBHA that was deduced from its gene. Lectins from the genus Eucheuma algae are generally obtained in the high yields. For example, ESA-2, an isolectin from Eucheuma serra, corresponds to about 0.9% on a dry weight basis of the alga (Kawakubo et al. 1997Go). ESA-2 shows various biological activities such as mitogenic activity for mouse and human lymphocytes (Kawakubo et al. 1997Go), in vitro growth inhibition of tumor cells (Sugawara et al. 2000) and antibacterial activity (Liao et al. 2003Go). Very recently, we further observed that the isolated lectin remarkably suppressed colonic carcinogenesis in mice when administered orally and inhibited growth inhibition in vitro of 35 human cancer cell lines (unpublished data; KH, AT, and NK). Hemagglutination by the lectin was strongly inhibited by glycoproteins bearing high-Man oligosaccharides such as yeast mannan, but not by any of monosaccharides. These interesting properties of ESA-2 led us to determine its carbohydrate-binding specificity in detail and its complete amino acid sequence. We found that ESA-2 has strict oligosaccharide-binding specificity for high-Man type (HM) N-glycans. The monomeric lectin possesses four carbohydrate-binding sites and four tandem repeat structures and, interestingly, shows high sequence similarity with a bacterium MBHA.


   Results
Top
 Abstract
 Introduction
 Results
Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Carbohydrate-binding specificity of ESA-2
The carbohydrate-binding profile of ESA-2 was evaluated by two approaches, i.e., an affinity-high performance liquid chromatography (HPLC) on an ESA-2-immobilized column and a centrifugal ultrafiltration-HPLC method in which the lectin is in the immobilized and free form. In the assays, we examined 45 different fluorescence-labeled (pyridylaminated, PA-) oligosaccharides including complex type (1–13), high-Man type (HM;14–26), hybrid type (32–34), and core pentasaccharides (35–37) of N-glycans, branched oligomannoses (27–31), and others (38–45), as represented in Figure 1.


Figure 1
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  		Fig. 1. The structures of PA-oligosaccharides used in this study. Forty-five oligosaccharides were selected to represent diverse carbohydrate structures: complex type N-glycans (1–13), HM N-glycans (14–26), branched oligomannoses (27–31), hybrid-type N-glycans (32–34), a core pentasaccharide and its relatives of N-glycans (35–37), and oligosaccharides originated from glycolipids (38–45).

 
Affinity-HPLC results with 31 different PA-oligosaccharides showed that all of the HM N-glycans tested (14–16, 24, 25) were retained on an ESA-2-immobilized column (4.6 x 50 mm, 2.1 mg of ESA-2 ml–1 of gel), and did not appear in the effluents and washings (Figure 2), although they passed through a reference column (4.6 x 50 mm) having no immobilized-ESA-2 (data not shown). None of the other N-glycans including complex type (1–8, 10–12), hybrid type (32–34), core structures (35, 37), as well as oligosaccharides from glycolipids (38–43) were retained either on the ESA-2 or on the reference columns (Figure 2). The peak heights of the non-adsorbed PA-oligosaccharides in the effluents were comparable with those from a reference column (data not shown), indicating that these oligosaccharides had no affinity for ESA-2. The fluorescence intensity of PA-oligosaccharides was not the same among the oligosaccharides, probably due to the difference in their structures. These results suggested that ESA-2 specifically bound HM N-glycans and recognized their branched moieties. However, free PA-mannobioses (27–29) did not adsorb to the ESA-2 column (Figure 2).


Figure 2
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  		Fig. 2. Representative chromatograms of affinity-HPLC of PA-oligosaccharides on an ESA-2-immobilized column. A 30 µL portion (0.9 pmol) of 30 nM PA-oligosaccharide was applied to an ESA-2-immobilized column (4.6 x 50 mm, 2.1 mg of ESA-2 mL–1 of gel) equilibrated with 50 mM Tris–HCl, pH 8.0, in an oven at 25°C. The column was eluted with the same buffer at 0.3 mL min–1 and the eluate was monitored for fluorescence at an excitation and an emission wavelength of 320 and 400 nm, respectively. The same amount of PA-oligosaccharide was applied to a reference column (4.6 x 50 mm) having no immobilized lectin (data not shown). The peak heights of a PA-oligosaccharide eluted from both columns were compared with qualitatively evaluate the affinity for ESA-2. The fluorescence intensity of PA-oligosaccharides was not the same among the oligosaccharides, probably due to the difference in their structures. The elution patterns in a box indicate that the PA-oligosaccharide applied was retained on the column and did not appear in the effluents.

 
Because it was possible that a part of the binding sites of the lectin could be hidden by immobilization due to non-uniform immobilization of multivalent ligands, the result from the affinity column may not reflect the intrinsic binding property of the lectin. To address this issue and more quantitatively evaluate the binding specificity of ESA-2, we employed a centrifugal ultrafiltration-HPLC method (Katoh et al. 1993Go) with 45 different PA-oligosaccharides as described in the Materials and methods section. This method also made it possible to determine the binding activity of ESA-2 in the soluble (free) form. In this assay, binding activity is defined as the percentage of bound PA-oligosaccharide to that added. We first determined the optimal condition of the ESA-2–carbohydrate interaction with a PA–heptasaccharide (M5, 14). Figure 3 shows that maximum reaction efficiency was achieved at pH 7.0–8.0 and at temperature near 0°C, and the lectin–carbohydrate interaction reached to equilibrium after 30 min. Binding experiments with the series of PA-oligosaccharides were performed at pH 7.0 on ice for 60 min. The binding activities of ESA-2 with 45 different PA-oligosaccharides are shown in Table I. In the assay system, ESA-2 exclusively bound with HM N-glycans (14–25) and did not bind with any of the other N-glycans including complex type (1–13), hybrid type (32–34), a core pentasaccharide and its relatives (35–37), and oligosaccharides (38–45) including asialo-GM1, asialo-GM2, globotriose, forssman antigen, or lacto-N-tetraose structure originated from glycolipids. Thus, ESA-2 bound selectively to HM N-glycans in both the free and immobilized form.


