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Trends in Glycoscience and GlycotechnologyVol. 30 No. 172 (January–May 2018) pp. SE137–SE153

Carbohydrate-Binding Specificity of Human Galectins: An Overview by Frontal Affinity Chromatography

Jun Iwaki*, †; and Jun Hirabayashi**National Institute of Advanced Industrial Science and Technology, Tsukuba, Ibaraki 305–8568, Japan

† Present address: Tokyo Chemical Industry, Co., Ltd.* E-mail: Jun.Iwaki@TCIchemicals.com

** FAX: +81–29–861–3125, TEL: +81–29–861–3124, E-mail: jun-hirabayashi@aist.go.jp

(Received on October 31, 2017, accepted on January 27, 2018)

Key Words: carbohydrate-recognition domain, dissociation constant (Kd), frontal affinity chromatography (FAC), molecular diversity, oligosaccharide specificity

AbstractTo understand the biological functions of lectins, it is important to investigate their sugar-binding specificity. Although galec-

tins are characterized as β-galactoside-binding proteins comprising evolutionarily conserved amino-acid sequences, they have sig-nificantly divergent specificities depending on their individual carbohydrate-recognition domains (CRDs). Of the various methods available to analyze lectin-glycan interactions, frontal affinity chromatography is unique in that it provides a quantitative set of dis-sociation constants (Kd’s) between immobilized lectins and a panel of (>100) fluorescently labeled oligosaccharides. In this article, we provide an overview of the features of galectin specificities with a focus on human galectins based on published data. From the data obtained, comprehensive features of individual CRDs can be systematically understood in terms of branching, and 3′-modifica-tions including sialylation, sulfation, αGal/GalNAc substitutions, β1-3Gal extension, and N-acetyllactosamine repeats. Additionally, we analyze evolutionarily more distant galectin molecules of non-human origins. These findings provide not only basic knowledge but also useful information for their applications: e.g., for engineering superior galectins improved in their specificity and affinity and developing galectin-targeted drugs.

A. Introduction: Transforming LectinomicsLectins are a group of carbohydrate-binding proteins with di-

verse structures and specificities. Recent analysis revealed that the number of protein families of lectins and those involving lectin do-mains is almost 50 (1). This is surprising considering that the num-ber of representative lectin families at the end of 20th century was only 10: R-type lectins (ricin B chain-like lectins) of β-trefoil fold, L-type lectins (legume lectin-like lectins) of β-sandwich fold (jelly-roll), GNA (Galanthus nivalis agglutinin)-related lectins of β-prism II fold, jacalin-related lectins (mJRLs and gJRLs) of β-prism I fold, hevein-like lectins (chitin-binding lectins) of hevein-type cystine-knot motif, C-type lectins (Ca-dependent-type animal lectins) of C-type α/β fold, galectins (β-galactoside-binding lectins), siglecs (sialic acid-binding immunoglobulin-like lectins), hyaladhesins (hyaluro-nan-binding proteins) of C-type α/β fold, and pentraxins (a class of pattern recognition receptors involved in innate immunity) of β-sandwich fold (jellyroll). The latest members of new lectin families include the 17-kDa α-D-galactose-binding lectin from the Mediter-ranean mussel Mytilus galloprovincialis termed MytiLec1) (2) and a

15.5-kDa mannose-specific lectin from the oyster Crassostrea gigas termed CGL1 (3). Lectin domains are also known as carbohydrate-recognition domains (CRDs) in hydrolytic enzymes, typically be-longing to the R-type lectin family (4). They are termed “carbohy-drate-binding modules” (CBMs) in the framework of Carbohydrate-Active enZymes (CAZy), with family numbers CBM 1-84 (http://www.cazy.org/Carbohydrate-Binding-Modules.html). Lectin do-mains are also found in enzymes involved in glycan synthesis, such as the R-type lectin domain in the polypeptide N-acetylgalactos-amine transferases. More recently, a stem region of protein O-linked mannose β1,2-N-acetylglucosaminyltransferase 1 was found to recognize the β-linked GlcNAc of O-mannosyl glycan, an enzymatic product of POMGnT1, by recruiting the enzyme to a specific site of α-dystroglycan to promote GlcNAc-β1,2-Man clustering (5).

Galectins, a theme of this special issue, form a large lectin family in the animal kingdom (6). However, they have also been found in more diverse organisms, such as the plant Arabidopsis thaliana and viruses (7). Historically, they have been character-ized as “developmentally regulated, soluble, metal-independent, β-galactoside-binding lectins of vertebrates” (8, 9; also see a chap-ter by Leffler in this issue, 10). Considering a long history of ga-lectin studies, they are now regarded as classic lectins as well as C-type lectins, which form another large family of animal lectins with multi-modular structures (11, 12). Distinct from C-type and R-type

MINIREVIEWdoi: 10.4052/tigg.1728.1SE

1) MytiLec was first reported as a novel R-type lectin but has no amino acid homology with the known R-type lectins. According to Pfam analysis (Ozeki, Y., personal communication), it was confirmed that MytiLec was not classified into R-type lectin and thus, is described here as a novel type lectin.

(Article for special issue on Galectins)

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lectins, however, galectin CRDs have never been found in complex proteins, such as multi-domain enzymes and membranous proteins. In fact, all the galectins so far investigated as well as galectin-re-lated proteins (see a chapter by Manning, 13) are soluble proteins. Thus, their protein architectures are simply classified into proto, chimera, and tandem-repeat types (14). Additionally, galectins are unique in that their carbohydrate-binding specificity is strictly re-stricted to β-galactosides, which made galectin functions a mystery for years. Apparently, an unsolved question remains: why galectin scaffolds confine their specificity to β-galactosides (15), as is dis-cussed by Hirabayashi et al. (16).

