Oligosaccharide specificity of galectins: a search by frontal affinity chromatography

Biochimica et Biophysica Acta 1572 (2002) 232 – 254 www.bba-direct.com

Review

Oligosaccharide specificity of galectins: a search by frontal affinity chromatography Jun Hirabayashi a,*, Tomomi Hashidate a, Yoichiro Arata a, Nozomu Nishi b,c, Takanori Nakamura b, Mitsuomi Hirashima c,d, Tadasu Urashima e, Toshihiko Oka f, Masamitsu Futai f, Werner E.G. Muller g, Fumio Yagi h, Ken-ichi Kasai a a Department of Biological Chemistry, Faculty of Pharmaceutical Sciences, Teikyo University, Sagamiko, Kanagawa 199-0195, Japan Department of Endocrinology, Faculty of Medicine, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kitagun, Kagawa 761-0793, Japan c Galpharma Co. Ltd., Kagawa 761-0703, Japan d Department of Immunology and Immunopathology, Faculty of Medicine, Kagawa Medical University, 1750-1 Ikenobe, Miki-cho, Kitagun, Kagawa 761-0793, Japan e Department of Bioresource Chemistry, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-8555, Japan f Institute of Scientific and Industrial Research, Osaka University, Osaka, Japan g Institut fur Physiologishe Chemie, Abteilung Angewandte Molekularbiologie der Universitat, D-55099 Mainz, Germany h Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan b

Received 24 May 2002; accepted 19 June 2002

Abstract Galectins are widely distributed sugar-binding proteins whose basic specificity for h-galactosides is conserved by evolutionarily preserved carbohydrate-recognition domains (CRDs). Although they have long been believed to be involved in diverse biological phenomena critical for multicellular organisms, in only few a cases has it been proved that their in vivo functions are actually based on specific recognition of the complex carbohydrates expressed on cell surfaces. To obtain clues to understand the physiological roles of diverse members of the galectin family, detailed analysis of their sugar-binding specificity is necessary from a comparative viewpoint. For this purpose, we recently reinforced a conventional system for frontal affinity chromatography (FAC) [J. Chromatogr., B, Biomed. Sci. Appl. 771 (2002) 67 – 87]. By using this system, we quantitatively analyzed the interactions at 20 jC between 13 galectins including 16 CRDs originating from mammals, chick, nematode, sponge, and mushroom, with 41 pyridylaminated (PA) oligosaccharides. As a result, it was confirmed that galectins require three OH groups of N-acetyllactosamine, as had previously been denoted, i.e., 4-OH and 6-OH of Gal, and 3OH of GlcNAc. As a matter of fact, no galectin could bind to glycolipid-type glycans (e.g., GM2, GA2, Gb3), complex-type N-glycans, of which both 6-OH groups are sialylated, nor Le-related antigens (e.g., Lex, Lea). On the other hand, considerable diversity was observed for individual galectins in binding specificity in terms of (1) branching of N-glycans, (2) repeating of N-acetyllactosamine units, or (3) substitutions at 2-OH or 3-OH groups of nonreducing terminal Gal. Although most galectins showed moderately enhanced affinity for branched N-glycans or repeated N-acetyllactosamines, some of them had extremely enhanced affinity for either of these multivalent glycans. Some galectins also showed particular preference for a1-2Fuc-, a1-3Gal-, a1-3GalNAc-, or a2-3NeuAc-modified glycans. To summarize, galectins have evolved their sugar-binding specificity by enhancing affinity to either ‘‘branched’’, ‘‘repeated’’, or ‘‘substituted’’ glycans, while conserving their ability to recognize basic disaccharide units, Galh1-3/4GlcNAc. On these bases, they are considered to exert specialized functions in diverse biological phenomena, which may include formation of local cell-surface microdomains (raft) by sorting glycoconjugate members for each cell type. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Galectin; Frontal affinity chromatography; Dissociation constant; Pyridylamination; Comparative glycomics

Abbreviations: CRD, carbohydrate-recognition domain; EDTA – MEPBS, 2 mM EDTA, 4 mM 2-mercaptoethanol, 20 mM Na-phosphate [pH 7.2], 150 mM NaCl; EDTA – PBS, 2 mM EDTA, 20 mM Na-phosphate [pH 7.2], 150 mM NaCl; FAC, frontal affinity chromatography; GST, glutathione S-transferase; HPLC, high-performance liquid chromatography; LN2, 3, 5, N-acetyllactosamine oligomers having repeating numbers of 2, 3, 5, respectively; LNT, lacto-Ntetraose; LNnT, lacto-N-neotetraose; LNFP-I, II, III, lacto-N-fucopentaose, I, II, and III, respectively; LNDFH, lacto-N-difucohexaose; NA2, 3, 4, biantennary, triantennary, and tetraantennary asialo type N-glycans, respectively; PA, pyridylaminated/pyridylamination * Corresponding author. Tel.: +81-426-85-3741; fax: 81-426-85-3742. E-mail address: [email protected] (J. Hirabayashi). 0304-4165/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 0 2 ) 0 0 3 11 - 2

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1. Introduction Galectins are widely distributed soluble metal-independent lectins, with sugar-binding specificity for h-galactoside via evolutionarily preserved carbohydrate-recognition domains (CRDs; for review, see Ref. [1]). Now it is evident that galectins form an extremely diverse protein family not only from a phylogenic viewpoint, but also from a structural viewpoint. As a result of recent progress of genomesequencing projects for various organisms, e.g., Caenorhabditis elegans [2], Drosophila melanogaster [3], Mus musculus [4], Homo sapiens [5], a number of genes that possibly encode galectin proteins having various structural types have been annotated in genome databases (described by Cooper [6] in this issue). As regard to the classification of galectins, Hirabayashi and Kasai [7] have categorized them into three types on the basis of their structural architecture (note that their classification system does not necessarily reflect sequence homology), i.e., ‘‘proto’’, ‘‘chimera’’, and ‘‘tandem-repeat’’ types. They are represented by galectin-1, -3, and -9, respectively. Critically, the system per se implies basic features regarding cross-linking aspects of lectins; i.e., the proto type, e.g., galectin-1, usually forms a noncovalent dimer consisting of two identical CRDs under nondenaturing conditions, and thus, should have two equivalent sugar-binding sites. On the other hand, the chimera type, solely represented by galectin-3 at the moment, has two distinct domains, i.e., an N-terminal collagen-like domain (non-CRD) and C-terminal galectin CRD, and thus, possibly cross-links nonsugar and sugar moieties. The remaining tandem-repeat type, e.g., galectin-9, has two homologous but significantly distinct CRDs on a single polypeptide. Therefore, galectins belonging to this type can have two distinct sugar-binding sites. From a theoretical viewpoint, this classification system specifies how each type of galectin exerts multivalency. To understand the in vivo functions of diverse galectins, it is essential to understand both basic features that are conserved through evolution and specialized features that individual galectins have acquired under given circumstances (e.g., species, cell types and states). For this purpose, elucidation of their detailed sugar-binding specificity and identification of their endogenous receptor glycans are necessary. Functional aspects of various galectins are described in other chapters of this special issue (i.e., ‘‘cancer and galectins’’ by Danguy et al. [8]; ‘‘immunomodulation and galectins’’ by Rabinovich et al. [9]; ‘‘intracellular functions of galectins’’ by Liu et al. [10]). For a better understanding of the molecular basis of these biological phenomena, discussion of the basis for interaction between multivalent galectins and complex carbohydrates is most important. The aspect of multivalent cross-linking between galectins and complex carbohydrates is described by Brewer and Dam [11] in this issue. In this chapter, we describe the fine carbohydrate specificity of galectins in terms of dissociation (Kd) or association (Ka) constant (‘‘not’’ I50 values often used in conventional

233

hemagglutination and solid-phase binding assays) by using galectins derived from an extensive array of organisms; i.e., mammals, chick, nematode, sponge, and mushroom, and 41 pyridylaminated (PA) oligosaccharides consisting of 12 complex-type N-glycans, glycolipid-type oligosaccharides, and three oligo-N-acetyllactosamines. For analysis of the sugarbinding specificity of lectins, various methods have been undertaken. These include hemagglutination assay [12 – 14], equilibration dialysis [15], binding assays using radiolabeled [16 – 19] or enzyme-labeled lectins [20], surface plasmon resonance using a biosensor BiaCore [21,22], and affinity capillary electrophoresis [23]. However, these methods present some difficulty with respect reliability, reproducibility, rapidity, sensitivity, economy, or various combinations of these items. More critically, kinds of available glycans are greatly limited in these methods. Recently, we have reinforced a system of frontal affinity chromatography (FAC), originally developed by Kasai et al. [24], by efficient combination of the merits of high-performance liquid chromatography (HPLC) and the properties of PA-oligosaccharides [25 – 27]. By the use of HPLC, rapid and reproducible analysis became possible with no need for special skill, while the adoption of the PA labeling method originally developed by Natsuka and Hase [28] expanded the repertoire of analyzed glycans, because more than 200 PA-oligosaccharides can be separated by the established 2-D mapping procedure developed by Tomiya and Takahashi [29]. In our latest system, the dissociation constant between a lectin and a PA-oligosaccharide can be determined in 10 min by using a 10 nM saccharide solution, when the flow rate is settled at 0.25 ml/min and a miniature column (4-mm diameter, 10-mm length) is used. PA-oligosaccharides are stably and sensitively detected by fluorescence (wavelengths for excitation and emission: 320 and 400 nm, respectively). As shown in Fig. 1, galectins described in this chapter have various origins, i.e., mammals (human galectin-1 (Gal1) [30 –33], rat galectin-2 (Gal-2) [34], human galectin-3 (Gal-3) [35], human galectin-7 (Gal-7) [36,37], human galectin-8 (Gal-8) [38] and human galectin-9 (Gal-9) [39 – 41]); bird (chicken 14-kDa (C14) [13,42] and 16-kDa galectins (C16) [43]), nematode (C. elegans 32-kDa galectin renamed LEC-1 [26,44,45] and 16-kDa galectin LEC-6 [25,46]), sponge (Geodia cydonium galectins designated in this paper GC1 and GC2 [47]), and mushroom (Agrocybe cylindracea galectin ACG [48]). Some properties of these galectins are summarized in Table 1. Among the described galectins, X-ray crystallographic studies have been accomplished for Gal-1 [49,50], Gal-2 [51], Gal-3 [52], Gal-7 [53], and C16 [54] with their counterpart ligand, i.e., either lactose or N-acetyllactosamine. Consistent with previous studies by site-directed mutagenesis [55,56], direct evidence was obtained that the eight most strongly conserved amino acid residues, i.e., His44, Asn46, Arg48, Val59, Asn61, Trp68, Glu71, and Arg73 (residue numbers are those of human galectin-1) form sugar-binding sites either by hydrogen bonds (His44, Asn46, Arg48, Asn61, Glu71 and Arg73) or van der Waals

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Fig. 1. Phylogenic distribution of the galectins described in this review. Approximate phylogenic positions are shown with their schematically represented structures. Galectin-1, -2, -3, -7, -8, -9 are from vertebrates belonging to deuterostomes, whereas galectins designated LEC-1 and -6 are from the nematode C. elegans, belonging to the protostomes. The two sponge isolectins, GC1 and GC2, are from G. cydonium belonging to the oldest metazoans, the Porifera. The prototype galectin, designated ACG, comes from the mushroon A. cylindrocea. For details, see text and Table 1.

contact (Val69 and Trp68). This sequence motif has proved to be a ‘‘signature sequence’’ to search for functional galectin genes in the C. elegans genome database (Hirabayashi et al., unpublished result). The above X-ray crystallographic studies also confirmed the previous observation based on binding assays [16 –20] that three hydroxy groups of N-acetyllactosamine (or lactose) are essential for recognition, i.e., the 4-OH and 6-OH of Gal and the 3-OH of GlcNAc (or Glc). They are required to form hydrogen bonds with side chains of the above six hydrophilic residues (i.e., His44, Asn46, Arg48, Asn61, Glu71, and Arg73).