Figure 3
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  		Fig. 3. Effects of pH and temperature on the binding activity of ESA-2 with a PA-heptasaccharide (M5). (A) To examine the effect of pH, 90 µL of 25 nM ESA-2 and 10 µL of 300 nM PA-heptasaccharide (M5, 14) were reacted at 37° C for 60 min in the following buffer: 50 mM MES buffer for pH 5.0 and 6.0 (white circle), 50 mM phosphate buffer for pH 7.0 (white triangle), and 50 mM Tris–HCl buffer for 7.0, 8.0 and 9.0 (black circle). The reaction mixture was then centrifuged at 10 000 g for 30 s in an ultrafiltration microtube (Nanospin Plus, USA) having an inner cell with a membrane filter (a cut off value of 10 kDa). The unbound PA-oligosaccharide was recovered in the filtrate and quantified by HPLC. As the blank, the mixture of 90 µL of each buffer and 10 µL of 300 nM PA-heptasaccharide was treated in the similar way to estimate the amount of added PA-oligosaccharide. The amount of PA-oligosaccharide bound (Obound) was calculated by subtracting the amount of unbound PA-oligosaccharide (Ounbound) from the amount added (Oadded). The binding activity (% binding) is defined as a ratio of Obound to Oadded. (B) To examine the effect of temperature, the reaction mixture of ESA-2 and PA-heptasaccharide in 50 mM Tris–HCl buffer, pH 7.0, at 0 (white circle), 37 (black circle), or 50°C (white triangle) was treated for the indicated incubation times. Each reaction was performed in duplicate.

 

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  		Table I. Binding activities of ESA-2 and ConA to PA-oligosaccharides

 
Slight differences in binding activities were observed dependent on the structures of the branched oligomannosides of HM N-glycans. Binding activities were slightly lower with oligosaccharides 16 (68.5%), 18 (66.4%), 20 (69.9%), 21 (68.2%), and 23 (52.8%), all of which attach the ({alpha}1-2)-linked mannose (Man) at the ({alpha}1-3)-linked Man residue branched from the ({alpha}1-6) arm of the core trimannose. On the other hand, the activity was over 79% with the oligosaccharides 14, 15, 17, 19, and 22, all of which have a non-reducing terminal ({alpha}1-3)-linked Man branched from the ({alpha}1-6) arm. These results revealed that ESA-2 specifically recognizes the branched oligomannosides of HM N-glycans with preference than those having the ({alpha}1-3)-linked Man at the non-reducing end in the D2 arm. Thus, this protein is a novel HM-binding lectin.

Interestingly, however, ESA-2 did not bind to free PA-oligomannoses such as Man({alpha}1-2)Man (27), Man({alpha}1-3)Man (28), Man({alpha}1-6)Man (29), Man({alpha}1-6)[Man({alpha}1-3)]Man (30), and Man({alpha}1-6)[Man({alpha}1-3)Man({alpha}1-6)[Man({alpha}1-3)]Man (31)] that are constituents of the branched oligomannosides. This result suggests that the portion of the core N-acetyl-D-glucosamine (GlcNAc) residue(s) of HM N-glycans is also essential for the interaction with ESA-2. The unique binding nature of this lectin was most obvious compared with the binding activities of Man5GlcNAc2 (M5) (14, 79.0%) and Man5 (31, 0%). Thus, the reducing terminal GlcNAc residue(s) appears to serve as a subsite in the recognition of HM N-glycans by ESA-2, although the primary recognition site(s) is likely to be present at the branched oligomannosides. ESA-2 did not bind to a PA-tetrasaccharide, Man({alpha}1-6)Man(ß1-4)GlcNAc(ß1-4)GlcNAc (26, 0%), although it bound to a PA-pentasaccharide, Man({alpha}1-3)Man({alpha}1-6)Man(ß1-4)GlcNAc(ß1-4) GlcNAc (25, 68.4%). Taken together, we conclude that ESA-2 has strict specificity for HM N-glycans and recognizes the extended carbohydrate structure with a minimum size of the tetra- or pentasaccharide, Man({alpha}1-3)Man({alpha}1-6)Man(ß1-4)GlcNAc or Man({alpha}1-3)Man({alpha}1-6)Man(ß1-4)GlcNAc(ß1-4)GlcNAc.