B. Priority: Elucidation of Carbohydrate-binding Specificity is a Key Approach to Understand Lectin Functions

To learn the basic properties of lectins, the hemagglutination inhibition assay and equilibrium dialysis have been the standard methods. However, various novel methods have been developed, which assure much higher throughput and sensitivity (described in section C). The first-priority approach taken to elucidate the bio-logical functions of a lectin is clarification of sugar-binding speci-ficity; it not only differentiates the lectin of interest from others but also provides useful clues to their counter receptor molecules (gly-coproteins and glycolipids). Glycosaminoglycans (GAG) and gly-cosylphosphatidylinositol (GPI)-anchor proteins can also be lectin counterparts. Unfortunately, the hemagglutination inhibition assay gives semi-quantitative data in terms of I50 (the concentrations of saccharides giving the half-maximal inhibition of hemagglutina-tion). The glycan microarray represents a more advanced method and provides high-content data on the sugar-binding preference of a broad panel (>400) of structure-defined oligosaccharides on a microarray platform (17). Nevertheless, it does not provide equilib-rium constants, in terms of dissociation constant (Kd) or association constant (Ka). Kd corresponds to Michaelis constant in enzymology, KM, providing essential information on the strength of the lectin-glycan interaction with the value being constant under the given conditions (e.g., temperature, pressure) regardless of the amounts and concentrations of immobilized lectins and eluted glycans. With the values obtained, we can discuss the issue of lectin specificity using this measure in a rigorous context and can compare them to the results obtained elsewhere.

In general, it is difficult to know how actual binding occurs between lectins and their counter-receptor molecules (e.g., glyco-conjugates) in physiological conditions. Identification of counter-part molecules on the cell surface is often difficult and may vary depending on source/origin of materials (e.g., type of cells, tissues, and organisms as well as developmental stages and malignancy

of the cells), although emerging technologies with high resolu-tion fluorescence microscopy, such as fluorescence recovery after photo-bleaching and fluorescence resonance energy transfer, are beginning to reveal the dynamics of galectin-glycoproteins inter-actions at the surface of live cells. Nevertheless, we require an understanding that the basic specificity of each lectin is consistent under simplified conditions. It should also be kept in mind that the Kd values will change significantly under special conditions, where simultaneous interactions are realized between multivalent lectins and multivalent oligosaccharides. It is well understood that lectin-glycan interactions become much stronger under the concept of the “clustering effect of glycosides” (18, 19). This specific feature was first noted for C-type lectins but was further demonstrated in many other lectins including galectins. Such enhanced interactions often occur on cell surfaces expressing high-density glycans or form-ing lattice architectures that achieve a tight binding, Kd <10−8 M (20). Even though this is true in complex life systems, we should recognize that lectin-oligosaccharide interactions are of basically low affinity (e.g., Kd>10−6 M) compared with other biomolecular interactions (21). Therefore, lectin-oligosaccharide interactions must first be analyzed with the simplified situation using purified glycans and lectins.

C. Technologies: Every Method Has Both SidesTable 1 summarizes the reported analytical methods avail-

able for lectin-oligosaccharide interactions (also see Table 1 in ref. 22 for a broader range of methodologies used to analyze protein-carbohydrate interactions). They include both semi-quantitative and quantitative methods. Though the former methods cannot determine the Kd values, they are generally easy to conduct with relatively cheap and simple equipment, such as red blood cells in a conventional hemagglutination inhibition assay (23) and various types of solid-phase binding assay (24, 25). From a current gly-comic viewpoint, however, the methods are required to be not only accurate and reproducible, but also of satisfactory throughput, con-tent, and speed. By the latter reason, the current glycan microarray is of the highest content with >400 synthetic glycans, and thus, the structure-defined glycans immobilized on the microarray plates can be scanned rapidly (26). Unfortunately, this powerful method does not provide quantitative data in terms of Kd. Equilibrium dialysis is the classic and simplest method to determine the Kd between free lectins and small oligosaccharide, which can freely permeate through the dialysis membrane, but lacks high throughput (27). Al-though modification using a micro-dialysis device has been report-ed (28), its use is substantially limited due to difficult manipulation and low reproducibility. Hori et al. developed a simple device of centrifugal ultrafiltration coupled with HPLC quantitation of la-

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Table 1. Analytical methods for lectin-glycan interactions.

Methods Glycan/Lectin states Strengths Weaknesses Ref.

Semi-quantitative methodsHemagglutination assay Glycan (expressed on cells,

+inhibition)Cheap and simple Limited availability of inhibitory

sugars23

Lectin (free) Lectins can be of non-purified states Depends on quality of red blood cells

Only semi-quantitative (with 50% errors)

Solid-phase binding assayBead-type Glycan (immobilized,

+inhibition)Conventional and wide applications

to glycoproteins in combination with labeled lectins or anti-lectin antibodies

Washing procedures disrupt equilib-rium

24

Lectin (labeled e.g., I125) Less applicable to oligosaccharides and determination of their specificity

ELISA-type Glycan (labeled, e.g., HRP, +inhibition)

Reversible (lectin immobilized) Washing procedures disrupt equilib-rium

25

Conventional and wide applications to glycoproteins in combination with labeled glycoproteins or neoglyco-proteins

Lectin (immobilized) Less applicable to oligosaccharides and determination of their specificity

Glycan microarray Glycan (immobilized) Reversible (lectin immobilized) Not quantitative if washing proce-dures are introduced

17, 26

High-throughput and comprehensive analysis

Lectin (labeled) A panel of structure-defined synthetic glycans immobilized on the micro-array plate

Amounts of immobilized glycans unknown

Quantitative methodsEquilibrium dialysis Glycan (labeled, e.g.,

PA, pNP)Simple principle and straightforward Requires a large amount of valuable

sugars27, 28

Lectin (free) Time-consuming and less reproducibleCentrifugal ultrafiltration HPLC Glycan (labeled, e.g., PA) Enables comprehensive analysis with

simple equipmentDepends on HPLC performance in

quantification of labeled glycans29

Lectin (free)Isothermal calorimetry (ITC) Glycan (free) Useful thermodynamic parameters

obtainedRequires a relatively large amount

of sugars30

Lectin (free) No need for labeling sugars, nor im-mobilization of sugars or lectins

Not direct determination of Kd

Surface plasmon resonance (SPR) Glycan (free or immobilized)

No need for labeling sugars Not direct determination of Kd 31

Lectin (immobilized or free)

Extensive application data available to other biomolecules

Not sensitive to measure small mol-ecules like mono and oligosaccha-rides

Fluorescence polarization (FP) Glycan (labeled, e.g., 2-AA, +inhibition)