Though galectins so far investigated have high affinity for N-acetyllactosamine (i.e., Galh1-4GlcNAc; called type-2 saccharide), they also show significant affinity for its linkage isomer, lacto-N-biose (i.e., Galh1-3GlcNAc, type-1 saccharide). Some galectins, e.g., Gal-3 and sponge galectins, have been shown to have strong affinity for a modified lactose structure or a saccharide other than lactose, A-tetrasaccharide (GalNAca1-3(Fuca1-2)Galh1-4Glc) [16,17,57] and Forssman disaccharide (GalNAca1-3GalNAc) [57], respectively; though other galectins, e.g., galectin-1, have no such particular feature. More exceptionally, galectin-10 has

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Table 1 Features of galectins used in this work Trivial name

Abbreviation

Origin (major tissues)

Structural type (subunit MW)

Conserved motif a

Reference

Galectin-1 Galectin-2 Galectin-3 Galectin-7 Galectin-8

Gal-1 Gal-2 Gal-3 Gal-7 Gal-8

human (placenta, lung, spleen, thymus) rat (stomach, intestine) human (breast cancer, macrophage) human (skin) human (liver, lung, kidney, brain, muscle)

proto (14.5 kDa) proto (14.5 kDa) chimera (29 kDa) proto (15 kDa) tandem repeat (34 kDa)

[30 – 33] [34] [35] [36,37] [38]

Galectin-9

Gal-9

human (kidney, thymus, lymphoma)

tandem repeat (35 kDa)

Chick 14 K Chick 16 K Nematode 32 K

C14 C16 LEC-1

chick (embryo skin, adult intenstine) chick (embryo muscle, adult liver) Caenorhabditis elegans (cuticle, pharynx)

proto (15 kDa) proto (15 kDa) tandem repeat (32 kDa)

Nematode 16 K Sponge clone 1 Sponge clone 2 Mushroom

LEC-6 GC1 GC2 AGG

Caenorhabditis elegans (testis, pharynx) Geodia cydonium Geodia cydonium Agrocybe cylindracea (fruiting body)

proto proto proto proto

H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R H.N.R...V.N...W..E.I H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R H.N.R. . .V.N. . .W..E.R H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R H.S.R...V.N...W..E.R H.N.R...I.N...W..E.R H.N.R...V.N...W..E.R H.N.R...V.N...W..E.H H.N.R...V.N...W..E.R H.N.R...V.N...W..E.R

(16 (15 (15 (17

N-CRD C-CRD N-CRD C-CRD

N-CRD C-CRD

kDa) kDa) kDa) kDa)

[39 – 41] [13,42] [43] [44,45] [46] [47] [47] [48]

a Critical amino acids which have been demonstrated to be involved in sugar binding function of galectin-1 [49,50], galectin-2 [51], galectin-3 [52] and galectin-7 [53].

recently been shown to bind to mannose [58]. In general, however, galectins are supposed to have enhanced affinity for poly-N-acetyllactosamine. This idea is based on several lines of evidence: (1), chicken 14-kDa galectin has endogenous insoluble glycoprotein receptor(s), the glycan moiety of which was sensitive to endo-h-galactosidase treatment [59]; (2) galectin-1 and -3 have previously been identified as nonintegrin laminin-binding proteins [60,61]; and (3) they actually have high affinity for poly-N-acetyllactosamine [62,63]. On the other hand, it is also possible that all types of galectins have high affinity for branched complex-type Nglycans to form a more rigid complex. However, these points have never been experimentally confirmed because of the lack of an appropriate assay system to assess precisely the binding affinity of extensive galectins for a series of naturally occurring complex oligosaccharides (‘‘not’’ simple saccharides). The reinforced FAC described above meets these requisites, and comprehensive analysis was performed to determine dissociation constants (Kd’s) between the 13 galectins and 41 PA-oligosaccharides. Parts of the data have already been published [25,26,64]. In this review, on the basis of thus obtained quantitative data, we will discuss, for the first time, the matter of how galectins have evolved their oligosaccharide specificity in relation to functional significance.

2. Technical backgrounds 2.1. PA-oligosaccharides Oligosaccharides, Gala1-3Galh1-4Glc (21 in Fig. 2, aGalLac), Gala1-3(Fuca1-2)Galh1-4Glc (37, B-tetrasaccharide), and Gala1-3(Fuca1-2)Galh1-4(Fuca1-3)Glc (38, B-pentasaccharide), were purified from the milk of the Japanese black bear, Ursus thibetanus japoncus, as described previously [65]. N-acetyllactosamine oligomers, Galh1-

4GlcNAch1-3Galh1-4GlcNAc (39, LN2), Galh1-4GlcNAc h1-3Galh1-4GlcNAch1-3Galh1-4GlcNAc (40, LN3), and Galh1-4GlcNAch1-3Galh1-4GlcNAch1-3Galh1-4GlcNAc h1-3Galh1-4GlcNAch1-3Galh1-4GlcNAc (41, LN5) were generous gifts from Dr. Kei-ichi Yoshida of Seikagaku Kogyo, Co. Ltd. (Tokyo Japan). These oligosaccharides were pyridylaminated by a standard procedure previously described [28]. Other PA-oligosaccharides (1 –36) and PA –Rha were purchased from Takara Breweries (Kyoto, Japan). 2.2. Preparation of galectins All of the galectin proteins used in this study were prepared as a form of recombinant protein except for the mushroom galectin, ACG. The recombinant proteins were produced in Escherichia coli BL21 (DE3) under the control of the T7 promoter as either intact forms or fusion forms. In the case of galectin-1, it was produced as a stable mutant, C2S, in which Ser was a substitute for the oxidationsensitive Cys2 [56]. Expression plasmids for intact proteins, i.e., Gal-1 (C2S), Gal-2, Gal-3, C14, C16, GC1, GC2, and LEC-32, were constructed by using pET expression systems (Novagen) essentially as described previously for LEC-32 [26]: briefly, (1) entire coding regions were amplified by the polymerase chain reaction using forward primers containing an NdeI site and reverse primers containing a termination codon, and original cDNA plasmids as templates. (2) The amplified fragments were cloned into pCRII vector (Invitrogen) by a TA cloning strategy according to the manufacturer’s instruction. (3) The inserts were cut out with relevant restriction enzymes, and were ligated into an appropriate pET vector. (4) E. coli BL21 (DE3) was transformed with the constructed expression plasmids, the nucleotide sequences of which were confirmed. (5) Production of galectin proteins was induced by the addition of 1 mM isopropyl-h-D-thiogalactoside. Optimal conditions for

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Fig. 2. Schematic representation of PA-oligosaccharides used in FAC analysis. Differently expressed hexagons, , and and NeuAc, respectively (anomeric carbon, i.e., position 1, is placed at the right side, and 2, 3, 4. . . are placed clockwise.). Thick (

represent pyranose rings of Glc, Man, Gal, GlcNAc, GalNAc, L-Fuc, ) and thin (——) bars represent a and h bonds, respectively.

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production of galectins, i.e., temperature (25 or 37 jC) and induction time (2– 16 h), were established for each case. (6) Soluble fractions were obtained for subsequent purification by affinity chromatography on an asialofetuin – agarose column (bed volume, 10 ml). The column was equilibrated with EDTA –MEPBS (2 mM EDTA, 4 mM 2-mercaptoethanol, 20 mM Na-phosphate, pH 7.2, 150 mM NaCl), and adsorbed galectins were eluted with 20 mM lactose dissolved in the same buffer. On the other hand, Gal-7, Gal-8, and Gal-9 were expressed as a fusion protein by using a glutathione S-transferase (GST) fusion vector, pGEX-4T-2 (Amersham Pharmacia Biotech). The resultant fusion proteins were purified by glutathione affinity chromatography followed by lactose affinity chromatography as described previously [66]. Production and purification of C. elegans galectin LEC-6 as a fusion with a-peptide of E. coli hgalactosidase was described previously [25,46]. Mushroom galectin, ACG, was purified from the fruiting bodies of A. cylindracea according to a previous procedure combining conventional chromatographies [48]. 2.3. Preparation of galectin columns Galectins were immobilized on Hi-Trap NHS-activated cartridge columns (Amersham Pharmacia Biotech. Co. Ltd.) as described previously for LEC-6 [25], LEC-1 [26], and Gal-9 [64]: in typical experiments, purified galectins were dissolved in 1 ml of coupling buffer (0.1 M NaHCO3, pH 8.3, 0.2 M NaCl, 0.1 M lactose), and was immobilized on Hi-Trap NHS-activated matrix (1 ml). To avoid exclusive immobilization on the uppermost layer of the column, the column was chilled on ice, and then galectin solution (also previously chilled on ice) was gradually injected over a period of at least 1 min. The reaction was allowed to proceed for 15 min on ice, and subsequently for another 15 min at room temperature (22 –23 jC). The column resin was blocked with an excess amount of ethanolamine for 1 h at room temperature, and was extensively washed with 0.5 M NaCl and then with EDTA – PBS (1 mM EDTA, 20 mM Na phosphate, pH 7.2, 150 mM NaCl). The resultant resin was taken after disruption of the cartridge column, and was packed into a stainless miniature column (i.d. 4  10 mm). 2.4. FAC Reinforced FAC was performed as described previously [26]. A scheme for total procedures is outlined in Fig. 3. Briefly, the prepared galectin columns were connected to a conventional HPLC system consisting of a Shimadzu LC10ADVP pump, a Shimadzu RF10AXL fluorescence detector, and a data processing system composed of a control interface module (Varian Chromatography Systems, USA) and a personal computer (Power Macintosh 7300/180). PAoligosaccharides (10 nM, 2 ml) were manually injected through a Rheodyne 7725 injector equipped with a 2-ml sample loop. The flow rate and the column temperature