As a reference, we also examined the oligosaccharide-binding property of concanavalin A (ConA), a known HM glycan-binding lectin, using the same assay. As shown in Table I, ConA bound with PA-oligosaccharides including a bi-antennary complex-type N-glycan (1), oligomannoses (27–31), a core pentasaccharide (35) in addition to HM N-glycans (14–23). Thus, the oligosaccharide-binding nature of ConA is not restricted toward HM N-glycans. In the assay, ConA showed affinity for mannobioses; Man({alpha}1-2) Man (27, 44.7%), Man({alpha}1-3)Man (28, 21.1%), and Man({alpha}1-6)Man (29, 13.9%), in this preference order, and for a core mannotriose (30, 32.5%) and a mannopentaose (31, 88.4%). The binding affinities of ConA determined with this assay were comparable with those reported previously (Gallego et al. 2004Go), indicating the validity of the method. ConA and ESA-2 had apparently distinct binding profiles for HM N-glycans. Unlike ESA-2, the binding preference of ConA for HM N-glycans was independent of the presence of the non-reducing terminal ({alpha}1-3)-linked Man residue in the D2 arm as revealed by the high binding activities with the PA-oligosaccharides 16 (100%) and 20 (100%) that attach the ({alpha}1-2)-linked Man at the ({alpha}1-3)-linked Man residue in the D2 arm. Con A appears to show the higher affinity for HM N-glycans (16, 17, 20, 22) bearing non-reducing terminal ({alpha}1-2)-linked Man in the D3 arm.

Kinetic analysis of oligosaccharide binding
The association constant of ESA-2 with a PA-heptasaccharide (M5, 14) was determined using a constant concentration of ESA-2 (4 nM). Figure 4 shows both the dose–response curve and the scatchard plot of the ESA-2–HM-heptasaccharide interaction. From the scatchard analysis, it was estimated that ESA-2 has a high association constant of 1.6 x 108 M–1 and four binding sites in the molecule. Thus, the existence of multivalent binding sites per polypeptide could allow the monomeric protein agglutination activity.


Figure 4
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  		Fig. 4. Dose–response curve and scatchard plot of the interaction of ESA-2 with a PA-heptasaccharide (M5). ESA-2 (4 nM) was incubated with various concentration of a PA-heptasaccharide (M5, 14) in 50 mM Tris–HCl buffer, pH 7.0, for 30 min, and the amount of bound HM-heptasaccharide was determined using the centrifugal ultrafiltration-HPLC method. The concentration of bound HM-heptasaccharide divided by the total concentration of ESA-2 is defined as r, and this value divided by the concentration of unbound HM-heptasaccharide is defined as r/c (µM–1). [(A) and (B)] represents the dose–response curve and the scatchard plot, respectively.

 
Primary structure of ESA-2
The amino acid sequence of ESA-2 was determined by a combination of sequential Edman degradation and electron spray ionization-mass spectrometry (ESI-MS) analyses of intact ESA-2, pyridylethylated (PE) ESA-2, and peptides produced by digestion of ESA-2 with lysylendopeptidase (Lys-C) or aspartylendoprotease (Asp-N). The molecular masses of intact ESA-2 and PE-ESA-2 were determined using ESI-MS to be m/z:27949.0 and 28055.0, respectively. The difference in molecular masses indicated that the lectin protein contains a single cysteine residue. The 64 N-terminal amino acids of ESA-2 were directly sequenced from the intact protein, whereas the C-terminal sequence was determined to be Val-Ala-Thr-Ser-COOH by a sequential digestion with carboxypeptidase Y (data not shown). Peptides produced by digestion of PE-ESA-2 with Lys-C and Asp-N were separated by reverse-phase HPLC, and the isolated peptides, L1–L12 (with Lys-C) and A1–A16 (with Asp-N), were subjected to ESI-MS analyses and sequential Edman degradation. Tables II and III shows the determined molecular masses and N-terminal sequences of L1–L12 and A1–16. The complete amino acid sequence of ESA-2 was deduced based on the overlapping sequences of the peptide fragments and the N- and C-terminal sequences of the intact proteins, as shown in Figure 5. The sequencing result showed that some peptides (L5, A6, A9, A10, and A13) of the enzymic digests were generated by non-specific cleavage. The molecular mass (27 950.3) calculated from the amino acid sequence of ESA-2 was in good agreement with that determined by ESI-MS (m/z: 27949.0). Thus, ESA-2 is a polypeptide composed of 268 amino acids, including a single cysteine residue, without posttranslational modifications such as carbohydrate addition.


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  		Table II. The N-terminal amino acid sequences and molecular masses of peptide fragments produced by digestion with Lys-C of PE-ESA-2

 

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  		Table III. The N-terminal amino acid sequences and molecular masses of peptide fragments produced by digestion with Asp-N of PE-ESA-2

 

Figure 5
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  		Fig. 5. The complete amino acid sequence of ESA-2. The complete amino acid sequence of ESA-2 was determined using the sequences of overlapping peptides generated by enzymic cleavages with Lys-C and Asp-N, and the N- and C-terminal sequences of the intact ESA-2. Amino acid residues identified by sequential Edman degradation are indicated by solid lines, and the non-identified amino acids are indicated by dashed lines. Abbreviations used: L, Lys-C peptides; A, Asp-N peptides.

 
Four tandemly repeated domains are present in ESA-2, each consisting of 67 amino acids and sharing 45.4% sequence identity (Figure 6). Clusters of identical amino acids among the four repeated domains were located on both N- and C-terminal regions of each domain. Hydropathy analysis suggested that the regions of conserved amino acids among the repeated domains are rather hydrophilic. The number of the repeats coincided well with the number of carbohydrate-binding sites in the monomeric molecule. BLAST search result revealed that ESA-2 shares significant sequence similarity with MBHA (Romeo et al. 1986Go). The bacterial hemagglutinin shares 59.7% of sequence identity and is of similar molecular size with ESA-2 and has four tandem repeats. Identical amino acids are located at both N- and C-terminal regions of each repeated domain (Figure 7), portions of which also correspond to the conserved regions among the four repeated domains of each lectin.