Direct observation of interacting lectin-glycan complexes

Needs to prepare fluorescently labeled glycan

32

Lectin (free)Capillary affinity electrophoresis

with laser-induced fluorescence (LIF) detection

Glycan (labeled) High-sensitivity, high-throughput and simultaneous analysis

Requires a special equipment of LIF and skill

33Lectin (free)

Frontal affinity chromatography (FAC)

Glycan (labeled) High-sensitivity and high-throughput analysis with simple equipment

Monoamine-coupled oligosaccha-rides with open ring structure

34Lectin (immobilized)

Utilizes commercial labeled-oligosac-charides

Immobilized lectins may have modi-fied properties

Reversible (glycan immobilized)

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beled oligosaccharides (29).Alternatively, isothermal calorimetry (ITC) analysis is now

much improved in terms of both sensitivity and resolution. The method is unique in that it enables the determination of various thermodynamic parameters, ΔH and ΔS and thereby ΔG (30). However, it requires substantial amounts of glycans, which can be either labeled or non-labeled. Therefore, the application of this method to lectin specificity analysis is still limited, considering the lack of availability of complex glycans. Analysis based on a sur-face plasmon resonance (SPR) principle has the widest application to many biomolecules in a manner using non-labeled free analytes (e.g., antibodies), which interact with the immobilized ligands (e.g., antigens), while its application to smaller molecules, e.g., oligosaccharides, is limited due to the low sensitivity (31). Fluores-cence polarization (FP) analysis requires prior preparation of ap-propriately labeled glycan probes to which non-labeled glycans are added as inhibitors (32). Unfortunately, preparation of a series of non-labeled glycans (e.g., high-mannose-type and highly branched/modified complex-type N-glycans) is not easy. Capillary-based lectin affinity electrophoresis (capillary affinity electrophoresis) enables high-throughput and systematic determination of the Kd us-ing a small amount of labeled oligosaccharides (33). However, this method requires technical expertise in the manipulation of capil-lary electrophoresis.

It should be noted that in any of the methods described above, large amounts of saccharides are required for which lectins have weaker affinity. Thus, it is difficult to determine such weak inter-actions precisely (i.e., larger Kd values), which is a major issue of lectin-glycan interactions. On the other hand, frontal affinity chro-matography (FAC) circumvents this difficulty (34; for details see below).

D. FAC: A Unique Method to Enable Both Quantita-tive and Comprehensive Analysis for Weak Interactions

Frontal affinity chromatography (FAC) was first invented for analysis of biomolecular interactions in a study of trypsin and its inhibitors (35, 36). Among the methods listed in Table 1, FAC is unique in that it has a range of methods for detection: radioiso-tope (RI; 37), mass spectrometry (MS; 38, 39), and fluorescence detection (FD; 40–45)2). To perform FAC-RI glycans must be pre-radio-labeled, e.g., with NaB[3H]4 for a series of N-glycans (37). Similarly, for FAC-MS, prior modification is necessary with an appropriate alkyl reagent to increase the ionization efficiency in MS, while it may interfere with lectin stereochemistry or have an additional (e.g., hydrophobic) artificial effect. Conversely, FAC-

FD is easily performed with a conventional isocratic system for high-performance liquid chromatography (HPLC). The most ad-vantageous part of FAC is the commercial availability of a series of (>100) oligosaccharides with relatively complex structures including N-glycans (Fig. S1). Pyridylaminated (PA)-glycans meet this requisite by providing a high enough sensitivity in the concentration range of approximately 1 nM and in the consump-tion range of 1 pmol (46, 47). Other fluorescent-labeling reagents, e.g., 2-aminobenzamide (2-AB) and 2-aminobenzoic acid (2-AA), can work for this purpose, whereas larger (i.e., more hydrophobic) reagents are unfavorable, because they have a tendency to be ad-sorbed in an HPLC flow system (41). With all these considerations, the PA-oligosaccharide is the best selection for FAC-FD to perform systematic determination of the Kd for immobilized lectins and a series of standard glycans. Conducted FAC analyses for various lectins are listed in Table S1. The lectin-oligosaccharide interaction data determined by FAC are also accessible via a public data base named Lectin frontier Data Base (LfDB; 34) under the framework of the Asian Community of Glycoscience and Glycotechnology (ACGG; http://acgg.asia/db/lfdb/).

In Fig. 1, a diagram of the FAC apparatus is shown. For de-tails of principle and experimental procedures, see references (48), and (40–45), respectively. The denoted advantages of FAC-FD are summarized in Table 2 (originally from Kasai; 48). Especially, it should be noted that in FAC analysis accurate concentration of analyte, [A]0, is not necessarily known if it is negligibly small compared to Kd (also see equation in Fig. 1).

It should be kept in mind that current FAC using PA-oligosaccharides has a few drawbacks. One is that reduced termi-nal monosaccharides take an open structure as a result of mono-amine coupling (Fig. 2). This is common to other methods using the same chemical procedure, e.g., 2-AA, 2-AB, ethyl p-amino-benzoate, 2-aminoacridone, and 8-aminopyrene-1,3,6-trisulfonic acid. Another concern of FAC is that the necessity to immobilize ligands (lectins), which may modify or reduce lectin functions. It is anticipated that some lectins are inactivated upon immobilization, which may occur via the ε-amines of lysine residues involved in the carbohydrate-binding site and N-hydroxysuccinimide-activated agarose. In such cases, the addition of a competitive saccharide to the immobilization reaction often results in successful immobili-zation without loss of lectin activity. Although there is no evolu-tionarily conserved lysine residue in galectins that is involved in the direct binding, it is strongly recommended to add a relatively high concentration of lactose (>10 mM) upon immobilization. As in methods using solid phase materials (i.e., immobilized lectins), glycans can be immobilized instead of lectins, and labeled lectins may be eluted (i.e., reverse FAC). In this case, either intrinsic UV 2) For details of individual references listed here, see Table S1.