Fig. 3. A schematic of the total procedures for the present comprehensive FAC analysis. The scheme consists of (1) preparation of galectin-immobilized affinity column, (2) evaluation of the prepared column by determination of effective ligand content, Bt, and availability of the immobilized ligands (galectins). (3) FAC analysis to determine Vf
V0 for various PA – oligosaccharides; and (4) calculation of dissociation constant Kd between each lectin and PA – oligosaccharide according to Eq. (1). Since [A]0 (kept at 10 nM in this work) is in most cases much smaller than Kd, Eq. (1) can be simplified to Eq. (2).

were kept at 0.25 ml/min and 20 jC, respectively. The volume of the elution front (Vf) of each saccharide was determined semi-automatically as described previously [67]. Retardation of the elution front compared with that of PA – Rha, i.e., Vf
V0, was used to obtain Kd according to a basic equation of FAC [24], Eq. (1), where Bt is the effective ligand content (expressed in mol), and [A]0 is the initial concentration of PA-oligosaccharide. Bt is calculated by concentration-dependence analysis using appropriate saccharides (for details, see Table 2), followed by preparation of either a Lineweaver – Burk type plot, i.e., 1/[A]0 vs. 1/ (Vf
V0)[A]0, or a Woolf –Hofstee type plot, i.e., (Vf
V0) vs. (Vf
V0) [A]0. Elution of various concentrations of paminophenyl-h-lactoside was monitored by absorbance at

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Table 2 Specifications of galectin-immobilized columns used in this study Galectin immobilized

Expressed form

Immobilized (mg/ml gel)

Bt (nmol)

Availability (%)a

r2b

Sugars used for concentration analysis

Gal-1 (C2S) Gal-2 Gal-3 Gal-3C Gal-7 Gal-8 Gal-8N (R233H) Gal-8C (R 69H) Gal-9 Gal-9N Gal-9C C14 C16 LEC-1 LEC-1N LEC-1C LEC-6 GC-1 GC-2 ACG

intact (oxidation resistant) intact intact CRD only fusion with GST fusion with GST functionally N-CRD only functionally C-CRD only fusion with GST N-CRD only C-CRD only intact intact intact N-CRD only C-CRD only fusion with a-peptide intact intact natural productc

0.92 5.3 0.55 0.69 1.5 2.0 2.0 2.0 1.4 1.0 1.0 2.9 0.99 5.9 6.6 1.9 7.4 1.2 2.2 0.7

3.3 4.0 0.19 1.6 5.1 2.2 1.8 1.4 0.33 0.31 1.2 12 2.0 11 18 7.1 30 3.0 5.4 2.3

41 9 8 28 100 55 54 33 11 11 42 50 24 47 35 47 68 30 39 39

0.96 0.83 0.95 1.00 0.99 1.00 0.99 0.95 0.93 0.95 0.99 0.96 0.83 0.98 0.99 1.00 1.00 1.00 1.00 0.87

PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I PA-NA3 + NA3 PA-LNFP-I + LNFP-I p-aminophenyl-h-lactoside PA-LNFP-I + LNFP-I PA-LNFP-I PA-LNFP-I PA-LNFP-I p-aminophenyl-h-lactoside PA-LNFP-I + LNFP-I PA-LNFP-I + LNFP-I p-aminophenyul-h-lactoside

a b c

Reference

[64] [64]

[26] [26] [26] [25,27]

Availability is calculated by the following fomurm: Bt (mol)/immobilized galectin (g)/molecular weight (g/mol)  100%. Reliability of lines obtained as a result of Woolf – Hofstee-type plot in each concentration analysis. A. cylindracea galetin purified from fruiting bodies of the mushroom [48] was used for immobilization.

280 nm, while that of PA-oligosaccharides was monitored by fluorescence (excitation and emission wavelengths, 320 and 400 nm, respectively) in the presence of various concentrations of unlabeled saccharides. Eq. (1) can be simplified to Eq. (2), where [A]0 (10
8 M) is negligibly small compared with Kd (e.g., > 10
6 M). Kd ¼ Bt =ðVf
V0 Þ
½A0

ð1Þ

Kd ¼ Bt =ðVf
V0 Þ;

ð2Þ

if Kd H½A0

It is often favorable to discuss the matter of lectin – oligosaccharide interactions in terms of affinity constant (Ka) instead of Kd. The two equilibrium constants are in the following relationship: Ka ¼ 1=Kd

ð3Þ

2.5. Evaluation of columns Specifications of the columns used in this study are listed in Table 2. As a result of concentration-dependence analysis using an appropriate oligosaccharide and subsequent Woolf –Hofstee type plot, the effective ligand content, Bt (0.19 –30 nmol), was determined for each column. Linearity of the regression lines was obtained in the range of 0.78 – 1.00 (average, 0.94), whereas availabilities for the most columns exceeded 30% (average, 39%). To those evaluated galectin-columns, 41 PA-oligosaccharides (for structures, see Fig. 2) were applied, and Kd’s were obtained by using the observed Vf
V0 values according to Eq. (1) or Eq. (2).

3. Conserved features for all galectins Obtained Kd’s for all combinations between 13 galectins, (Gal-1, 2, 3, 7, 8, 9, C14, 16, LEC-1, 6, GC1, 2 and ACG) plus 7 individual CRDs (Gal-3C, Gal-8N, Gal-8C, Gal-9N, Gal-9C, LEC-1N, and LEC-1C) and 41 PA-oligosaccharides are summarized in Table 3 (data obtained at 20 C unless otherwise mentioned). As conserved features, the following three points can be made: (1) All galectins and all of their CRDs recognized to a certain degree the basic structure Galh1-4GlcNAc, such as found in lacto-N-neotetraose (26, LNnT). However, affinity for it differed considerably among the galectins investigated. The highest affinity was observed for Gal-3 (Kd = 1.7 AM), whereas the lowest was observed for a sponge galectin, GC2 (1400 AM). A linkage isomer, Galh1-3GlcNAc, contained in lacto-N-tetraose (27, LNT) was also commonly recognized. Again, the highest affinity was observed with Gal-3 (Kd = 2.6 AM) and the lowest, with GC2 (130 AM). These two disaccharide units, i.e., type 1 (Galh1-3GlcNAc) and type 2 (Galh1-4GlcNAc), are known to take almost equivalent conformation as regard to configurations of the three OH groups essential for galectin recognition [16,17]. Affinity for lactose (13) is considerably reduced, probably because the pyranose ring of the reducing terminal glucose is opened upon pyridylamination. (2) Substitution at 4-OH and 6-OH groups of Gal of the above three h-galactosides (i.e., LNT, LNnT, and lactose) was by no means acceptable. In other words, no galectins could bind to N2 (12), GA2 (14), 6V-sialyllactose (17), GM2 (18), or Gb3 (22). From a general viewpoint, galectins never bind to nonreducing terminal mannosides or glucosides with few

Table 3 Kd’s determined by frontal affinity chromatography for PA-oligosaccharuides and galectinsa No.

Trivial Name

NA2 NA3 NA3 NA4 NA3 NA4 NA2 NA3 NA4 NA2 NA2 NA2

13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37

GA3 (lactose) GA2 GA1 GM3 6V-SiaLac GM2 GM1 GD1a GD1b Gb3 Gb4 Forsmann pentasaccharide Galili pentasaccharide LNnT LNT LNFP-I LNFP-II LNFP-III LNDFH A-hexasaccharide A-heptasaccharide 2V-FucLac a-GalLac B-tetrasaccharide B-pentasaccharide

38

A-tetrasaccharide

39 40 41

LN2 LN3 LN5 a

(type 1) (Lex) (Lex) (1-6Fuc) (1-6Fuc) (1-6Fuc) (monosialo) (monosialo) (disialo)

Gal-2

Gal-3

Gal-7

Whole

Gal-3C

38 19 16 18 52 27 42 23 16 160 160 –

1.4 0.78 0.92 0.69 1.5 1.1 1.5 0.82 0.71 4.0 5.6 –

3.2 1.8 2.1 1.7 3.6 2.8 4.0 1.9 1.7 13 14 –

– – – – – – – – – – – – 33 49 62 58 – – – 240 – – 290 – –

1100 – 180 – – – 240 – 260 – 290 290 56 130 68 23 1100 – – 33 – 350 230 81 –

26 – 30 26 – – 20 23 26 16 12 6.2 0.68 1.7 2.6 1.5 4.3 2.4 3.5 0.63 4.4 36 9.4 1.6 –

260 – 180 230 – – 130 83 160 – 61 27 2.5 7.9 9.9 5.1 20 9.1 12 1.8 17 93 29 5.9 –

460 – 1200 – – – 580 770 770 – 1200 – 82 130 48 49 220 510 – 270 660 920 160 200 –

–

150

3.8

14

1500

50 45 39

140 90 85

1.3 0.35 0.19

7.6 4.5 4.7 4.8 8.5 7.8 8.3 4.5 4.7 48 66 –

4.8 0.87 0.43

45 9.2 8.5 7.4 44 28 45 9.7 < 8.4 420 180 –

Gal-8

Gal-9

C14

Whole

Gal-8N

Gal-8C

Whole

Gal-9N

Gal-9C

85 23 14 14 120 51 81 27 18 – – –

– 290 47 180 – – – 440 290 – – –

120 52 26 34 150 76 91 59 38 – – –

0.70 0.16 0.22 0.16 0.95 0.26 0.87 0.29 0.17 55 170 –

3.1 0.38 0.59 0.18 9.3 0.47 3.5 0.45 0.23 26 44 –

1.2 0.43 0.39 0.35 2.0 0.81 1.6 0.50 0.44 31 44 –

150 – 52 1.5 – – 39 0.64 47 – 120 18 8.8 12 43 26 33 3.3 120 3.3 96 120 76 9.6 –

130 – 46 0.62 – – 30 0.50 35 – 130 – 5.9 7.6 39 32 29 1.6 580 – 440 88 56 5.9 –

– – 97 – – – 97 230 150 – 330 8.8 28 52 68 27 54 100 91 1.6 76 – 230 31 –

– – 21 – – – 17 24 33 – 37 0.41 7.0 10 8.7 7.2 16 41 10 1.2 10 – 13 26 –

103 – 5.4 309 – – 3.8 4.2 7.4 – 9.7 0.09 4.4 4.1 4.8 3.8 3.9 9.4 2.6 0.26 3.2 – – – –

12 120 44 – – – 39 84 79 200 59 59 6.7 15 12 9.0 34 38 27 7.7 26 150 – – –

–

25

6.4

47

3.0 0.81 0.12

17 2.7 0.41

49

47 60 31

3.5 9.6 1.6

1.7 12 1.8

23 9.0 2.1

22 8.3 1.6 0.09

C16

LEC-1 Whole

9.1 5.2 4.4 5.4 9.1 10 10 7.0 8.5 140 200 –

LEC-1N

LEC-6

GC1

GC2

ACG

LEC-1C

4.9 3.0 3.1 3.2 5.4 6.0 5.7 3.4 3.6 21 32 –

220 100 80 110 230 230 230 120 110 960 – –

680 370 260 380 920 780 580 370 370 – – –

150 84 52 77 210 150 200 82 78 950 1300 –

93 40 36 27 100 54 82 46 38 590 710 –

68 20 5.5 10 70 26 140 21 11 – – –

190 96 45 70 270 140 190 100 69 1400 – –

< 3.0 < 3.0 < 3.0 < 3.0 3.7 < 3.0 2.7 < 3.0 < 3.0 1.8 3.0 –

750 – – 920 – 2400 – – 2000 1700 1700 750 89 110 69 140 – – – 920 – 2000 540 – –

57 – – 150 – – – – – – 330 400 9.3 17 17 29 – – – 57 – – 20 – –

– – 680 – – – 880 – – – 530 – 54 210 91 60 300 780 240 58 250 – 290 190 –

– – – – – – – – – – 2900 – 72 1000 440 130 – – – 48 1800 – 420 350 –

– – 670 – – – 1100 – – – 500 – 100 200 77 65 240 890 190 810 170 – 97 270 –

2000 – 580 3400 – – 630 – 1600 – 1300 3400 160 320 190 170 – – – 1600 – 3800 850 1500 –