Figure 6
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  		Fig. 6. Comparison of tandem-repeated sequences of ESA-2. Identical amino acids among the four repeated domains in the lectin molecule are shaded.

 

Figure 7
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  		Fig. 7. Sequence alignment of ESA-2 and the MBHA. Identical amino acids are shaded. The amino acid sequence of MBHA was deduced from its DNA (Romeo et al. 1986Go; GenBank accession no. M13831).

 

   Discussion
Top
 Abstract
 Introduction
 Results
 Discussion
Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
Most lectins from marine algae, especially red algae, are monomeric proteins with no affinity for monosaccharides but with strong hemagglutination activities. These properties suggest that they belong to a new category of lectins (Hori et al. 2000Go). Here, we show that a representative of such algal lectins, ESA-2 from the red alga E. serra, has strict oligosaccharide-binding specificity for HM N-glycans and four carbohydrate-binding sites per polypeptide that satisfies multivalency required for all agglutinins. The oligosaccharide-binding specificity verifies the previous results obtained from hemagglutination-inhibition test with glycoproteins (Kawakubo et al. 1997Go). We pointed out previously that algal lectins with affinity only for glycoproteins have low MW and are monomeric, whereas algal lectins with affinity for monosaccharides have a large molecular size or oligomeric structures (Hori et al. 1990Go, 2000; Rogers and Hori 1993Go). As demonstrated here for ESA-2, restricted binding, only with oligosaccharides, might be due to the monomeric structure. The strict specificity for HM N-glycans with a high association constant in the nanomolar range has not been reported for other known high-Man-binding lectins except the HIV-inhibiting proteins from some algae. Thus, ESA-2 may be useful as a specific probe for N-glycans and, potentially, as a potent anti-HIV agent.

Because of the high affinity for HM glycans, it was impossible to elute the HM-oligosaccharides bound to an ESA-2-immobilized column with any of the reagents tested including simple sugars, chaotropic ions, detergents, and denaturants such as urea and sodium dodecyl sulfate (SDS), except HCl. The same is true for the interaction of ESA-2 with HM-linked glycoproteins immobilized on affinity columns or on the sensor chips of BIAcore in a surface plasmon resonance study (data not shown). The centrifugal ultrafiltration-HPLC method used in this study enabled semi-quantitative binding assays of ESA-2 with PA-oligosaccharides. Using this assay, we showed that ESA-2 distinguishes HM N-glycans with different branched structures. ESA-2 showed relatively low affinity for HM N-glycans bearing non-reducing terminal ({alpha}1-2)-linked Man in the D2 arm. Additionally, it is notable that ESA-2 requires the reducing terminal GlcNAc residue(s) for binding to HM N-glycans, although it does not bind the core pentasaccharide containing the same residues. Enhancement of binding affinity by the addition of GlcNAc residues at the reducing end has been reported for the high mannose-binding lectins from land plants such as Allium sativum (Dam et al. 1998Go; Bachhawat et al. 2001) and Artocarpus integrifolia (artocarpin; Misquith et al. 1994Go) and from the cyanobacteria, N. ellipsosporum (cyanovirin-N; Sandstrom et al. 2004) and M. viridis (MVL; Bewley et al. 2004Go; Williams et al. 2005Go). However, unlike ESA-2, the two land plant lectins have affinity for Man, oligomannoses, and the core pentasaccharide, and cyanovirin-N and MVL bind oligomannoses and the core pentasaccharide, respectively. Unlike MVL that can bind structural unit of Man({alpha}1-6)Man(ß1-4)GlcNAc(ß1–4)GlcNAc as the smallest ligand (Bewley et al. 2004Go), the minimum unit bound by ESA-2 is the tetra- or pentasaccharide, Man({alpha}1-3)Man({alpha}1-6)Man(ß1-4)GlcNAc or Man({alpha}1-3)Man({alpha}1-6)Man (ß1-4) GlcNAc(ß1-4)GlcNAc that contains non-reducing terminal ({alpha}1-3)-linked Man. To our knowledge, ESA-2 is the only lectin that shows strict specificity for HM N-glycans including the reducing terminal GlcNAc residue(s) without binding to monosaccharides and the core pentasaccharide. Therefore, a future goal of our research is to define the molecular basis of the strict specificity of ESA-2 for HM N-glycans. In addition, it will also be interesting to investigate the relationships between the strict binding specificity and the biological activities of the lectin, because lectin suppressed the growth in vitro of human cancer cells independently of the specific cell line used and also of colonic carcinogenesis in mice (unpublished data; KH, AT, and NK).