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Fig. 1. Outline of the FAC system and procedures. Isocratic elution implements reproducible results based on the HPLC analysis. Flow rate of buffer including sample injection is employed at 0.125 mL/min for a reliability of the dynamic equilibrium state in a miniature lectin-immobilized column (lectin conc.; approximate 1.0–10 mg/mL gel, column size; inner diameter 2 mm×length 10 mm) with the minimum liner flow (e.g., 0.6 mm/s). If [A]0 is negligibly small rather than Kd, the Kd value is determined by two types of parameters: the delayed volume (V-V0) of oligosaccharide-dependent binding by comprehensive analyses and the binding total (Bt) of immobilized ligands by a dose-dependent analysis. Thus, there is no influence by improper sample preparation for oligosaccharide concentration whereas small Bt value causes moder-ate susceptibility to analytical error. To require a proper Kd value, the Bt should be set within sufficient extent. Elution profiles of pNP-glycan, which is diluted to appropriate concentrations (approximate 5.0–100 µM), compared with a control glycan (e.g., pNP-βMan) are acquired in a prescribed cut-off value (5 µL) and the resulting data are plotted by the use of the Woolf-Hofstee plot.

Table 2. Unique points to FAC.

1. Simple and straightforward theoretical basisFAC deals with only an equilibrium state (dynamic equilibrium state) established between an immobilized ligand and a soluble analyte, where formed equilibrium is not disrupted.

2. Enabling weak interaction analysis in contrast to all other available methodsIt is not necessary to raise the concentration of the analyte (e.g., PA-oligosaccharides) in order to enhance complex formation and signal intensity. Regardless of binding strength, the analyte concentration can be kept at the lowest level that allows drawing of its elution profile.

3. Requiring only simple equipments and proceduresIsocratic elution is conducted using a relatively cheap HPLC apparatus, and thus, robust.

4. Accurate elution volumes by calculating integrated elution signalsThis minimizes the influence of noise and resulting in acquisition of reliable and reproducible Kd values. Frontal procedure is superior to zonal one, while the both methods are based on the same principle.

5. Simultaneous determination of dissociation constant (Kd)Once a Bt value is determined for a given column, Kd can be determined automatically by the basic equation of FAC; Kd=Bt/(V-V0)−[A]0, or Kd=Bt/(V-V0), where Kd≫[A]0.

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absorbance or fluorescence based on Trp is utilized for detection, thus, no chemical modification is necessary (49, 50).

E. Galectin Specificity: Universality and VarietyIt should be noted that under normal FAC conditions,

where galectins are immobilized at a relatively high density (i.e., 1 mg/mL) and glycans are diluted at a nano molar level, individual glycans have difficulty in interacting with multiple CRDs simulta-neously, where the clustering effect is unlikely to occur. Therefore, in FAC analysis, simple glycan-galectin interaction events are generally observed. However, it is worth mentioning essence of the features of galectin specificity has been demonstrated by preceding works by a Hakon Leffler’s group (24, 51), where they found the basic galectin-binding “epitopes” on disaccharides, and demon-strated what additions prevent binding and what additions enhance binding for some galectins. In short, these epitopes consist of three hydroxyl groups of the recognition disaccharides like lactose (Galβ1-4Glc) and LacNAc (Galβ1-4GlcNAc); namely C4-OH and C6-OH of non-reducing terminal Gal and C3-OH of reducing terminal Glc/GlcNAc. They also found that Galβ1-3GalNAc could be a ligand for galectins, though the affinity is considerably lower when compared with its isomer, LacNAc both in galectin-1 and 3 (24, 51).

Detailed binding profiles (affinities for a series of glycan structures) of human galectin-1–9 and their individual domains for chimera (galectin-3) and tandem-repeat types (galectin-4, 8 and 9) are shown in Fig. 3 from which we can conclude the followings:1) Binding profiles of full-length galectin-3 and the C-terminal

CRD are almost the same.2) Binding profiles of tandem-repeat type galectins (-4, 8 and 9)

are almost the same as the sum of the individual domains of each galectin.

3) N-terminal and C-terminal CRDs of tandem-repeat type galectin (-4, 8 and 9) show significantly distinct binding specificities. Apparently, this is attributed to significant diversion during the course of molecular evolution after an assumed duplication

event having occurred for an ancestral CRD.In fact, N-terminal and C-terminal CRDs deviate substantially

in terms of amino acid sequences. In the most typical case, the C-terminal CRD of galectin-12 has as many as 6 amino acid substitu-tions among an evolutionarily conserved eight, resulting in a loss of sugar-binding activity (Fig. 4A left). In the same way, more than 2 substitutions of the consensus motif found in placenta protein 13-like protein (PPL-13), galectin-related protein (HSPC159), and galectin-related inter-fiber protein (GRIFIN) abolish their ability to bind β-galactoside (also see a chapter by Caballero et al. in this issue, 52). Therefore, preservation of the consensus amino acids is a good primary criterion to predict galectin function. However, sub-site specificity of each galectin is often attributed to other amino acids; those located on different strands of the same sheet, e.g., S2 and S3 strands. Note that an additional hydrogen bond (1.9–6.9 kcal/mol), e.g., formed between -OH and -O- groups (5 kcal/mol), contributes significantly to the increase in affinity by approximately one order of magnitude.

According to a systematic analysis of galectins including those of non-human origins, it is evident that all the galectins share common features and bind any saccharide, while fulfilling the structural requirement of “Galβ-equatorial” (Fig. 4B left; also see 40, 44). This consensus rule seemed to apply for all disaccharides to which so far investigated galectins have been shown to bind. Based on the configuration of the glycosidic linkages regarding the reducing terminal monosaccharides, they are further catego-rized into two cases of configuration pattern: one is the “typi-cal” pattern comprising Galβ1-4Glc (lactose), Galβ1-4GlcNAc (LacNAc), GalNAcβ1-4GlcNAc (LacdiNAc), GalNAcβ1-4GlcA (chondroitin component), Galβ1-4Man (Leishmania epitope), and Galβ1-3GlcNAc (lacto-N-biose), where the equatorial 3-OH or 4-OH of the reducing terminal monosaccharides form a hydrogen-bond network with the commonly conserved Glu and Arg residues located on S6 (Fig. 4A right). The other is the “atypical” pattern comprising GalNAcβ1-3Gal (non-reducing terminal disaccharide in globotetraose, Gb4) and Galβ1-3GalNAc (T antigen and non-

Fig. 2. A reaction scheme of monoamine coupling between a reducing sugar and a coupling reagent. In the case of pyridylamination (PA), use of 2-aminopyridine (2-AP) is shown. After the coupling reaction, the derived Schiff base is reacted with NaBH3CN to stabilize the chemi-cal structure.