750 – 42 – – – 38 750 48 – 83 < 0.1 8.3 600 64 20 – – – < 0.1 54 200 11 0.2 –

– – 58 – – – 48 1100 58 – 140 0.13b 11 1400 130 25 – – – 0.10b – 600 25 1.6 –

400 – 15 23 – – 9.4 < 3.0 15 – – 46 1.9 26 70 190 1200 – – 7.5 – 770 NDc NDc

2000

250

–

–

–

–

< 0.1

0.23b

110 81 41

19 19 19

120 68 31

940 590 350

97 51 18

370 230 140

120 25 8.3

550 430 320

J. Hirabayashi et al. / Biochimica et Biophysica Acta 1572 (2002) 232–254

1 2 3 4 5 6 7 8 9 10 11 12

Gal-1

18 NDc NDc NDc

Data obtained at 20 jC otherwise mentioned. Dissociation constants (Kd’s) are given in AM.

b

Data obtained at 37 jC.

c

ND, not determined.

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exceptions (described later). In this context, the requirement for the intact 4-axial OH group of galactose is quite reasonable. On the other hand, the critical requirement of the 6-OH group of Gal should be meaningful because its a2-6 sialylation completely abolished the binding of all galectins examined, whereas a2-3 sialylation had no such catastrophic effect (described in detail later). (3) Substitution at 3(4)-OH group of penultimate Glc(NAc) of Galh14(3)GlcNAc also abolished the binding of most galectins. In this case, however, the detrimental effect was not perfect, because a1-3 fucosylation of the penultimate GlcNAc of LNnT (26), resulting in LNFP-III (30), reduced the affinity of some galectins only partially: e.g., Gal-3 (Kd’s for 26 and 30, 1.7 and 2.4 AM, respectively), Gal-7 (130, 510 AM), Gal-9 (10, 41 AM). Rather, a1-3 fucosylation of LNnT significantly increased the affinity of Gal-8 (i.e., 12, 3.3 AM). However, these incomplete effects can be attributed to enhanced residual affinity for lactose at the reducing terminal, since all of these exceptional galectins showed detectable affinity for lactose (13). In fact, no galectin showed significant affinity for B-pentasaccharide (37), Fuca1-2(Gala1-3)Galh1-4(Fuca1-3)Glc, even though the above galectins could recognize B-tetrasaccharide (36), Fuca1-2(Gala1-3)Galh1-4Glc. Therefore, the requirement of the third OH group (3-OH of GlcNAc of type-2 saccharides, or 4-OH of GlcNAc of type-1 saccharides) is consistent. These observations let us conclude that no galectins can recognize Le-type structures, such as Lex (30), Lea (29), and Leb (31). However, this never means that the presence of the 3(4)-equatorial OH group of the penultimate Glc(NAc) is essential for galectin recognition, because a significant number of galectins could bind to disaccharide units other than Galh1-3(4)GlcNAc, such as Galh1-3GalNAc found in GA1 (15) and GM1 (19), the penultimate GalNAc of which has an axial 4-OH group instead of the equatorial one of GlcNAc. Probably, these galectins can recognize both types of disaccharides. To summarize, all of the investigated galectins derived from diverse origins proved to preserve significant affinity for lactosamine-type disaccharides, Galh1-3(4)Glc(NAc), via recognition of 4OH and 6-OH of galactose and 3(4)-OH of Glc(NAc), as has previously been indicated for several galectins [16 – 20]. This observation implies that the galectins have diverged from a common ancestor sharing essentially the same structural and functional properties with their descendants; and, hence, we can discuss the matter of diverged features that specify individual galectins from a comparative viewpoint.

4. Specialized features of individual galectins To grasp the specialized features of galectins, we plotted affinity constants (Ka’s) for a set of 41 glycans as a vertical bar graph (Fig. 4). Glycans which showed distinguished binding are depicted with trivial names in the figure. 4.1. Galectin-1 (Gal-1) Gal-1 was the first recognized member of the mammalian galectin family [30,31,33], and shows the widest distribution in mammalian tissues, where it is relatively abundant. Previous studies showed that Gal-1 forms a noncovalent dimer under nondenaturing conditions, and thus, exerts significant hemagglutination activity [14]. On the other hand, this galectin is also known to be highly susceptible to oxidative inactivation possibly through oxidation of a few reactive cysteine residues [68,69]. To circumvent such a situation, we employed an oxidation-resistant mutant, previously designated C2S [56]. As a result of FAC analysis, Gal-1 showed extensive affinity for complex-type N-glycans, as has previously been shown [70]. The affinity increased with an increase in the branching number up to triantennary N-glycans, i.e., NA2 (1, Kd = 7.6 AM), NA3 (2, 4.5 AM); whereas affinity for functionally monovalent N-glycans, i.e., NA2, of which either of the two nonreducing terminal Gal is a2-6 sialylated, was low (10 and 11, Kd’s = 48 and 66 AM, respectively). Significant increase in affinity to such multivalent glycans is understood under the concept of the ‘‘clustering effect’’ [70 – 73] (also mentioned in this issue by Kilpatrick [74], Weigel and Yiki [75], and East and Isacke [76]). The idea was first demonstrated for C-type lectins [71], another representative family of animal lectins. However, Gal-1 did not show yet higher affinity to the tetraantennary glycan NA4 (4, 4.8 AM). a2-6 Sialylation completely abolished the affinity, since Gal-1 had no affinity to N2 (12), in which both galactose residues are a2-6 sialylated. Gal-1 showed significant but somewhat lowered affinity for a type-1 saccharide, LNT (27, 62 AM) than for a type-2 saccharide, LNnT (26, 49 AM). Other than these basic saccharides, Gal-1 bound to some modified lacto/ neolacto-series glycans, such as Galili pentasaccharide (25, 33 AM), LNFP-I (28, 58 AM), A-hexasaccharide (32, 240 AM), and a-GalLac (35, 290 AM). However, the effects of these modifications were modest or rather detrimental, when compared with some other galectins (described below). As far as examined by the present system, Gal-1 did not show significant affinity for ganglioside and globoside-type oligosaccharides, though GM1 was once suggested to be a major

Fig. 4. Bar graph representation of affinity constants (Ka’s) between 13 galectins and 41 PA – oligosaccharides. For features of galectins, specifications of galectin columns used, and structures of PA-oligosaccharides, see Tables 1 and 2, and Fig. 3, respectively. Note that ordinate scales are variable, reflecting overall binding strengths of galectins. Several saccharides showing particular affinity for each galectin are denoted by their trivial names. Asterisks (*) mark saccharides that do not interact with galectins, because they have a detrimental substitution at 1 of the 3 critical OH groups required for the recognition (see text, for details). Very strong binding affinities are not precisely drawn because their values are out of the range of each graph. To determine accurate Ka values, use Eq. (3). Only preliminary data are shown for the mushroom lectin ACG. Strong affinities of some glycans for ACG are shown with upper arrows (e.g., Ka for GD1a>3 AM). For structures of numbered sugars on the abscissa, see Fig. 2.

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endogenous ligand for Gal-1 [77]. Moreover, Gal-1 did not show any particular preference for repeated N-acetyllactosamine structures, i.e., LN2 (39, 50 AM), LN3 (40, 45 AM), and LN5 (41, 39 AM), despite the fact that this galectin was previously identified as a binding protein of laminin [60], which is known to have poly-N-acetyllactosamine chains. From a general viewpoint, Gal-1, as well as some other prototype galectins (e.g., Gal-7, C14, and LEC-6; described below), shows rather simplified profiles as regard to sugarbinding specificity. Such a feature should be reflected on functional divergence from other members of the galectin family, e.g., Gal-3 [78]. 4.2. Galectin-2 (Gal-2) Gal-2 used in this work was identified as a rat homologue [34] to human Gal-2, which was first investigated in 1986 [79] and further characterized thereafter [80]. This galectin is exclusively localized in digestive tissues, such as gastrointestinal epithelial cells. Gal-2 shows the highest homology to Gal-1 (45% in amino acid identity) among the members of the galectin family. However, as far as examined, rat galectin-2 shows considerably lower hemagglutinating activity toward trypsinized rabbit erythrocytes than Gal-1. Consistently, Gal-2 appears to exist as a monomer in our gel-filtration analysis (Hirabayashi et al., unpublished result). Much about the functions and biological significance remains to be elucidated for Gal-2. FAC analysis revealed that Gal-2 exerts only 20– 50% affinity relative to that of Gal-1 for a whole set of glycans examined. The highest affinity was observed for NA3, which contains a single type-1 chain (3, 16 AM), and a16 fucosylated NA4 (9, 16 AM). To these oligosaccharides, Gal-1 bound more tightly (Kd = 4.7 AM). However, several points unique to Gal-2 can be made: (1) Gal-2 bound more preferentially to a type-1 saccharide (27, 68 AM) than to a type-2 one (26, 130 AM). As a result, the affinity of binding to the latter saccharide, LNT, reached a level almost identical to that of Gal-1 (62 AM). (2) Though relatively weak, Gal-2 can recognize extensive saccharides other than lactosamine-type glycans, e.g., GA1 (15, 180 AM), GM1 (19, 240 AM), Gb4 (23, 290 AM), and Forssman pentasaccharide (24, 290 AM). (3) a1-2 Fucosylation of the nonreducing terminal Gal of LNT considerably enhanced its affinity for Gal-2, whereas this substitution had only a little effect on that for Gal-1. As a result, Gal-2 showed much higher affinity for LNFP-I (28, 23 AM) and also for A-hexasaccharide (32, 33 AM) than did Gal-1 (58 and 240 AM, respectively). On the other hand, Gal-2 showed moderately enhanced affinity for oligolactosamines, i.e., LN2 (39, 140 AM), LN3 (40, 90 AM), and LN5 (41, 85 AM). 4.3. Galectin-3 (Gal-3) Gal-3 is the sole member of the chimera type. However, owing to its multi-talented features, this galectin has been