Moreover, the anti-HIV activity of ESA-2 remains to be investigated. It is relevant to discuss the possibility that the HIV-inhibiting activity of ESA-2 is related to its strict specificity for HM N-glycans, because there is increasing evidence that the high-Man-binding lectins inhibit HIV replication (Botos and Wlodawer 2005Go). Many of the HIV-inhibiting proteins reported so far appear to act by binding HM N-glycans on the envelope glycoprotein, gp 120; thereby interfering with virus entry into the host cells (Botos and Wlodawer 2005Go). Among these HIV-inhibiting proteins, plant lectins such as Hippeastrum hybrid agglutinin (HHA) and Galanthus nivalis agglutinin (GNA), which are classified into a family of monocot Man-binding lectins, bind to monosaccharide, core mannotriose and pentasaccharide of N-glycans in addition to HM N-glycans (Barre et al. 1996Go). The same is true for a legume lectin, ConA whose binding profile has been confirmed in this study. A C-type animal lectin, dendritic cell-specific intercellular adhesion molecule-3 grabbing nonintegrin, which also binds to the HIV envelope, recognizes the internal core of HM glycans (Feinberg et al. 2001Go). In contrast, ESA-2 did not bind monosaccharide, core mannotriose and pentasaccharide of N-glycans, and branched oligomannoses. Its binding specificity was restricted to HM N-glycans. Such binding specificity of ESA-2 for HM N-glycans somewhat resembles those of the recently discovered anti-HIV proteins, cyanovirin-N (Bewley et al. 1998Go), MVL (Bewley et al. 2004Go) and griffithsin from the red alga, Griffithsia sp. (Mori et al. 2005Go), which are not classified into established lectin families. These algal lectins commonly showed high affinity in the nanomolar range for HM N-glycans. The striking finding is that ESA-2 mimics the mode of HM N-glycan recognition by a human antibody 2G12, which neutralizes a broad range of HIV-1 isolates by binding a dense cluster of oligomannoses on the "silent" face of gp120 (Calarese et al. 2003Go). Biochemical and crystallographic studies have demonstrated that 2G12 binds to the non-reducing terminal Man({alpha}1-2)Man in both the D1 (Man({alpha}1-2)Man({alpha}1-2)Man) and D3 arm (Man({alpha}1-2)Man({alpha}1-6)Man) of branched oligomannosides but not to that in the D2 arm (Man({alpha}1-2)Man({alpha}1-3)Man) (Calarese et al. 2005Go). Similarly, the binding activity of ESA-2 was considerably impaired when ({alpha}1-2)-linked Man is attached at the terminus of D2 arm (as observed with oligosaccharide 16, 18, 20, 21, and 23), whereas it was not affected but rather enhanced by the presence of ({alpha}1-2)-linked Man at the terminus of D1 or D3 arm (oligosaccharide 15, 17, 19, and 22). In addition, ESA-2 resembles 2G12 in its reduced interaction with free oligomannoses, because 2G12 has been reported to have the 50-fold less affinity for Man({alpha}1-2)Man and much weaker interactions with Man({alpha}1-3)Man or Man({alpha}1-6)Man than for Man9GlcNAc2 (Calarese et al. 2003Go). ESA-2 did not bind appreciably to these mannobioses. In this context, it is of particularly interest that cyanovirin-N recognizes both D1 and D3 arms but not the D2 arm in analogy with ESA-2 and 2G12 (Bewley et al. 2001, 2002). The crystal structure of cyanovirin-N with Man9GlcNAc2 revealed that the binding interface is present on D1 arm (Botos et al. 2002Go). Unlike ESA-2, however, cyanovirin-N does not bind HM-glycans bearing heptamannosides (M7) or lower mannooligomers (Shenoy et al. 2001Go). The lack of structural similarity between ESA-2 and these proteins, including 2G12, suggests that the mode of carbohydrate recognition is different between these groups of the HM-binding proteins. It has been reported that the binding sites of 2G12 are formed by its characteristic assemblage, interlocked VH domain-swapped dimer and involves the two deep primary combining sites and the two shallow secondary binding sites (Calarese et al. 2003Go). The multivalent interaction accounts for the high affinity of 2G12, i.e., more than two HM oligosaccharides on gp120 are trapped by one 2G12 molecule in the proposed model (Calarese et al. 2003Go). In contrast, kinetic binding assay of ESA-2 with PA-heptasaccharide (M5) suggests the presence of four binding sites per a monomeric molecule. Unlike 2G12, it seems likely that the high affinity of ESA-2 for a HM glycan may stem from the multiple contacts with a glycan via the four binding sites, because ESA-2 recognizes a long carbohydrate sequence from the non-reducing terminal mannose to the reducing terminal GlcNAc residue with the minimal length of a tetra- or pentasaccharide. This observation also suggests that the carbohydrate-binding pocket of ESA-2 molecule must be deep enough to accommodate the extended carbohydrate structure. Ongoing crystal structural analysis of ESA-2 will provide additional insight into the detailed mode of high-Man oligosaccharide recognition. Cyanovirin-N, MVL, and griffithsin also bind HM glycans with very high affinity, despite the absence of multivalent interactions with more than two HM oligosaccharides (Chang et al. 2002; Bewley et al. 2004Go; Ziolkowska et al. 2006Go). Cyanovirin-N (Yang et al. 1999Go; Botos et al. 2002Go) and griffithsin (Ziolkowska et al. 2006Go) exist as domain-swapped dimers with four and six binding sites, respectively, and MVL as a homodimer with four binding sites (Williams et al. 2005Go). Although ESA-2, a monomeric lectin with four possible binding sites, has no sequence similarity with these cyanobacterial lectins, the strict specificity for HM oligosaccharides with very high binding affinities seems to be a common feature among the lectins from lower plants such as cyanobacteria and algae.