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Fig. 4. Common features of β-galactoside recognition by galectins. (A): Conserved amino-acid sequences of human galectins for β-galactose binding (there are no human homologues for galectin-5, 6, 11, 14, and 15. Galectin-15 was an incorrectly assigned its number and is identi-cal to galectin-11). Essential amino acids form a hydrogen-bond network and hydrophobic interaction. (B): “Galβ-eq. rule”, a consensus rule regarding the configuration of hydroxyl groups of galectin-recognition disaccharides. Distortion of glycosidic bonds composed of φ and Ψ ro-tation is limited in galectin-binding disaccharides (C): Three types of galectin-recognition disaccharides are classified based on the reducing terminal monosaccharides of the galectin-binding disaccharides. For original structural data, refer to PDB ID: 2EAK (human galectin-9N and lactose), 2EAL (human galectin-9N and Forssman pentasaccharide), and 2WKK (fungal CGL2 and Galβ1-4-L-Fuc), respectively, which were rendered by PyMOL. The three types of hydroxyl groups from various penultimate monosaccharides (including predicted) are shown in Newman projection view. R and R’ represent individually different directions of the pyranose ring against the hydroxyl group recognized by galectin.

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reducing terminal disaccharide, GA1). As described, these disac-charides have been reported as galectin ligands, while the affinities for the isolated glycans were relatively low in comparison with LacNAc (24, 51). In 1999, Choufani et al. found that galectin-1 and 3 could bind to the Thomsen-Friedenreich (T) antigen with clustering effect on glycopeptide under physiological conditions from correlation with oncological aspects (53). In this instance, however, recognition of several galectins for characteristic axial coordination of the 4-OH group projecting from the penultimate monosaccharide is limited to Gal(NAc), L-Ara, and L-Rha3) (Fig. 4C middle, dotted-circle drawn in red), resulting in somewhat dif-ferent angles when making hydrogen bonds (compare Fig. 4C left and middle). As has been described (54), it is possible to note that addition of an amino acid at the reducing end appears to enhance affinity. Thus, an explanation for binding of galectins to T-antigen within glycoproteins may be attributed to an extended mining-epitope (GalNAc-Ser/Thr) rather than cluster effect. Anyway, these two patterns (i.e., typical and atypical) are common in that the non-reducing terminal galactose is linked to a penultimate residue with a glycosidic bond to an equatorial hydroxyl group; e.g., C3/4-OH of Glc(NAc)/GlcA/Man in typical and C3-OH of Gal(NAc) in atypical cases, respectively.

However, the above rule turned out to be incomplete with the finding of a unique Galβ1-4Fuc structure in the nematode Caenorhabditis elegans, the “novel” pattern (55, 56). As a re-vised consensus rule, each galectin requires a gauche OH group (against an oxygen atom of glycosidic bond) of the reducing ter-minal monosaccharide included in the binding in addition to the axial 4-OH and the primary 6-OH groups of βGal (Fig. 4B right). As previously revealed by NMR analysis using C-lactose (57) as well as follow-up studies by the same group (58, 59), the 3-OH or 4-OH of the reducing terminal monosaccharide, e.g., Glc(NAc)/Gal(NAc)/Man(NAc), always takes a gauche position in the case of Galβ-equatorial when the glycosidic bond is in a syn configu-ration. When galectins bind to their ligands (e.g., lactose), the glycosidic bond of lactose shows two types of rotations φ and Ψ. The φ rotation is regulated by exo-anomeric effect with each lone pair between the hemiacetal oxygen (endocyclic oxygen O5) and glycosidic oxygen (Fig. 4B right). On the other hand, the Ψ rota-tion is flexible compared with φ rotation in solution. Based on this observation, Asensio et al. suggests that the galectin-binding disac-charide may take a syn/syn-form among the three possible combi-nations (syn φ/syn Ψ, syn φ/anti Ψ, anti φ/syn Ψ; 57).

Therefore, galectin-binding disaccharides are systemati-

cally classified into typical (e.g., Galβ1-4Glc), atypical (e.g., Galβ1-3GalNAc), and novel (e.g., Galβ1-4Fuc) patterns based on a configuration of a reducing terminal saccharide (Fig. 4C). How-ever, modifications of the three essential hydroxyl groups of the disaccharides result in complete loss of binding activity. These include: i) βGalNAc or αGal substitutions (GA2: 702 and Gb3: 715 in Fig. S1) at 4-OH of βGal; ii) sialylation (503, 704), sulfa-tion (922, 924), and GlcNAc branching (733, 734) at the 6-OH of βGal; and iii) fucosylation at the 3-OH (726, 909) or 4-OH (730) of Glc(NAc), known as Lewis epitopes, Lex and Lea, respectively, often combined with each other (727) or with ABO epitopes (721, 723, Leb; 731, Ley; 910). For structures containing multiple recog-nition units, caution is necessary to interpret the result obtained. For instances, the glycan 726 contains the Lex epitope at the non-reducing terminus to which galectin cannot bind but still preserves a recognition unit in its reducing terminus (Galβ1-4Glc-PA) with permitted 3-OH substitution by GlcNAc. Notably, such structures as those with the 3-OH modified βGal often gain an enhanced af-finity to some galectins, possibly by increasing sub-site specificity (described below).

F. Specific Features of Sugar-binding Specificity of Human Galectin-1–9

In this section, one of the most distinguished features of ga-lectins, a variety of their binding to 3′-modified β-galactosides, is described. Most galectins accommodate substitutions at this posi-tion, some of them showing an enhanced affinity to the resultant saccharides unlike other galactose-binding lectins, such as R-type lectins, legume lectins, and C-type lectins, which require 3-OH group of Gal for recognition (44). It is of special note that even though legume lectins have the same β-sandwich structural fold as galectins they show no apparent sequence homology and evolved individual sugar-binding sites.