studied so far by investigators with various research backgrounds, for it was shown to be a metastasis-associated cancer antigen [81], an activated macrophage antigen, Mac2 [82], a component of ribonucleoprotein complex [83] (also described in this issue by Liu et al. [10]), a nonintegrin type laminin-binding protein [61] and an IgE-binding protein [84]. With no doubt, such unique features of Gal-3 are partly attributable to the presence of its N-terminal non-CRD region, which consists of repeating segments rich in Pro and Gly. The N-terminal non-CRD region is susceptible to collagenase treatment, whereas the C-terminal CRD is resistant to it. Previous research suggested that the Nterminal non-CRD contributes to higher affinity for glycoconjugates by positive cooperativity [85]. Another notable feature of this lectin is its particular preference for blood group A saccharides [16,17,86 –88], though affinity for Bsaccharides has not been systematically compared with other galectins. To confirm these points, we also compared the affinity of a deletion product designated Gal-3C, in which the N-terminal non-CRD was removed by Clostridium histolyticum collagenase digestion as described previously [89] and the resultant CRD moiety was recovered by asialofetuin – agarose chromatography. Sequencer analysis indicated that thus derived Gal-3C was a mixture of two polypeptides beginning with Ser96 (approximately 20%) and Gly108 (80%). FAC analysis revealed that both intact Gal-3 and Gal-3C showed the highest affinity for a panel of oligosaccharides investigated in this study (Fig. 3; note difference in the scales of ordinates with the others). However, intact Gal-3 showed 2.3– 4.4 times (on the average, 3.8 times) higher affinity than Gal-3C. Notably, the increase in affinity over Gal-3C was more evident with respect to glycolipid-type glycans (13 – 38; on the average, 4.7 times) than to Nglycans (1 –11; on the average, 2.5 times). This difference suggests that the N-terminal non-CRD contributes to enhanced affinity of Gal-3 for extended structures of basic recognition units, such as lactose or N-acetyllactosamine. In fact, Gal-3 showed distinguishably high affinity (i.e., Kd < 1 AM) for Galili pentasaccharide (25, 0.68 AM) and Ahexasaccharide (32, 0.63 AM). As described, Gal-3 has long been known to show the highest affinity among all galectins for blood group A-saccharides, e.g., A-hexasaccharide (32) and A-tetrasaccharide (38). However, the present analysis revealed still higher affinity for a B-saccharide: compare Kd’s for A-tetrasaccharide (38, 3.8 AM) and B-tetrasaccharide (36, 1.6 AM). Therefore, if B-hexasaccharide were available, it would show in theory a Kd as low as 0.27 AM (i.e., 0.63  1.6/3.8). It is also noteworthy that Gal-3 could bind even to the Le-containing saccharides with relatively high affinity, i.e., to LNFP-II (29, 4.3 AM), LNFP-III (30, 2.4 AM), and LNDFP (31, 3.5 AM). As discussed above, this can be attributed to somewhat enhanced affinity for lactose moiety in the reducing terminal, since Gal-3 also showed significant affinity toward lactose (13, 26 AM). In contrast to Gal-1 and Gal-2, Gal-3

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showed a drastic response to an increase in repeating lactosamine units; i.e., LN2 (39, 1.3 AM), LN3 (40, 0.35 AM), and LN5 (41, 0.19 AM). This may explain how macrophage Gal-3 could bind to h1-2-linked oligomannosides derived from Candida albicans [90]. Such an inherent nature of Gal-3, but not of Gal-1, should be of biological relevance to the recent report by Demetriou et al. [91]. They proposed possible involvement of Gal-3 in negative regulation of T cell activation probably via clustering poly-N-acetyllactosamine chains expressed on T cell receptor glycoproteins, which are generated by the action of N-acetylglucosaminyltransferase V (Mgat5). So far, an X-ray crystallographic study has been successful only on the C-terminal CRD (Gal-3C) [53], whereas no study of intact Gal-3 or Gal-3 complexed with a highaffinity glycoligand, such as A-tetrasaccharide, has not been made. 4.4. Galectin-7 (Gal-7) Gal-7 is a newly recognized member of the galectin family. It was identified as a stratified epithelium-specific protein by differentiation expression screening [36,37], and hereafter its sugar-binding activity was confirmed. As regard to physiological function of Gal-7, it has been implicated to be associated with p53-induced apoptosis of keratinocytes [92,93]. X-ray crystallographic study on Gal-7 and its complexed forms with galactose, lactose, and Nacetyllactosamine revealed that the protein exists as a noncanonical dimer [53], possibly as a result of the increased protein concentration required for crystallization. Consistent with the above speculation, Gal-7 showed relatively low affinity for all kinds of saccharides, even toward lactosamine-containing saccharides, e.g., NA2 (1, 45 AM) and LNnT (26, 130 AM). These Kd values are almost identical to those of Gal-2 (1, 38 AM; 26, 130 AM), which is also characterized as a monomeric protein. On the other hand, the effect of branching number on affinity is more evident in the case of Gal-7: affinity for a triantennary saccharide, NA3 (2, 9.2 AM), became about 5 times higher than that for NA2 (45 AM), and 16 times higher than that for functionally monovalent NA2 (10 and 11, on the average, 300 AM). However, the effect of the fourth branch in NA4 (4) was only partial (7.4 AM), as with most other galectins. Another distinct feature of Gal-7 is that it showed a particular preference for a type-1 saccharide, LNT (27, 48 AM), than for a type-2 saccharide, LNnT (26, 130 AM). Like Gal-1, Gal-7 showed only poor affinity toward other types of saccharides, whereas the effect of repeating lactosamine units was moderate. 4.5. Galectin-8 (Gal-8) Gal-8 is also a newcomer, having been found serendipitously in the course of expression cloning of an insulin receptor substrate probably by the use of glycosylated

243

antibody as a probe [38]. By subsequent studies, Gal-8 was found to show a much broader distribution in mammalian tissues (e.g., liver, heart, muscle, kidney, brain, etc.) than other tandem-repeat type galectins, such as galectin-4, -6 [94], -9 (described below), and -12 [95]. Gal-8 exerts strong hemagglutinating activity and binds to lactosyl – agarose. N-terminal and C-terminal CRDs of Gal-8 show approximately 40% amino acid identity. As regards its biological functions, Gal-8 was shown to inhibit adhesion of human carcinoma cells to integrin-coated plates, resulting in the induction of apoptosis of the cells [96]. Gal-8 has also been shown to influence migration of colon carcinoma [97]. However, the molecular basis for this phenomenon has not been fully understood in the context of sugarbinding properties. In this work, to investigate roles of individual Gal-8 CRDs, we prepared functionally monovalent Gal-8 proteins by introducing amino acid substitutions of either the conserved Arg233 or Arg69 with His to produce R233H (i.e., Gal-8N) and R69H (Gal-8C), respectively. FAC analysis revealed that overall sugar-binding profile of Gal-8 resembled that of Gal-3 in the following points: (1) Both galectins showed relatively low affinity for complextype N-glycans, to which other galectins (e.g., Gal-1 and Gal-7) exerted high avidity. (2) In contrast, both galectins showed extremely high affinity toward repeated structures of N-acetyllactosamine (e.g., Kd’s of Gal-3 and Gal-8 for LN5, 0.19 and 1.6 AM, respectively). (3) Both Gal-3 and Gal-8 showed particular preference for some glycolipid-type glycans. These included Galili pentasaccharide (25, 0.68 and 8.8 AM, respectively), A-hexasaccharide (32, 0.63 and 3.3 AM, respectively), and B-tetrasaccharide (36, 1.6 and 9.6 AM, respectively). On the other hand, completely distinct features between them can also be noted: Gal-8 bound most strongly to acidic saccharides, GM3 (16, 1.5 AM), and GD1a (20, 0.64 AM), whereas Gal-3 showed only limited affinity toward them (i.e., 26 AM and 23 AM, respectively). FAC analysis of individual CRDs of Gal-8 (i.e., Gal-8N and Gal-8C) revealed that these denoted features are attributable to the N-terminal CRD, because Gal-8N retained high affinity for GM3 (16, 0.62 AM) and GD1a (20, 0.5 AM), whereas Gal-8C showed only poor affinity for these saccharides (not detectable for GM3 and 230 AM for GD1a; Fig. 5). Interestingly, Gal-8N exerted rigorous selectivity for a blood group B saccharide: it showed relatively high affinity for B-tetrasaccharide (36, 5.9 AM), whereas it did not bind at all to A-tetrasaccharide (38). On the other hand, Gal-8C had moderate affinity toward both A- and Btetrasaccharides (25 and 31 AM, respectively). High affinity toward oligolactosamines (LN2, LN3 and LN5) was maintained by either CRD. However, the sugar-binding property of Gal-8N was apparently abnormal, because it showed comparably high affinity for both LN2 (39, 1.7 AM) and LN5 (41, 1.8 AM), whereas the affinity to the medial LN3 (40, 12 AM) was much lower. Moreover, a similar sugarbinding property was also observed for the intact Gal-8, i.e.,

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Kd’s of Gal-8 (whole) for LN2, LN3, and LN5 are 3.5, 9.6, and 1.6 AM, respectively. On the other hand, Gal-8C showed a normal behavior toward LN2 (23 AM), LN3 (9.0 AM), and LN5 (2.1 AM). Another distinguishing feature of Gal-8N (but not Gal-8C) was its high affinity for LNFP-I (30, 1.6 AM). As a possible explanation for this, Gal-8N can also recognize a reducing terminal lactose unit (13, 130 AM), the affinity toward which is strongly enhanced by substitution with Galh1-4(Fuca1-3)GlcNAch1-3, as described for Gal-3. 4.6. Galectin-9 (Gal-9) Gal-9 is a new member of the tandem-repeat type galectins, but it is unique in that it was found by studies on divergent research topics unexpectedly, i.e., as an antigen reactive with autologous serum of a patient with Hodgkin’s lymphoma [39], as a new tandem-repeat type galectin screened from a kidney cDNA library using degenerate primers [40], and as a novel eosinophil chemoattractant produced by T lymphocytes, previously designated ‘‘ecalectin’’ [41]. A recent molecular biological study proved that both N-terminal and C-terminal CRDs of Gal-9 were required for this lectin to exert eosinophil chemoattraction activity [66]. As regard to oligosaccharide specificity, we have performed FAC analysis on the two separate domains of Gal-9 in the form of GST fusion protiens [64]. In the present study, oligosaccharide specificity of the whole protein (intact Gal9) was also examined. The result shown in Fig. 4 indicates that Gal-9 had striking affinity for both branched N-glycans and repeated oligolactosamines. In fact, affinities toward biantennary (1, Kd = 0.7), triantennary (2, 0.16 AM), and tetraantennary N-glycans (4, 0.16 AM) were more than 100

times higher than those towards the average of functionally monovalent N-glycans, i.e., a2-6 silalylated NA2 (10 and 11, Kd = 55 and 170 AM, respectively); and those toward oligolactosamines drastically increased as the repeat number increased, i.e., LN2 (39, 8.3 AM), LN3 (40, 1.6 AM), and LN4 (41, 0.09 AM). Further, Gal-9 showed particular preference for two glycolipid-type glycans, i.e., Forssman pentasaccharide (24, 0.41 AM) and A-hexasaccharide (32, 1.2 AM). Apparently, this special ability is attributable to the N-terminal CRD, because it retained or showed still higher affinity toward these saccharides (0.09 AM and 0.26 AM, respectively; Fig. 5). Gal-9N also showed significant affinity for other glycolipid-type glycans, i.e., GA1 (15, 5.4 AM), GM1 (19, 3.8 AM), GD1a (20, 4.2 AM), GD1b (21, 7.4 AM), and Gb4 (23, 9.7 AM). On the other hand, enhanced affinity toward branched N-glycans and oligolactosamines was shared by both CRDs. This finding that Gal-9 showed particular preference for Forssman saccharide and A-hexasaccharide as well as repeated oligolactosamines should give a clue to elucidation of the molecular mechanism underlying the ability of this unique galectin to exert its various biological functions not restricted to chemoattracting activity. 4.7. Chicken 14-kDa (C14) and 16-kDa galectins (C16) C14 and C16 represent well-known isolectins in the chick. Although they belong to the same proto type and share moderate sequence similarity (i.e., 47% amino acid identity), their tissue distributions [98,99] and hemagglutinating activity [13] are fairly distinct. Apparently, the lower hemagglutinating activity of C14 can be attributed to the monomeric feature of this lectin in contrast to C16, which exists as a stable dimer as far as examined by gel-filtration