Recently, Balzarini et al. demonstrated that HIV treated with Urtica dioica agglutinin (UDA), a N-acetylglucosamine-binding lectin from U. dioica, deleted the highly conserved glycosylation sites in the gp120 envelope, possibly to escape constrains caused by UDA binding (Balzarini, Van Laethem, Hatse, Froeyen, Peumans, et al. 2005). Deletion of glycosylation sites results in higher susceptibility of HIV to neutralizing antibodies, because the hidden immunogenic epitopes are exposed after the removal of the carbohydrate shields. The advantage of using GlcNAc-specific lectins instead of Man-binding lectins is assumed to be that the GlcNAc moiety is invariably present at the core structure of N-glycans in gp120. The fact that ESA-2 recognizes both core GlcNAc residue(s) and branched oligomannosides in HM glycans suggests its potential in creating novel form of trimmed HIV, which might be applicable for vaccine development. Trimming of the glycosylation site of HIV without changing the tertiary structure by using a novel lectin, such as ESA-2, could result in the exposure of HIV epitopes that are normally shielded by HM glycans, the counterparts (binding sites) to Chemokine receptor 4, chemokine (c-c motif) receptor, and chemokine (C-x-C motif) receptor in the distinct fashion from other class of HM-binding lectins (Balzarini, Van Laethem, Hatse, Froeyen, Van Damme, et al. 2005). Thus, compared with the known HM-binding lectins, ESA-2 should be an intriguing candidate as an anti-HIV agent, because its binding specificity is restricted toward HM N-glycans.

As described in the Introduction section, ESA-2 is a member of a new family of lectins found in lower organisms including marine red algae, a freshwater cyanobacterium, and a soil bacterium. This family also includes lectins from the Solieriacea, a family of marine red algae: Solieria robusta (Solnins A–B) (Hori et al. 1988Go), Eucheuma serra (ESA-1–3) (Kawakubo et al. 1997Go), E. amakusaensis (EAA-1–3) (Kawakubo et al. 1999Go), E. cottonii (revised later as Kappaphycus alvarezii) (ECA-1–2) (Kawakubo et al. 1999Go), Gracilaria busra-pastoris (Okamoto et al. 1990); a freshwater cyanobacterium, O. agardhii (Sato et al. 2000Go); and a soil myxobacterium, M. Xanthus (Romeo et al. 1986Go). This family had been defined primarily on the basis of the limited criteria of similarities of N-terminal sequence and hemagglutination-inhibition profile. The complete amino acid sequences of lectins in this family have not yet been elucidated, except for MBHA. In this study, we have determined the complete amino acid sequence of ESA-2 and confirmed that it strongly resembles that of MBHA, including its molecular size and the presence of four tandemly repeated motifs. Very recently, we have elucidated the primary structure of a HM N-glycan-specific lectin from the cyanobacterium, O. agardhii, that is also homologous to ESA-2 and MBHA (Y Sato and K Hori, in preparation). The carbohydrate-binding specificity of MBHA has not been well characterized, although hemagglutination was inhibited by the addition of ConA. Based on similarities in their primary structures, MBHA is also predicted to be specific for HM N-glycans. The occurrence of a novel lectin family in the lower organisms raises an interesting question about their biological significance. Although the biological role of ESA-2 is unknown, this lectin is present at a high level in the alga and is easily extracted when algal tissues are soaked in aqueous ethanol (Kawakubo et al. 1997Go). Immunochemical staining using anti-ESA-2 polyclonal antibodies showed that the lectin is located primarily on cell walls through the whole portion of the algal thallus (unpublished data; KH, AK, and YH). On the other hand, MBHA is thought to be involved in mediating social behaviors of myxobacteria. M. xanthus grows in a complex life cycle that includes fruiting body formation (Cumsky and Zusman 1979Go). Under starvation conditions, a developmental program triggers the cellular aggregation that results in fruiting body formation. MBHA is induced during the aggregation phase, suggesting its involvement in the fruiting body formation (Cumsky and Zusman 1979Go). The understanding of the physiological role of ESA-2 would also give some insight to clarify the biological significance of the novel lectin family in the lower organisms that are evolutionally and geographically distinct.

Algae of the genus Eucheuma, including ESA-2, that are high-yielding sources of this new family of lectins, have been widely cultivated as edible seaweeds or the sources of carrageenans in subtropical costal areas. These algae will draw considerable attention as the source of not only carageenans but also a valuable protein for biochemical and medicinal uses.


   Materials and methods
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
Conflict of interest statement
 Acknowledgments
 References
 
Material
E. serra lectin (ESA-2) was extracted using 20% ethanol and purified as described previously (Kawakubo et al. 1997Go). The purified lectin was stored at –20°C until used. ConA was purchased from Seikagaku Corporation (Japan). The powdered resin of TSKgel Tresyl-5PW and HPLC columns of TSKgel ODS80TM, TSKgel NH2-60 and TSKgel ODS-120T were obtained from Tosoh Corporation (Japan). An YMC PROTEIN-RP column was from YMC (Japan). PA-oligosaccharides were obtained from TAKARA (Japan) or AJINOKI Corporation (Japan). Mannobioses (Man({alpha}1-2)Man, Man({alpha}1-3)Man, Man({alpha}1-6)Man), mannotriose (Man({alpha}1-6)[Man({alpha}1-3)]Man) and mannopentaose (Man({alpha}1-6)[Man({alpha}1-3)]Man({alpha}1-6)[Man({alpha}1-3)]Man) were from FUNAKOSHI (Japan). Asp-N and Lys-C (Achromobacter protease I) were obtained from TAKARA (Japan) and WAKO (Japan), respectively. All other chemicals used in this study were of the highest purity available.