F-1. Sialylation and SulfationIn the case of 3′-sialylation, galectin-8N shows a dramatic

increase in glycan binding affinity to any glycoform; i.e., N-glycan (sialylated biantennary glycan; 602), O-glycan (sialyl-core 1; 1108 shown below), milk oligosaccharide/ganglioside (sialyl lactose, GM3; 705), and GAG (sialylated keratan sulfate; 948), while many other galectins do not favor this type of modification (Fig. 5A). Interestingly, galectin-8N further enhances its affinity by α2-8Sia extension, as can be seen in gangliosides, GD3 (707). However, 3′-sulfation moderately reinforces binding of many galectins’ CRDs (galectin-3, 4N, 7, 8N, 9N, 9C). Of particular note is that both ga-lectin-4N and 9N bind well to sulfated lactose (918), but not at all to 3′-sialyllactose (GM3; 705), which is in contrast to galectin-8N. Consistently, Ideo et al. reported that galectin-8N binds well to an-

3) In case the penultimate monosaccharide is L-Rha, 2-OH group takes an axial configuration in analogy to 4-OH of D-Gal and L-Ara.

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ionic gangliosides regardless of sialylation and sulfation (60), for which detailed recognition mechanism has been elucidated (61). On the other hand, galectin-4N shows selective binding to sulfated glycans as well as 3-sulfo-cholesterol (62). In this context, Ideo et al. found that mutation at Arg45 (conserved in galectin-3, 4N, 7, 8N, 9N, 9C) of galectin-4N diminished binding toward sulfated ligands (63). Interestingly to note, this Arg is conserved in some of other galectin CRDs (galectin-3, 4N, 7, 8N, 9N, 9C), and all of them show enhanced affinity for 3′-sulfated lactose as described above.

F-2. α1-3Gal/GalNAc ModificationModification of LacNAc with either αGal or αGalNAc is as-

sociated with blood group B and A epitopes, respectively, when accompanied with 2′-fucosylation. Particularly high affinity to blood group A oligosaccharide was first demonstrated for galec-tin-3 (51); however, it is now evident that some other galectins show apparently enhanced affinity to either type A or type B sac-charides based on comparison between 2′-fucosyllactose (718) and B-tetrasaccharide (722) in galectin-3, 4N, 8N, 9N and 9C; between 2′-fucosyllactose (718) and A-tetrasaccharide (719) in galectin-3,

Fig. 5. Specific features in terms of the 3′-modifications of galectin binding. A: negative charged sialylation/sulfation. B: αGal/αGalNAc glycolipid epitopes. (C): polyβGal/polyLacNAc extension by sugar-binding specificity of human galectin CRDs 1–9.

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4N and 9N; and between LNFP-I (729) and A-hexasaccharide (720) in galectin-3, 8C, 9N and 9C. On the other hand, the effect is relatively modest on proto-type galectins (Fig. 5B). Notably, sole modification with αGal is less affective in almost all galectins; compare between LnNT (724) and Galili pentasaccharide (725). Thus, the preceding modification with 2′-fucose is important to make up strong interaction.

Jin et al. previously reported that galectin-3 plays a key role

in immune response to xenoantigen in porcine-derived organiza-tion transplantation (64). 3′-αGalNAc modification of Gb4 results in Forssman pentasaccharide (717), which dramatically reinforces the binding of galectin-9N. The reason for this unique feature was elucidated by X-ray crystallography analysis (65). It was revealed that extended recognition of αGalNAc by galectin-9N was caused by the presence of a unique Asn137 on S2 and Arg77 on S5 (Fig. 6A). Note that in this case the subsequent GalNAcβ1-3Gal is the

Fig. 6. A: Sequence alignment of CRDs (S3–S6 β-sheets) of human galectins. Conserved residues that seem to be participating in the bind-ing to the 2′-fucosylation of oligosaccharides and the N-acetyl group of GalNAc were highlighted in black followed by small Ala residues that are highlighted in gray. B: Enhancement of galectin binding with 2′-fucosylation and N-acetylation. C: Structural findings of conserved Ser and Arg residues on S3 of Gal-4C and Gal-9N, respectively and the extra-loop between S4 and S5 of Gal-1. The structural data are rendered by PyMOL with PDB ID: 1GZW (galectin-1 and lactose), 4YM1 (galectin-4C and 2′-fucosyllactose with a water molecule depicted by red sphere), and 2EAL (galectin-9N and Forssman pentasaccharide).

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recognition disaccharide but not reducing terminal lactose, which loses the essential 4-OH group of βGal by glycosidic linkage.

F-3. Galβ1-3 ExtensionSome galectins not only bind but also prefer the “inner” disac-

charide unit in polymerized oligosaccharides extending from the 3′-position of Lac (Fig. 5C left). A series of poly-β1-3-galacto-sides, defined by the formula (Galβ1-3)n-Lac, have been identi-fied as major milk oligosaccharides in marsupials (66; also see a chapter by Urashima et al. in this issue, 67) and are preferably recognized by most galectins (735–738). Of specific note, is that galectin-2, 3, 4N, 4C, 7, 8C, 9N, and 9C increase binding affinity accompanied by β1-3Gal extension. From the fact that these poly-saccharides are found as exogenous epitopes in pathogenic Leish-mania major, several galectins are expected to participate in the innate immune system as represented by galectin-3 and 9 (68, 69; also see a chapter by Sato, 70).

F-4. LacNAc ExtensionThe polylactosamine is one of the well-known endogenous

galectin ligands (71–73), which are elongated on either the N-, O-, or glycolipid glycans. Close correlation between physiologi-cal phenomena and specific interaction of galectins has been described. The FAC analysis provides quantitative data for sys-tematic discussion (Fig. 5C right). Apparently, galectin-2, 3, 8C, 9N, and 9C show an enhanced affinity with the increasing number of lactosamine repeats; i.e., n=1 (901), 2 (902), 3 (903), and 5 (905). Among them, chimera type galectin-3 and tandem-repeat type galectin-9, which are extensively involved in both innate and acquired immunity, exhibit prominent affinity to elongated polylac-tosamine structures.

Previously, Iwaki et al. reported that keratan sulfate, which has a framework identical to polylactosamine, showed binding pro-files similar to polylactosamine in the case of low sulfation (74). In contrast, another recognition unit categorized as “typical” pat-tern, chondroitin/dermatan sulfate composed of GalNAcβ1-4GlcA/IdoA, selectively interacts with galectin-3, 7, 9N, and 9C. Among them, galectin-3, 7, and 9N show proportionally increased binding to desulfated chondroitin-disaccharide in accordance with the num-ber of repeating units. From a structural viewpoint, the N-acetyl group of βGalNAc of Forssman pentasaccharide interacts with Arg44 on the S3 strand of galectin-9N (65). Although detailed rec-ognition mechanism is not known, the conserved features among galectin-3, 4N, 7, 8N, 9N, and 9C may be related to the observed specificity to desulfated glycosaminoglycans (Fig. 6A).F-5. α1-2 Fucosylation and N-Acetylation

α1-2 fucosylation also contributes to the enhancement of ga-lectin affinity, though its effect is rather limited, because the 2-OH group is oriented away from the protein surface of galectins (Fig.