Fig. 5. Bar graph representation of affinity constants, Ka’s, of individual N-terminal and C-terminal CRDs of three tandem-repeat type galectins. Ka values of Nterminal CRDs are shown at the left side of central sugar numbers, and those of C-terminal ones are shown at the right side. Trivial names of some saccharides that showed particular specificity for each CRD are denoted as in Fig. 4.

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analysis [98]. Therefore, C16 is considered to form a canonical dimer like mammalian galectin-1 under physiological conditions. In this context, C16 has been implicated to be a growth regulator of activated T cells [100]. A solidphase binding assay using synthetic lactose derivatives also suggested their fine sugar specificities to be different [100 – 102]. However, detailed quantitative analysis using complex oligosaccharides has not been carried out. The present FAC analysis confirmed the previous observation that the two isolectins showed different hemagglutinating activities. That is, C16 showed significantly higher affinity to the entire range of PA-oligosaccharides compared with C14. However, the overall profiles of their sugar-binding specificity apparently resembled each other and those of other proto-type galectins, Gal-1 and Gal-7. In short, both C14 and C16 showed moderately enhanced affinity for branched N-glycans (Kd’s in the range of 3 –10 AM), whereas affinity toward functionally monovalent N-glycans, 10 and 11, was much lower in C14 (i.e., on the average, 170 AM for C14 and 27 AM for C16, respectively). Therefore, the effect of branching was more evident for C14. On the other hand, enhancement of affinity for oligolactosamines LN2 (39), LN3 (40), and LN5 (41) was relatively poor for C14 (110, 81, and 41 AM, respectively) as in the case of Gal-1. Notably, repeating units had no enhancing effect on the affinity of C16 (19 AM for each of LN2, LN3, and LN5). If the number of lactosamine units is considered (in terms of mol lactosamine unit), repeating lactosamine units had a rather detrimental effect on these two chicken galectins, in particular on C16. Significantly distinct features between C14 and C16 include the preference of C14 for a type-1 saccharide, LNT (27, 69 AM) over a type-2 saccharide, LNnT (26, 110 AM); whereas C16 showed completely the same affinity for these saccharides (17 AM). In addition, C16 showed relatively high affinity toward Galili pentasaccharide (25, 9.3 AM), toward which C14 had much lower affinity (89 AM). 4.8. Nematode 32-kDa galectin (LEC-1) C. elegans 32-kDa galectin was the first recognized example of an invertebrate galectin as well as of a tandem-repeat type galectin in the animal kingdom. As previously reviewed [103,104], the nematode 32-kDa galectin was renamed LEC-1 along with finding of many other galectin genes in this organism (designated lec-1-11). Among thus identified genes, lec-1-5 encodes tandemrepeat type galectins showing 40 –80% amino acid identities to one another. N-terminal and C-terminal CRDs of LEC-1 share approximately 40% amino acid identities, similar to those of mammalian tandem-repeat-type galectins. Our previous study using individual domains of LEC-1 showed that the N-terminal CRD (LEC-1N) had only weak ability to bind to asialofetuin-agarose compared with the C-terminal CRD (LEC-1C) [105]. However, a subsequent

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study [26] demonstrated that LEC-1N was not just an inactive lectin but rather had developed a specialized function to recognize a distinct type of saccharide, i.e., A-hexasaccharide (32, 48 AM), which was only poorly recognized by LEC-1C (810 AM; Fig. 4). In the present study, we found that Galili pentasaccharide (25) was also well-recognized by LEC-1N (72 AM) as well as by LEC1C (100 AM). In this context, it is noteworthy that LEC-1N could also recognize B-tetrasaccharide (36, 350 AM), whereas it could not bind to A-tetrasaccharide (38, affinity not detectable). Therefore, we highly suggest that LEC-1N would show stronger affinity toward B-hexasaccharide than toward A-hexasaccharide. On the other hand, LEC-1C proved to show much increased affinity for repeat oligolactosamines, LN2 (39, 97 AM), LN3 (40, 51 AM), and LN5 (41, 18 AM); whereas affinity for them remained much lower in LEC-1N, i.e., LN2 (940 AM), LN3 (590 AM), and LN5 (350 AM). Nevertheless, it should be noted that both LEC-1N and LEC-1C preferred a type-1 saccharide, LNT (27, 440 and 77 AM, respectively) to a type-2 saccharide, LNnT (26, 1000 and 200 AM, respectively). If this LNT was a1-2 fucosylated at the nonreducing terminal galactose, the affinity for the resultant LNFP-I (28) was considerably increased in the case of LEC-1N (130 AM), whereas only a slight increase in affinity was observed for LEC-1C (65 AM). Notably, further modification with a13GalNAc at the nonreducing terminal galactose enhanced much the affinity for the resultant A-hexasaccharide (32, 48 AM) as mentioned above, whereas it greatly diminished the affinity of LEC-1C (810 AM). From a global viewpoint, however, affinities determined for LEC-1, LEC-1N and LEC-1C were somewhat low compared with those for other galectins. This can partly be attributed to a significant difference in glycan structures between PA-oligosaccharides used in this study (all from mammals) and the endogenous glycans that C. elegans LEC-1 specifically recognizes. Until now, however, no structures of complex-type N-glycans have been reported for C. elegans, while the occurrence of high-mannose type N-glycans has been confirmed recently [106]. 4.9. Nematode 16-kDa galectin (LEC-6) LEC-6 represents the sole proto-type galectin identified in the C. elegans genome database. It was purified from the lactose extract of the worm together with LEC-1 as major galectins, both of which showed significant hemagglutination activity [107]. According to gel filtration analysis, LEC-6 existed as a dimer consisting of 2 16-kDa subunits [46]. Preliminary FAC analysis of LEC-6 was already performed by using a fusion protein with E. coli h-galactosidase a-peptide [25]. In the present study, the same approach was taken for this galectin and the use of many more PA-oligosaccharides. As depicted previously [25], LEC-6 showed significantly enhanced affinity for branched N-glycans, i.e., functionally

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monovalent NA2 (10 and 11; on the average, Kd = 650 AM), NA2 (1, 93 AM), NA3 (2, 40 AM), and NA4 (4, 27 AM). The slightly higher affinity toward NA3 including a type-1 chain (3, 36 AM) reflects the preference of this lectin for a type-1 saccharide, LNT (27, 190 AM) over a type-2 saccharide, LNnT (26, 320 AM). Further modification of the nonreducing terminal Gal of LNT with a1-2Fuc slightly increased the affinity (28, 170 AM) as in the case of many other galectins. In contrast, a1-3 galactosylation of LNnT more evidently enhanced the affinity; i.e., compare LNnT (26, 320 AM) with Galili pentasaccharide (25, 160 AM). These observations suggest that LEC-6 would still have higher affinity for LNT modified with an aGal epitope, i.e., Gala1-3Galh1-3GlcNAch1-3Galh1-4Glc, if it were available. On the other hand, LEC-6 showed moderately enhanced affinity toward oligolactosamines, i.e., LN2 (39, 370 AM), LN3 (40, 230 AM), and LN5 (41, 140 AM). Another notable feature of LEC-6 is that it could weakly but significantly recognize some glycolipid-type glycans other than lacto/neolacto-series ones, i.e., GA1 (15, 580 AM) and GM1 oligosaccharides (19, 630 AM). Since both glycans include a T-antigen structure (Galh1-3GalNAc) in the nonreducing terminal, also known as core-1 structure of Oglycans, LEC-6 can bind to not only N-glycans but also Oglycans to some degree. However, mainly due to technical difficulty in preparation of O-glycans (e.g., no PA-O-glycans are commercially available), much remains to be determined for analysis of O-glycans. As in the case of LEC-1, overall affinities of LEC-6 for the PA-oligosaccharides tested in this assay were relatively low. It is not clear at the moment whether these galectins would have much higher affinities to glycans produced in C. elegans or not. However, our recent structural analysis of C. elegans Nglycans that were affinity-purified by using a LEC-6-agarose column showed significant divergence from those derived from mammals (Hirabayashi, Kaji, Isobe, and Kasai, unpublished result). 4.10. Sponge galectins clone 1 (GC1) and clone 2 (GC2) Pfeifer et al. [47] reported the presence of 2 cDNA clones, both of which encoded proto-type galectins of the sponge G. cydonium. The two isolectins showed particular resemblance to each other, having 65% amino acid identity. However, similarity to vertebrate galectins was fairly low (i.e., approximately 20% identity). In addition, the sponge galectins have some distinguishing features not found in other animal galectins [108]: (1) they form a large complex molecule in the presence of Ca2 + ; (2) they are glycoproteins comprising 15% carbohydrates; (3) they show extremely high affinity for GalNAc-containing saccharides. According to Hanish et al. [57], Geodia galectin purified from the sponge showed extremely high affinity toward Forssman disaccharide (GalNAca1-3GalNAc) and blood group A disaccharide (GalNAca1-3Gal) in the range of 10
7 M in terms of Ki in a conventional lectin-binding