Pyridylamination of oligomannoses
PA-oligomannoses used in this study were prepared using a pyridylamination reagent kit (TAKARA) and a semi-automated PA-derivatization apparatus (PALSTATION, TAKARA) according to the manufactures' protocol. Briefly, 20 µL of 2-aminopyridine in acetic acid was added to 50 nmol of lyophilized oligomannoses (mannobioses, mannotriose, and mannopentaose) and heated at 90°C for 60 min. A volume of 20 µL of borane dimethylamine–acetic acid was then added, and the mixture was heated at 80°C for 60 min. Excess reagents were removed by normal phase HPLC on a TSKgel NH2-60 column (4.6 x 250 mm) using a linear gradient of acetonitrile in 50 mM acetic acid–triethylamine buffer, pH 7.3, at a flow rate of 1.0 mL min–1 at 40°C. The eluate was monitored for fluorescence of PA-oligosaccharide at an excitation wavelength of 310 nm and an emission wavelength of 380 nm, and the peak fractions of PA-oligosaccharide were recovered. The amounts of PA-oligosaccharides prepared were quantified as follows. An aliquot of each PA-oligosaccharide preparation was dried with a Speed Vac and subjected to gas phase acid-hydrolysis in 4 N HCl–4 M trifluoroacetic acid; (TFA (1:1, v/v)) at 100°C for 4 h. An aliquot of the hydrolysate was applied to reverse-phase HPLC on a TSKgel ODS-80TM column (4.6 x 150 mm). Elution was done with 10% methanol in 0.1 M ammonium acetate buffer at a flow rate of 1.0 mL min–1 at 40°C and the peak area of PA-Man was measured. At the same time, various amounts of PA-Man (TAKARA) were subjected to the same HPLC to make a standard curve for quantification. Thus, the free PA-Man liberated from PA-oligomannose by acid hydrolysis was quantified using a standard curve of the authentic PA-Man.

Preparation of ESA-2-immobilized column for HPLC
ESA-2 (7.8 mg) was dissolved in 2 mL of 1 M sodium phosphate buffer, pH 7.5. To the lectin solution, solid powder of TSKgel Tresyl-5PW (0.3 g) was added and stirred for 8 h at 25°C. The lectin-immobilized gel was packed to a stainless-steel column (4.6 x 50 mm) at a flow rate of 0.5 mL min–1. The remaining reactive groups of the gel were blocked by washing the column with 100 mL of 0.1 M Tris–HCl buffer, pH 8.0, and the column was equilibrated with 50 mM Tris-HCl buffer, pH 8.0. The amount of immobilized lectin was estimated by subtracting the sum of absorbance at 280 nm of the effluent and washes from that of the original lectin solution. In the similar way, a reference column (4.6 x 50 mm) was also prepared without the addition of lectin solution.

Affinity chromatography on an ESA-2-immobilized column
Binding experiments of PA-oligosaccharides on an ESA-2-immobilized column were performed at 25°C as follows. A volume of 30 µL (0.9 pmol) each of 30 nM PA-oligosaccharide was injected separately to a lectin (ESA-2) and a reference column that had been equilibrated with 50 mM Tris–HCl buffer, pH 8.0. Both columns were eluted at a flow rate of 0.3 mL min–1 with the same buffer and the eluate was monitored for fluorescence at an excitation wavelength of 320 nm and an emission wavelength of 400 nm for PA-oligosaccharide. Binding affinity was qualitatively evaluated by comparing peak heights of non-adsorbed PA-oligosaccharides in eluates from both columns.

Binding assay by centrifugal ultrafiltration-HPLC method
Oligosaccharide-binding activity of free ESA-2 was determined using a centrifugal ultrafiltration-HPLC method as described by Katoh et al. (1993). A volume of 90 µL of 500 nM ESA-2 and 10 µL of 300 nM PA-oligosaccharide were reacted in 50 mM Tris–HCl buffer, pH 7.0 in an ice bath for 60 min. The reaction mixture was then centrifuged (10 000 g x 30 s) using a centrifugal ultrafiltration tube (Nanospin Plus, Gelman Science, Ann Arbor, MI) having an inner cell with a membrane filter (a cut off value of 10 kDa). An aliquot of the filtrate was applied to a TSKgel ODS 80TM column (4.6 x 150 mm) equilibrated with 10% methanol in 0.1 M ammonium acetate buffer and eluted at a flow rate of 1.0 mL min–1 with the same solvent. HPLC was performed at 40°C. The eluate was monitored for PA-oligosaccharide as described in Affinity chromatography on an ESA-2-immobilized column section, and the amount of unbound PA-oligosaccharide (Ounbound) was estimated from the peak area. To determine the amount of PA-oligosaccharide added (Oadded) in the reaction system, 90 µL of 50 mM Tris–HCl buffer, pH 7.0 with no lectin and 10 µL of 300 nM PA-oligosaccharide in the same buffer were combined and treated. The amount of bound PA-oligosaccharide (Obound) was calculated by subtracting the amount of unbound PA-oligosaccharide from that of the added (Oadded–Ounbound). The binding activity is defined as the ratio of Obound to Oadded and denoted as percent binding. The binding assay was performed in duplicate for each PA-oligosaccharide and the activity was expressed as the average value from duplicate assays. As a reference lectin, the binding activity of ConA was examined in the same way except the reaction buffer used was 50 mM Tris–HCl buffer, pH 7.4 containing 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, and 1 mM MnCl2.