6B left). This type of modification can be found in the ABO-blood type epitopes, such as H-antigens in various glycoforms (H type-1: Fucα1-2Galβ1-3GlcNAc, H type-2: Fucα1-2Galβ1-4GlcNAc, H type-3: Fucα1-2Galβ1-3GalNAc). For example, α1-2 fucosylation of LNT results in an H type-1 glycan, LNFP I (729), which shows a 2-3-fold increase in affinity for galectin-2 and 8C. Galectin-4C also shows a slight but significantly increased affinity to this oligo-saccharide that is found in high levels in human milk. This feature possibly correlates with symbiosis of bifidobacteria, which effec-tively uptakes fucosylated milk oligosaccharides in the intestinal environment (75, 76; also see a chapter by Urashima et al. in this issue, 67).

In the case of galectin-4C and 8C, Ser220 and Ser214 are con-served on S3, respectively. From structural analysis of galectin-4C with 2′-fucosyllactose, the fucose residue is not directly involved in hydrogen bonding to the Ser residue, while the space between fucose and Ser is filled by a water molecule (Fig. 6C middle; 77). The Ser residue probably possesses potential for hydrogen-bonding with the 2-fucosylated epitopes. Galectin-1 also has this Ser30, but the His52 residue on the extra loop between S4-S5 is prob-ably a refuse modification at the 2′-position of βGal (Fig. 6C left). LacdiNAc (GalNAcβ1-4GlcNAc-pNP, 1110) enhanced the binding of galectin-7, 8N, 9N, and 9C. Interestingly, these galectins possess Arg44 (residue number, galectin-9N) on S3, which is involved in enhanced binding with 3′-sulfo-Lac (Fig. 6B right, 6C right).F-6. Lac/LacNAc Preference

By comparing lactose-β-pNP and LacNAc-β-pNP, it is evident that galectin-1, 3, and 9C, show substantially enhanced affinity to the latter, while lactose-pNP is more preferably recognized by others including galectin-2, 4N, 7, 8N, and 9N (Fig. 6B right). Of particular note is that neither CRD of galectin-4 nor 8 shows sub-stantial affinity to LacNAc-β-pNP, whereas binding well to lactose-β-pNP. This observation is possibly associated with their physi-ological functions in light of counterpart ligands as discussed later.

F-7. Branched OligosaccharidesBy comparing mono-, bi-, tri-, and tetra-antennary N-glycans

(304, 307, 313 and 323, respectively), we find that most galec-tins had an increased affinity for the branched glycans, while the increase is additive or at the most modest (Fig. 7A). In fact, the multivalent effect is limited to tri-antennary N-glycans except galectin-9 because its binding to tetra-antennary N-glycans is al-most the same as that of the tri-antennary glycans, which is thought to be due to the steric hindrance. In this respect, galectin-9N is unique in that it solely shows higher affinity to tetra-antennary N-glycan (323). It should be noted that CRDs of neither galectin-4 nor 8 could bind to these N-glycans, regardless of their ability to bind N-acetyllactosamine (901).

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F-8. Type I/II PreferenceIn general, galectins recognize both type I and type II lactos-

amine structures though most galectin CRDs shows a preference for any of these isomers. Comparisons between N-glycans con-taining only type II LacNAc (313) and those containing both type I and type II (314) as well as between the milk oligosaccharides LNnT (type II, 724) and LNT (type I, 728) indicate that galectin-1, 3, 8N, and 9N preferentially bind to type II lactosamine, while galectin-2, 4N, 4C, 7, and 9C favor type I (Fig. 7B). Essentially the

same results are obtained for the branched milk oligosaccharides LNH (type I, 733) and LNnH (type II, 744): galectin-1, 2, and 3 prefer LNnT, while galectin-1, 2, 3, 7, and 9N prefer LNH, though the difference is subtle. Obviously, branching is disfavored by galectin-4N, 8N, and 9C by comparison of LNT/LNnT and LNH/LNnH. The multivalent effect in these branched glycans is less sig-nificant compared to that in LacNAc repetition.

F-9. O-linked OligosaccharidesThus far, only short O-linked glycans are available as four p-

Fig. 7. Binding characteristics of human galectin 1–9 to A: mono-, di-, tri-, and tetra-branched N-glycans; B: preferences to type I/II oligo-saccharide isomers included in N-glycan and milk oligosaccharide; and C: anomer-defined core 1, core 2, core 6, and sialyl core 1 O-glycans.

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nitrophenyl derivatives of which three showed significant binding to galectins, while core 6 did not because it is not compatible with the Galβ-equatorial manner (Fig. 7C). As a galectin binding disac-charide, the core 1 disaccharide (1103) is a good atypical ligand (Fig. 4C) for galectin-9N, followed by galectin-2, 3, 4N, and 9C. Galectins which have the Glu-water-Arg-water motif composed of Glu residue in L4 loop and Arg residue on S6 sheet are capable of recognizing endocyclic oxygen O5 and N-acetyl group of the penultimate GalNAc of core 1 disaccharide via water molecules (54). Consequently, deficient interaction of galectin-1 and 8N is caused by the extra-loop accompanied by steric hindrance and the lack of Glu-water-Arg-water motif, respectively. The core 2 structure (1104), a branched form of core 1 (Galβ1-3GalNAcα) with GlcNAcβ1-6 linkage, is also recognized, but its binding af-finity is somewhat diminished in comparison with core 1. The α2-3sialylated form of core 1 (1108) shows an enhanced affinity for galectin-8N and 9N, while the affinity increase is modest or almost the same as other galectins.

