three-dimensional structures of galectins
TRANSCRIPT
Trends in Glycoscience and Glycotechnology 30(172): SE41-SE50
(2018)© 2018 FCCA (Forum: Carbohydrates Coming of Age)SE41
Trends in Glycoscience and Glycotechnology Vol. 30 No. 172 (January–May 2018) pp. SE41–SE50
Three-Dimensional Structures of Galectins
Shigehiro Kamitori Life Science Research Center and Faculty of Medicine, Kagawa University, 1750–1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761–0793, Japan
FAX: +81–87–891–2421, E-mail: [email protected]
(Received on August 18, 2017, accepted on October 5, 2017)
Key Words: carbohydrate recognition domain, oligosaccharide, prototype galectin, tandem-repeat type galectin, X-ray crystal structure
Abstract The galectins are a family of β-galactoside-specific animal lectins that contain a conserved carbohydrate recognition domain
(CRD) with approximately 140 amino acid residues. There are 14 members in the mammalian galectin family (galectin-1–10, and 11– 15), and they have different specificities for oligosaccharides. X-ray structures of the galectin CRD in complexes with oligosaccha- rides have provided important clues about the oligosaccharide-recognition mechanisms of galectins giving the different specificities. Galectin is divalent in glycan binding due to the association of two CRDs that crosslink with oligosaccharides. The spatial arrange- ment of the two CRDs is very important for elucidating the biological functions of galectins. Several different spatial arrangements of CRDs are found in X-ray structures of galectins. I herein examined the three-dimensional structures of galectins relevant for biological functions, based on the protein–ligand interactions related with oligosaccharide-specificity, the cross-linking structure by galectin and oligosaccharides, and the spatial arrangements of CRDs.
A. Introduction The galectins are a family of β-galactoside-specific animal
lectins that contain a conserved carbohydrate recognition domain (CRD) with approximately 140 amino acid residues, and have at- tracted much attention as novel regulators of the immune system (1, 2). There are 14 members in the galectin family (galectin-1–10, and 12–15), which are classified into three subtypes based on structure. The prototypes (galectin-1, 2, 5, 7, 10, 13, 14 and 15) have a single CRD. The chimera type (galectin-3) has a single CRD and a non-lectin N-terminal domain. The tandem-repeat types (galectin-4, 6, 8, 9, and 12) have two different CRDs in the N- and C-terminal regions (N-CRD and C-CRD) that are joined by a linker peptide (Fig. 1A). The prototype galectins mostly form homo-dimers, and the chimera type galectin is expected to form an oligomer based on its non-lectin N-terminal domain. Thus, ga- lectins are divalent and/or multivalent in glycan binding. The most well-characterized role of galectins is crosslinking with oligosac- charides in the extracellular space, which is involved in cell–cell and cell–matrix interactions. Recently, additional roles of galectins in the cytosol have attracted interest (3, 4).
Galectin CRDs have different specificities for oligosaccha- rides. The N-CRD of galectin-8 (galectin-8_N-CRD) exhibits a strong affinity for α(2-3)-sialylated oligosaccharides, but the C- CRD does not. The N-CRD of galectin-9 (galectin-9_N-CRD) has high affinity for oligolactosamines with a linear structure, but the C-CRD does not. Both the galectin-9_N-CRD and C-CRD have high affinities for N-glycan-type branched oligosaccharides (bi- anntenary oligosaccharides) (5). Many X-ray structures of galectin CRDs in complexes with oligosaccharides have been deposited into PDB (6), and have provided important clues about the oligo-
saccharide-recognition mechanisms of galectins giving the differ- ent specificities (7–18). Selected oligosaccharides and a synthetic ligand used in structural studies of galectin CRDs are listed in Fig. 1B with their abbreviations.