assay. However, since they used natural galectin protein (possibly a mixture of multiple isolectins), it is not known whether one or both of the two isolectins are responsible for these unique properties. Moreover, specificity for more complex oligosaccharides remains to be determined. The present FAC analysis confirmed striking high affinity of both GC1 and GC2 for three GalNAc-containing oligosaccharides, i.e., Forssman pentasaccharide (24), Ahexasaccharide (32), and A-tetrasaccharide (38). However, the affinity of GC1 toward these saccharides was considerably higher than that of GC2. Actually, Kd’s of GC1 for these saccharides could not be determined under the present conditions because of too tight binding (i.e., < 0.1 AM), whereas Kd’s of GC2 could be determined at higher temperature (37 jC): Forssman pentasaccharide (24, 0.13 AM), Ahexasaccharide (32, 0.10 AM), and A-tetrasaccharide (38, 0.23 AM). Since lectin affinity for saccharides is known to be considerably increased by lowering the temperature [25,109], actual Kd’s (at 20 jC) of GC2 for these saccharides should become much lower. Interestingly, both GC1 and GC2 also showed high affinity for B-tetrasaccharide (36), i.e., 0.20 and 1.6 AM, respectively (data at 20 jC). This result indicates that GC2 prefers A-tetrasaccharide (38, 0.23 AM at 37 jC) >7 times more than B-tetrasaccharide (36, 1.6 AM). If the same situation is applied to GC1, the Kd of GC1 for A-tetrasaccharide (32) at 20 jC would be < 0.029 AM (i.e., 0.20/7), and that for A-hexasaccharide (32) would be as low as < 0.013 AM (i.e., 0.029  0.10/ 0.23). This would be the highest affinity (in the order of 10
8 M) found so far in our FAC analysis. Since both GC1 and GC2 showed rather low affinity for aGalLac (35, 11 and 25 AM, respectively), the contribution of a1-2Fuc in the binding to B-tetrasaccharide is evident. In contrast to such extraordinary high affinity for aGalNAc-containing glycolipid-type glycans, Geodia galectins showed relatively low affinity for complex-type N-glycans, i.e., at most 5.5 AM for GC1 and NA3 (3, containing a type-1 chain). Nevertheless, both GC1 and GC2 preserved all features of the lactose/Nacetyllactosamine recognition patterns described above, i.e., showing the critical importance of the 4-OH and 6-OH of Gal as well as of the 3-OH of Glc(NAc). In the case of GC1, their importance is evident by comparison between lactose (13, 750 AM) and GA2 (14, affinity not detectable), between NA2 (1, 68 AM) and N2 (12, affinity not detectable), and between LNnT (26, 600 AM) and LNFP-III (30, affinity not detectable). As in the case of C. elegans, structures of sponge glycans remain to be clarified. 4.11. Mushroom galectin (ACG) The occurrence of galectins in fungi was first demonstrated by Cooper et al. [19], who identified two galectin cDNAs, designated Cgl-I and Cgl-II, from the mushroom Coprinus cinereus; these cDNAs encoded similar 16-kDa galectin proteins. Though these galectins retained only 18% amino acid identities to human galectin-1, critical amino

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acid residues involved in the sugar binding function were completely conserved except for Asn46 in both Cgl-I and II (residue number is that of human galectin-1). We also investigated and characterized a lectin from the fruiting bodies of the mushroom A. cylindracea [48]. The lectin is composed of 17-kDa subunit, and as a result of structural analysis, we found it to be a member of the galectin family sharing 36% amino acid identity to the Coprinus galectin (unpublished result). The A. cylindracea galectin (designated ACG hereafter) showed particular affinity toward acidic saccharides, such as those containing sialic acid. Physiological roles of these mushroom galectins remain to be elucidated. Therefore, using the same set of PA-oligosaccharides as above, we performed FAC analysis to obtain a clue to their biological functions. Though still preliminary at the moment, our results are conclusive in the following points: ACG showed extremely strong affinity toward both branched N-glycans and a few acidic glycolipid-type glycans. It showed the highest affinity for branched N-glycans, NA2-NA4 (1– 9, < 3.0 AM; Kd’s not precisely determined), unless terminal Gal was masked with a2-6NeuAc. It also showed such high affinity for an acidic glycan, GD1a (20, < 3.0 AM). Affinity for GM1 (19, 9.4 AM) was second. This particularly high affinity toward these ganglioside-type saccharides is apparently due to modification by a2-3 sialylation of the nonreducing terminal Gal. However, in these cases, ACG is considered to specifically recognize a2-3NeuAc linked to nonreducing terminal Gal of Galh1-3GalNAc (T antigen) in GD1a or Galh1-4Glc (lactose) in GM3. Since ACG recognized neither GA2 (14, GalNAch1-4Galh1-4Glc) nor GM2 (18, GalNAch1-4(NeuAca2-3)Galh1-4Glc), the significant affinity for GA1 is attributed to recognition of T-antigen. Consistently, the affinity toward several saccharides having this recognition unit (Galh1-3GalNAc) were almost comparable: i.e., GA1 (15, 15 AM), GM1 (19, 9.4 AM), and GD1b (21, 15 AM). Therefore, the highlighted affinity for GD1a is considered to be a result of enhanced affinity for this Galh1-3GalNAc recognition unit by a2-3 sialylation. On the other hand, a2-6 sialylation of both lactose (13, 400 AM) and biantennary N-glycan NA2 (1, < 3.0 AM) completely abolished the affinity. ACG also showed relatively high affinity to other aGalNAc-containing neutral oligosaccharides, such as A-tetrasaccharide (38, 18 AM) and A-hexasaccharide (32, 7.5 AM). Another notable feature is that ACG also showed particular preference for Galili pentasaccharide (25, 1.9 AM). Compared with LNnT (26, 26 AM), this a1-3 galactosylation enhanced the affinity by as much as 13.6 times. Apparently, ACG has acquired a unique property by specialization for recognizing modification of the 3-OH group of Gal.

5. Directions for specialized functions In this section, several ways for specialized features of sugar-binding functions of galectins are discussed. For this,

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however, it is important to confirm the observed fact that all galectins recognize two basic disaccharides, i.e., Galh13GlcNAc and Galh1-4Glc(NAc). Then, further discussion on the effects of branching (Section 5.1), repetition (Section 5.2), and substitutions (Section 5.3) becomes possible. To discuss the former two subjects, i.e., branched N-glycans and repeated oligolactosamines, we will use FAC data for the 13 whole galectin proteins, because there is possibility that some of these mutilvalent glycans interact in an extended manner; i.e., they may interact simultaneously with both N-terminal and C-terminal CRDs of analyzed tandem-repeat-type galectins (Gal-8, Gal-9, and LEC-1). On the other hand, discussion about the effect of substitution should be made with respect to individual CRDs. 5.1. Effect of branching of the recognition unit As mentioned, many galectins showed increased affinity for branched N-glycans; i.e., when the branching number increased from mono- (i.e., functionally monovalent 10 and 11), to bi- (1), tri- (2), and teraantennary (3) saccharides, affinities toward them increased not simply additively but rather synergistically (Fig. 6A). Such an enhanced affinity for branched glycans is understood along with the concept of the ‘‘glycoside clustering effect’’, which was first demonstrated for C-type lectins [71 –76]. However, compared with the drastic cluster effect for some C-type lectins, that observed for galectin-1 (i.e., bovine spleen galectin-1) was not very strong [70]. In the present study, it became evident that there is a broad range of enhancing effects of branching (Fig. 6A; note that relative affinity is calculated in comparison with LNnT as well as in Fig. 6B). The highest group is formed by Gal-9 and GC1: NA4 (4) (closed bar) as compared with LNnT (26) showed as large as 60 times higher affinity for Gal-9 and GC1. However, in terms of Kd, the affinity of GC1 for NA4 (10 AM) was much lower than that of Gal-9 (0.16 AM), because its basic affinity to LNnT is fairly low (600 AM). On the contrary, Gal-3 showed only poorly enhanced affinity to branched N-glycans. Nevertheless, this galectin showed the highest level of affinity for both NA2 (1, 1.4 AM), NA3 (2, 0.78 AM), and NA4 (4, 0.69 AM). This is apparently due to the extremely high affinity for the basic saccharide, e.g., LNnT (26, 1.7 AM). In contrast, some tandem-repeat-type galectins did not show significantly enhanced affinity or significant absolute affinity for branched N-glycans. Such ‘‘blunt’’ galectins may include Gal-8 (Kd for NA4, 14 AM) and LEC-1 (Kd for NA4, 110 AM). In the case of Gal-8, however, the galectin showed relatively high affinity toward a basic saccharide, LNnT (26, 12 AM) probably because of residual affinity for reducing terminal lactose (see 4.4); and actually it did not show any affinity toward functionally monovalent NA2 (i.e., 10 and 11). Considering these facts, Gal-8 can be regarded as a galectin having moderate affinity to branched glycans. Similarly, other galectins can be categorized into a ‘‘moderate’’ affinity group.

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family (Fig. 6B). The most distinguished effect of repeating was observed for Gal-9 and GC1 as in the case of branching (Fig. 6A). In particular, Gal-9 showed the highest enhancement effect (i.e., 111 times compared with LNnT in the case of LN5) as well as the highest affinity toward these glycans; i.e., LN2 (39, 8.3 AM), LN3 (40, 1.6 AM), and LN5 (41, 0.09 AM). This enhancement effect was also distinguishable for GC1. However, this galectin show relatively poor affinity to a basic saccharide, LNnT, as mentioned above. Though not very evident from the figure, enhanced affinity for repeated lactosamine was fully significant for Gal-3 and Gal-8 (i.e., LN5/LNnT = 8.9 and 7.5, respectively, in terms of Ka), in particular, when considering their highest affinity for LN5 (0.19 and 1.6 AM, respectively). A significant enhancement effect was also observed for Gal-7 and LEC1 (i.e., LN5/LNnT = 4.2 and 6.8, respectively, in terms of Ka), though basic affinity of these galectins are relatively low (Kd’s for LNnT are 130 and 210 AM, respectively). On the other hand, only a poor enhancement effect (i.e., LN5/ LNnT < 2 in terms of Ka) was observed for Gal-1, Gal-2, and C16. In this context, it was recently implied that Gal-3, but not Gal-1, is involved in negative regulation of T cell activation, because the T-cell receptor is thought to have poly-N-acetyllactosamine chains by the action of Mgat5, to which Gal-3 binds to block the clustering that leads to T cell activation [91]. 5.3. Effect of substitution of the recognition unit Fig. 6. Effect of branching (A) and repeating (B) on affinity enhancement. In both bar graphs, relative affinities with respect to LNnt (26 Fig. 2) are shown. In A open bars ( ), shaded bars ( ) and closed bars ( ) represent biantennary NA2 (1), triantennary NA3 (2) and tetraantennary NA4 (4); whereas in B they represent oligolactosamines, LN2 (39), LN3 (40), and LN5 (41), respectively. Note that higher relative affinity to LNnT does not necessarily mean higher absolute affinity in terms of Ka.