G. Prospect for Development of Galectin InhibitorsGalectins are involved in extensive physiological phenomena

through binding with various forms of β-galactosides; they can be either endogenous or exogenous ligands, including glycoproteins, glycolipids, GAGs, and possibly even free oligo- and polysaccha-rides. In general, galectins are widely involved in immunological regulation mediated on cell surface with lattice formation between multiple glycans and oligomerized galectins (78, 79; also see a chapter by Demetriou et al. in this issue, 80). As a well-known example, T-cell activation is exquisitely modulated by tight as-sociation of galectin-3 with branched and extended N-glycans

on T-cell receptor complex (81). Following examples also relate galectin functions to immunity and carcinogenesis: galectin-1 suppresses endocytosis of the Ca2+ channel TRPV5 through the binding between Klotho-mediated desialylated N-glycan and the extracellular surface (82). Galectin-1 also promotes plasma cell differentiation from activated mature B cells and immunoglobulin production, while galectin-3 regulates plasma cell differentiation from peritoneal B1 cells (83, 84). Galectin-3 production is induced by MUC1 via binding of an N-glycan on Asn36 and regulates the acceleration of cancer malignancy depending on microRNAs (85). Furthermore, galectin-3 relates to an immuno-suppressive antigen on the CD8+ cytolytic T lymphocyte (CTL) by infiltrating leukocytes to suppress effector activity (86). Galectin-4 (but not galectin-3) specifically introduces signal induction to CD4+ T cells on the intestinal epithelial cell surface, affecting pathogenic T cell survival accompanied with IL-6 expression caused by galectin-4 located in a super-raft with cell-surface glycans (87). Galectin-8 specifically binds to CD44 and is mediated by specific glycan recognition, while inhibition of which causes activation of inflam-matory cells, resulting in inhibition of apoptosis of autoimmune inflammation in rheumatoid arthritis (88). Galectin-9 represses the Th1 immune-response through induction of apoptosis of CD4+/CD8+thymocytes, which is caused by binding of N- and O-glycans projected on Tim-3 (T-cell immunoglobulin- and mucin-domain-containing molecule; 89).

As shown above, each galectin is extensively involved in glycan structure-dependent physiological regulations. Therefore, galectins are good targets for the treatment of various immuno-logical diseases (9). If galectin isomer-specific glycan-mimetic compounds are developed for specific galectin members, they can

Fig. 8. Effective framework molecules containing β-galactose as the galectin inhibitor. TDG and lactobionic acid are available as galectin-binding molecules as a target framework for a galectin inhibitor. Essential hydroxyl groups for interaction with galectin are circled with a dotted line. Structural models of TDG and lactobionic acid with galectins are rendered by PyMOL with PDB ID: 3OY8 (galectin-3 and TDG), 3OYW (galectin-1 and TDG), and 4JC1 (galectin-1 and lactobionic acid).

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be used as drugs to inhibit specific galectin functions without del-eterious side effects. Based on the early observations by Leffler et al. (24, 51), thiodigalactoside (TDG) has been a potential seed of pharmaceuticals, where the following points are noted; i) possess-ing a symmetric disaccharide structure (Galβ1-S-1β-Gal), ii) com-posed of glycosidase-tolerant thioglycoside, and iii) making double aromatic substitutions introduced at both C3 and C3′ positions to enhance affinity and specificity (90, 91; and also see a chapter by Denavit et al. in this issue, 92). The introduced aromatic groups were found to interact with an Arg residue located in the S3 strand (Fig. 6A; 93). Later, Salameh et al. used click chemistry to modify TDG at the C3-position of both βGals in the TD139 framework for a galectin-3 inhibitor (94). More recently, extensive structural analyses have been made to investigate detailed binding mode of each galectin molecule and TD139 (95). On the other hand, as an unexpected finding, α-(3,4-dichlorophenyl)-thiogalactoside modi-

fied with 3,4,5-trifluorophenyl group at the C3-position strongly enhanced galectin binding (96). Notably, this is no longer β-galac-toside (also see a chapter by Hirabayashi et al. in this issue, 16).

Other inhibitors have also been developed with the third cri-terion above, but using naturally occurring scaffolds, e.g., lactobi-onic acid (97) and type II lactosamine (Fig. 8; 90, 91, 97–100). In all these cases, the described empirical rule “Galβ-equatorial” is preserved. Actually, development of these galectin inhibitors show potential pharmaceuticals in vivo (97, 101) and eventually achieve practical use for clinical trial (102–104; also see a chapter by Gi-rard and Magnani, 105). Thus, in the future, the “Galβ-equatorial” rule would be a basic standard for comprehensive development of seeds as drug targets and, furthermore, sub-site specific chemical modification adapted to each galectin CRD that would be required for individual diseases administrated by specific galectin members based on our insight from FAC analysis.

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Jun Iwaki graduated his doctorate degree in Graduate School of Science and Engineering, Faculty of Science, Yamagata University and obtained his PhD in 2006 from the Yamagata University. He partici-pated as a postdoctoral fellow in the Dr. Hirabayashi’s Lab. at Biotechnology Research Institute for Drug Discovery (from the former Research Center for Medical Glycoscience), National Institute of Ad-vanced Industrial Science and Technology (AIST) and researched the molecular recognition of human galectins toward oligosaccharides. From 2014, he works for a chemical reagent company “Tokyo Chem-ical Industry Co., LTD” and belongs to Department of Glycotechnology to develop advanced reagents for glycoscience. His research interest is the structure-function relationships of molecular recognition between lectin and oligosaccharides and the industrial application of lectins.

Jun Hirabayashi Born in Feb 28, 1958 in Tochigi prefecture in Japan. Education: Bachelor of Science, Tohoku University (Mar., 1980), Master of Science (chemistry), Tohoku University Graduate School (Mar., 1982). Professional Carrier: Assistant professor, Faculty of Pharmaceutical Sciences, Teikyo Uni-versity, Sagamiko, Kanagawa, Japan (Apr., 1982), Lecturer (Apr., 1989), PhD in science (chemistry), Tohoku University (Dec., 1989); Team leader, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan (Nov., 2002); Visiting professor, Kagawa University, Kaga-wa, Japan (Sep., 2003); Deputy director of Research Center for Medical Glycoscience, AIST (Dec., 2006), Prime senior researcher, AIST (Apr., 2012). Main research fields: Biochemistry, Glycoscience, Glycotechnology, Lectin Engineering, evolutionary chemistry. Councilor, The Japanese Society of Car-bohydrate Research (JSCR) Director, Japan Consortium for Glycoscience and Glycotechnology (JCGG)

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