Most of the reported X-ray structures of galectins contained only a single type of CRD. The homo-dimer structures of the prototype galectins were found to show the spatial arrangements of CRDs. However, there was no information about the spatial ar- rangement of the CRDs of chimera type and tandem-repeat type galectins. Tandem-repeat type galectins are inherently divalent in glycan binding with different specificities, and structural informa- tion about the spatial arrangement of the two CRDs is very impor- tant for elucidating their biological functions. Tandem-repeat type galectins are sensitive to proteases due to the long linker. To carry out X-ray crystal structure determinations of tandem-repeat type galectins with two CRDs, the galectins with a short linker(19) and/ or the protease-resistant mutant forms with a modified linker pep- tide were used (20–22).
In this review, I examined three-dimensional structures of galectins based on the protein–ligand interactions related with oligosaccharide-specificity, the cross-linking structure by galectin and oligosaccharides, and the spatial arrangements of CRDs of the prototype and tandem-repeat type galectins. Galectin-1, 2, 7, 8, 9, 10 stand for human galectin-1, 2, 7, 8, 9, 10, respectively (unless stated otherwise). Figures 2, 3, 4, 5 were drawn with the program PyMOL(23).
B. Three-Dimensional Structure of Galectin CRD The overall structure of galectin-9_C-CRD in complex with
LacNAc (galectin-9_C-CRD/LacNAc (PDB ID: 3NV2)) (17) is
MINIREVIEW doi: 10.4052/tigg.1731.1SE(Article for special issue on Galectins)
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Fig. 1. Three subtypes of galectins and ligands used in structural studies of galectins. (A) Schematic diagrams showing the three subtypes of galectins are illustrated. (B) Chemical structures of oligosaccharides and a synthetic ligand are illustrated with their abbreviations.
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shown in Fig. 2A. The galectin-9_C-CRD adopts a β-sandwich structure formed by two anti-parallel β-sheets consisting of six (S1–S6) and five (F1–F5) β-strands, respectively. The strand S6 is divided into two strands (S6a and S6b). A short helix (H1) exists between F5 and S2. The carbohydrate-binding sites are exposed to the solvent-accessible surfaces of the molecule, and a LacNAc binds to the concave surfaces formed by S3, S4, S5 and S6, via non-reducing and reducing ends located at S3 to S6. The overall structure is well conserved among galectin CRDs, and can be represented by a tetragonal prism. For clarity, the face, back, top, bottom and sides of the CRD are defined as in Fig. 2A (right). The
carbohydrate-binding site is on the face of the CRD, and the oppo- site side is the back. The short helix (H1) is on the bottom and the opposite side is the top. The β-strand of S1 is on the right side and S6 is on the left side.
Three structures of galectin CRDs in complexes with oligo- saccharides are shown in Fig. 2B. Sugar units of the bound oligo- saccharides are numbered −1, +1, and +2, from the non-reducing end to reducing end (Fig. 1B). Gal+1 occupies the same position in each complex.
In galectin-9_C-CRD/LacNAc (3NV2) (17), the galactose moiety (Gal+1) forms stacking interactions with Trp255, and forms
Fig. 2. X-ray structures of galectin CRDs with the bound oligosaccharides. (A) Overall structure of galectin-9_C-CRD/LacNAc (3NV2) is illustrated as viewed from the face (left) and the left side (middle). The β-sheet on the back of the CRD is shown in a dark color, and a short helix (H1) is shown in red. A schematic diagram of tetragonal prisms showing the CRD is illustrated (right). The back, bottom and right sides of tetragonal prisms are shown in gray. (B) Galectin-9_C-CRD/LacNAc (3NV2) (left), Galectin-8_N-CRD/SiaLacNAc (3VKO) (middle) and Galectin-9_N-CRD/ LN3 (2ZHM) (right) are illustrated with the protein–ligand interactions.