Prior to measuring binding activities using this method, the optimum pH and temperature for binding were determined using a PA-oligosaccharide (M5, 14). To determine the optimal pH, 90 µL of 25 nM ESA-2 and 10 µL of 300 nM PA-heptasaccharide were reacted at 37°C for 60 min in buffers at different pH; 50 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 5.0, 6.0), 50 mM sodium phosphate buffer (pH 7.0), 50 mM Tris–HCl buffer (pH 7.0, 8.0, 9.0). To determine the optimal temperature, the same mixture of lectin and PA-oligosaccharide were incubated in 50 mM Tris–HCl buffer, pH 7.0, at varying temperatures (0, 37, and 50°C) for 1, 5, 10, 30, 60 and 90 min. Each reaction mixture was treated as described in the Affinity chromatography on an ESA-2-immobilized column section to determine binding activities.

Kinetic analysis of binding
The association constant and the number of carbohydrate-binding sites in ESA-2 were determined with a PA-heptasaccharide (M5, 14) as follows. A volume of 90 µL of 4 nM ESA-2 was reacted with 10 µL of PA-heptasaccharide (14) at various concentration (100~500 nM) in 50 mM Tris–HCl buffer, pH 7.0, in an ice bath for 30 min. The amount of bound PA-oligosaccharide in each reaction mixture was determined by the centrifugal ultrafiltration-HPLC method, and data were used for scatchard plot analysis.

Preparation of S-pyridylethylated (PE-) ESA-2
ESA-2 (1 mg) was dissolved in 500 µL of 250 mM Tris–HCl, pH 8.5, containing 6 M guanidine HCl and 1 mM EDTA. An amount of 1 mg of dithiothreitol was then added to the lectin solution and allowed to stand at room temperature for 2 h under nitrogen gas. Subsequently, 2 µL of 4-vinyl pyridine was added and incubated for another 2 h. The resulting PE-ESA-2 was purified by reverse-phase HPLC on a YMC PROTEIN-RP column (6.0 x 250 mm) using a linear gradient of acetonitrile in 0.1% TFA. The peak containing PE-ESA-2 was recovered and dialyzed against distilled water to remove excess reagents.

Enzymic cleavage and separation of peptides
An amount of 200 µg of PE-ESA-2 was digested with Lys-C [E/S = 1/100 (w/w)] in 50 mM Tris–HCl, pH 8.5 at 37°C for 24 h, and with Asp-N [E/S = 1/100 (w/w)] in 50 mM Tris–HCl, pH 7.5, at 37°C for 18 h, respectively. Peptides generated by each digestion were separated by reverse-phase HPLC on a TSKgel ODS-120T column (4.6 x 250 mm) using a linear gradient of acetonitrile in 0.1% TFA.

Analyses of N- and C-terminal sequence and amino acid composition
The N-terminal amino acid sequences of PE-ESA-2 and the peptides generated by enzyme digestion of PE-ESA-2 were determined by an automated-protein sequencer (Applied Biosystems 477A, Foster City, CA) connected to phenylthiohydantoin analyzer (120 A) or alternatively by a protein sequencer (Hewlett-Packard G1005A, Palo Alto, CA). The C-terminal amino acid sequence of ESA-2 was determined as follows. ESA-2 (5 nmol) was denatured by heating at 60°C for 20 min in the presence of 0.5% SDS in 50 µL of 0.1 M pyridine–acetic acid, pH 5.6. The denatured protein was digested with 3 µL of carboxypeptidase Y (1 mg mL–1), and the digest at each time point (0, 1, 2, 5, 10, 20, 30, and 60 min) was subjected to amino acid analysis calibrated with the internal standard of norleucine (5 nmol). Amino acid analysis was performed as described previously (Hori et al. 2000Go).

Molecular mass determination of protein and peptides
The molecular masses of intact ESA-2, PE-ESA-2 and peptide fragments were determined by ESI-MS (LCQ, Finingan).


   Conflict of interest statement
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
Acknowledgments
 References
 
None declared.


   Acknowledgments
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
References
 
We thank Prof. John J. Harada (University of California, Davis) for his critical reading of the manuscript. This work was supported in part by Grant-in-Aid for Scientific Research (B) from Japan Society of the Promotion of Science. The protein sequence data of ESA-2 reported in this paper will appear in the Swiss-Prot and TrEMBL knowledgebase under the accession number P84331 [GenBank] .


   Abbreviations
 
Asp, aspartylendoprotease; ConA, concanavalin A; EAA, Eucheuma amakusaensis lectin; ECA, Eucheuma cottonii (Kappaphycus alvazerii) lectin; ESA, Eucehuma serra lectin; ESI-MS, electron spray ionization-mass spectrometry; GlcNAc, N-acetyl-D- glucosamine; GNA, Galanthus nivalis agglutinin; HHA, Hippeastrum hybrid agglutinin; HIV, human immunodeficiency virus; HM, high-mannose type; HPLC, high performance liquid chromatography; Lys, lysylendopeptidase; Man, mannose; MBHA, Myxococcus xanthyus agglutinin; MES, 2-(N-morpholino)ethanesulfonic acid; MVL, Microcystis viridis lectin; MW, molecular weight; PA, pyridylaminated; PE, pyridylethylated; SDS, sodium dodecyl sulfate; TFA, trifluoroacetic acid; UDA, Urtica dioica agglutinin.


   References
Top
 Abstract
 Introduction
 Results
 Discussion
 Materials and methods
 Conflict of interest statement
 Acknowledgments
 References
 
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