5.2. Effect of repetition of the recognition unit Increasing the repeat number of N-acetyllactosamine units is another way to acquire efficiently stronger avidity to lectins. However, there has been only little discussion on this matter based on experiments [62,63], certainly because of the lack of appropriate methodologies and saccharides. Poly-N-acetyllactosamine is extensively found in various forms of glycoconjugates, i.e., in glycoproteins (both O- and N-glycans), glycolipids, and keratan sulfate proteoglycans. In the present study, oligolactosamines, i.e., LN2, LN3, and LN5, were used as model saccharides for poly-N-acetyllactosamine, and the relative affinity with respect to LNnT was plotted versus the repeat number. Although it has long been believed that poly-N-acetyllactosamine is a most probable endogenous glycan ligand for galectins based on several lines of evidence [59 – 61], the present analysis revealed that enhanced affinities toward these oligolactosamines were observed for only rather particular members of the galectin

As described, substitution of OH groups did significantly enhance the affinity of many galectins. However, it is important to point out that such favored substitutions are restricted to only a few positions, because 4-OH and 6-OH groups of Gal and 3(4)-OH of Glc(NAc) must be intact; and therefore, substitutions at none of these critical OH groups are permitted. As a result, galectins would have evolved their detailed sugar-binding specificity by modifying the remaining OH groups, which are allowed to be substituted. From a molecular structural viewpoint, 2-OH and 3-OH groups of Gal meet this requirement, because if substitution occurred at these positions, no detrimental effect such as steric hindrance would be expected. Moreover, the 3-OH group is directed to a widely open space forming a possible subsite as an extension from the major binding site [50 – 54] (also mentioned in this issue by Gabius et al. [110] and Loris [111]. Hence, we focused our attention on substitution occurring at these two positions of Gal. The results are summarized in Fig. 7, where relative affinity to some basic saccharides is plotted versus 16 galectin CRDs (not the whole proteins in case of Gal-8, 9 and LEC-1). To make comparisons clear, we chose only pairs of saccharides, in which only one chemical change was made. There are four cases for such a purpose; i.e., (1) the effect of substitution with a1-2Fuc (comparison of LNFP-I with LNT; Fig. 7A); (2) effect of substitution with a1-3Gal (comparison of Galili

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Fig. 7. Effect of substitution of lactose/lactosamine units on galectin affinity. Four types of substitutions were examined in terms of relative affinity: (A) LNFP-I (28) to LNT (27; effect of substitution with a1-2Fuc); (B) Galili pentasaccharide (25) to LNnT (26; effect of substitution with a1-3Gal); (C) A-hexasaccharide (32) to LNFP-I (28; effect of substitution with a1-3GalNAc ); (D) GM3 (16) to GA3 (13; effect of substitution with a2-3NeuAc).

pentasaccharide with LNnT (Fig. 7B); (3) effect of substitution with a1-3GalNAc (comparison of A-hexasaccharide with LNFP-I; Fig. 7C); (4) effect of substitution with a23NeuAc (comparison of GM3 with GA3; Fig. 7D). The following points are notable: First, a1-2 fucosylation is favored by all galectins but only slightly. The highest enhancement was recorded for GC2 (5.2 fold increase), followed by Gal-2 (3.0). Second, in contrast, substitutions at 3-OH group of Gal resulted in drastically enhanced affinity of particular galectin CRDs. Third, in the case of tandemrepeat-type galectins, i.e., the effects of substitutions on affinity enhancement toward particular saccharides were not always comparable. For example, Gal-8N showed 200 times

higher affinity when there was a substitution with a2-3 NeuAc, whereas this substitution had no effect on Gal-8C. Affinity enhancement was most evident for the two sponge galectins, which showed extremely high affinity for Ahexasaccharide compared with that for LNFP-I (>200-fold increase in affinity). Molecular modeling study along with X-ray structural study and/or NMR study should reveal more details about the molecular mechanisms involved in their enhanced affinities. More importantly, such great differences in sugar-binding specificity should be understood in the light of specialized functions of individual galectins. The effects of the four types of permitted substitutions are summarized in Table 4.

Table 4 Sugar-binding preference in individual galectin CRDsa CRD

Gal-1 Gal-2 Gal-3C Gal-7 Gal-8N Gal-8C Gal-9N Gal-9C C14 C16 LEC-1N LEC-1C LEC-6 GC1 GC2 ACG

type 1/type 2

A/B

a1-2Fuc

a1-3Gal

a1-3GalNAc

a2-3NeuAc

LNT/LNnT

A-tetra/B-tetra

LNFP-I/LNT

Galili/LNnT

A-hexa/LNFP-I

GM3/GA3

0.79 1.9 0.65 2.7 0.19 0.76 0.85 1.3 1.6 1.0 2.3 2.6 1.7 9.4 11 0.37


/
b AbB (0.54) AbB (0.42) A < B < (0.13) AbBc A>B (1.2) AHBd AHBd
/
b
/
b A>B A>B
/
b A>B AHB (7.0) NDe

1.1 3.0 1.7 0.98 1.2 2.5 1.3 1.3 0.49 0.59 3.4 1.2 1.1 3.2 5.2 0.37

1.5 2.3 2.5 1.6 1.3 1.9 0.93 2.2 1.2 1.8 14 2.0 2.0 72 130 14

0.24 0.70 2.4 0.18 0 17 15 1.2 0.15 0.51 2.7 0.08 0.11 >200 >250 25


/
b
/
b 1.0
/
b 210
/
b 0.33 0 0.82 0.38
/
b
/
b 0.59
/
b
/
b 170

a In order to quantitatively evaluate binding preference for linkage (i.e., types 1 or type 2), A/B groups, or particular types of substitutions (i.e., a1-2Fuc, a1-3Gal, a1-3GalNAc and a2-3NeuAc), relative affinities for relevant pairs were calculated based on Kd’s presented in Table 2. b Relevant galectins showed only poor affinity to both A-tetrasaccharide and B-tetrasaccharide. c Affinity to A tetrasaccharide not detectable. d Afinity to B tetrasaccharide not detectable. e Not determined.

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5.4. Preference Table 4 also summarizes the sugar-binding preference for common pairs of saccharides; i.e., type 1 or type 2 (i.e., comparison of LNT/LNnT), and blood groups A or B (Atetrasaccharide/B-tetrasaccharide). Notably, many galectin CRDs much preferred a type-1 saccharide to a type-2 one (i.e., LNT/LNnT>2): these included Gal-7 (2.7), LEC-1N (2.3), LEC-1C (2.6), GC1 (9.4), and GC2 (11). Gal-2, C14, and LEC-6 also preferred the type-1 saccharide over the type-2 saccharide. On the contrary, strong type-2 favorers (i.e., LNT/LNnT < 0.5) were represented by Gal-8N (0.19) and ACG (0.37). The remaining were in between, but Gal-1, Gal-3, Gal-8C, and Gal-9N rather preferred type-2, whereas Gal-2, Gal-9C, C14, and LEC-6 preferred type-1. This preference may be the result of balance between favored and unfavored interactions as regards two distinguished counterpart groups of GlcNAc, i.e., the N-acetyl group and 6-CH2OH group. On the other hand, discussion on A/ B preference cannot be perfect, because many galectins did not show significant affinity for A-tetrasaccharide or Btetrasaccharide, possibly because the basic recognition unit (lactose) of these saccharide is partially broken (Glc is open). Nevertheless, we can conclude that Gal-9N, Gal9C, and GC2 form a group of strong A-favorers, whereas Gal-3, Gal-7, and Gal-8N form a group of strong B-favorers. Since the N-acetyl group is bulky, it may interfere with binding between a basic recognition unit (in this case, lactose) and side chains of galectin proteins, or it may add further favorable interaction. The former case would tend to generate B-favorers; and the latter, A-favorers.

6. Concluding remarks By using the established FAC system, it became possible for the first time to analyze a wide range of lectin – oligosaccharide interactions in a rapid, sensitive, systematic, reliable, and quantitative manner. Based on thus obtained comprehensive data on Kd (or Ka), we discussed how galectins have evolved their fine and dynamic sugar-binding specificities. Our conclusions are as follows: the basis for lactosamine recognition has been maintained throughout evolution. On the other hand, specialized features can be understood from various aspects: first of all, they have fundamental protein architectures (i.e., either proto, chimera or tandem repeat types), which basically define their crosslinking properties. Next, selectivity and avidity to enhance affinity of individual galectins for certain types of saccharides should have been developed by fine-tuning of their ancestral CRDs. Further, strategies for this specialization processes can be categorized into three aspects, i.e., enhancement of affinity toward (1) branched saccharides (i.e., N-glycans), 2) repeated saccharides (i.e., oligolactosamines), and (3) substituted saccharides (various modifications). From a practical viewpoint, possible substitutions of

lactosamine were restricted to either 2-OH or 3-OH groups of nonreducing terminal Gal. Substitution at 2-OH group had only one variety (i.e., a1-2Fuc) with a relatively weak effect on affinity, whereas that at 3-OH group had three variations (i.e., a1-3Gal, a1-3GalNAc, and a2-3NeuAc), which led to drastic enhancement of affinity of some galectins for some oligosaccharides. By all these strategies for diversification, individual galectins may be considered to have evolved their present specificity and affinity for certain types of complex oligosaccharides. It is of value for argument that all of the tandem-repeattype galectins investigated in this work showed completely distinct sugar-specificity between their N-terminal and Cterminal CRDs (Fig. 5). In this context, some of such tandem-repeat type galectins may function to mediate the formation of microdomains (rafts) on cell surfaces by selecting (or sorting) glycoprotein and glycolipid members. In fact, Gal-4 has been found in an extremely insoluble fraction, and now is regarded as a raft marker of enterocytes [112]. Chimera-type galectin-3 has been implicated to function in negative regulation of T cell receptor activation possibly by binding to poly-N-acetyllactosamine chains expressed on this glycoprotein receptor [91]. Proto-type galectin-1 induces apoptosis of activated T cells in a sugar-dependent manner [113]. For a possible explanation, a new paradigm has been given, that multivalent protein – carbohydrate interactions trigger signal transduction [114,115]: galectin-1 binding to surface glycoproteins (CD3, CD7, CD43, and CD45) of activated T cells results in a dramatic redistribution of these constituents into ‘‘segregated’’ membrane microdomains consisting of CD45, CD3, and galectin-1. The present result provides a molecular basis for this idea, because galectin-1 was found to prefer branched N-glycans. Therefore, future studies on galectins will be focused on identification of endogenous receptors on certain types of cells, where individual galectins actually function. Also, elucidation of their cross-linking features from various aspects is needed, based on detailed sugar specificity analysis, as described here, supermolecular assemblage analysis by X-ray crystallographic study, and electron microscopy. For the former purpose, we have already developed a novel affinity technique named the ‘‘glyco-catch’’ method [27,104,116]. The described FAC system is versatile and serves various purposes, not only for studies on galectins. In this context, it is important to note that glycans from an extensive number of biological species are required for future post-genome scientific study, and model organisms such as C. elegans are extremely useful for genetic, proteomic analysis, and, possibly, glycomic analysis. Though, presently, only little is known for glycan structures of these model organisms, we should learn much more from them than from only mammals, because not only nucleic acids and proteins, but also glycans are descendant molecules from our common ancestor [117]. In this context, we need to study various glycans of various organisms under the concept of ‘‘comparative glycomics’’.

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Acknowledgements This work was supported in part by Grants-in-Aid for Scientific Research on Priority Area ‘‘Genome Science’’ (no. 13202058 to J.H.) and Grants-in-Aid for Scientific Research (no. 12680617 to J.H. and 11771453 to K.K.) from the Ministry of Education, Science, Sports and Culture of Japan and by the Mizutani Foundation for Glycoscience.

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