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six hydrogen bonds with the protein: O4-His235, O4-Asn237, O4-Arg239, O5-Arg239, O6-Asn248 and O6-Glu258. The axial conformation of the O4 of Gal+1 is strictly recognized by three hy-
drogen bonds from His235, Asn237 and Arg239. The glucosamine moiety (GlcNAc+2) forms hydrogen bonds with the protein by O3: O3-Arg239, O3-Glu258, and O3-Arg260. As Arg239 and Glu258
Fig. 3. Cross-linking structures by galectins and oligosaccharides. (A) Bovine galectin-1/BIOS (1SLA) is illustrated. The S4–S5 loop is shown in red. A schematic diagram showing the cross-linked structure is illustrated (right). (B) Galectin-9_C-CRD/BIOS (3NV3) is illustrated. The modeled GlcNAc+5 and Man+4 are shown in blue. (C) Galectin-7/D2 (4UW5) is illustrated.
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form bifurcated hydrogen bonds with both Gal+1 and GlcNAc+2, they efficiently recognize the β (1-4) glycoside bond of LacNAc. The protein–ligand interactions found in galectin-9_C-CRD/ LacNAc are conserved in galectin CRD/oligosaccharide complex structures.
In galectin-8_N-CRD/SiaLacNAc (3VKO) (21), in addition to the conserved protein–ligand interactions, Arg59 forms ef- ficient salt–bridge interactions with the carboxyl group of Sia−1, and Gln47 and Trp86 hold the carboxyl group from both sides via hydrogen bonds. Arg59 is unique to galectin-8_N-CRD, and may be responsible for the strong affinity for α(2-3)-sialylated oligosac- charides.
In galectin-9_N-CRD/LN3 (2ZHM) (16), the bound LN3 has a linear structure which enables GlcNAc−1 to form a hydrogen bond with Asn48. Furthermore, Ala46, which is unique to galec- tin-9_N-CRD, is proposed to be one of the residues responsible for the high affinity for oligolactosamines (13). This is because an amino acid residue with bulky side chain group at the position of Ala46 (His223 in galectin-9_C-CRD and/or Gln47 in galectin-8_ N-CRD) causes steric hindrance with GlcNAc−1 of LN3.
C. Cross-Linking Structure by Galectin and Oligosac- charides
Galectin crosslinks with oligosaccharides, and the N-glycan-
Fig. 4. Spatial arrangements of CRDs of prototype galectins. (A) The homo-dimer of galectin-1/LacNAc (1W6P) is illustrated with a sche- matic diagram showing the spatial arrangement of the two CRDs. The β-sheet on the back of the CRD is shown in a dark color. (B) The homo-dimer of galectin-7/Gal (2GAL) is illustrated. (C) The homo-dimer of galectin-10/Man (1QKQ) is illustrated. (D) The homo-tetramer of Xenopus laevis skin galectin Va (3WUC) is illustrated.
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Fig. 5. Spatial arrangements of CRDs of tandem-repeat type galectins. (A) Galectin-9Null_R221S/Lac (3WV6) is illustrated. The N-CRD, C-CRD and linker are shown in blue, pink and yellow, respectively, and the β-sheet on the back of the CRD is shown in a dark color. (B) Porcine Ad- enovirus Type 4 galectin domain/Lac (2WSV) is illustrated. The C-CRD is shown in salmon pink. A LN3 molecule binding to the groove (2WT2) is superimposed. (C) Galectin-8Null/SiaLac/Lac (3VKM) is illustrated. The C-CRD is shown in green. (D) The homo-dimer of galectin-8Null/SiaLac/ Lac is illustrated. (E) Galectin-8Null/NDP52-peptide (4HAN) is illustrated. (F) The homo-dimer of galectin-8Null/NDP52-peptide is illustrated.
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type branched oligosaccharide is also able to crosslink with galec- tins.
The X-ray structure of bovine galectin-1 in complex with
BIOS was reported with three crystal forms, hexagonal, trigonal and monoclinic, and the structure in the hexagonal form (bovine galectin-1/BIOS (1SLA)) is shown in Fig. 3A (7). In this structure,
Table 1. Geometrical parameters for spatial arrangements of the two CRDs.
CRD orientation Solvent-accessible
Solvent-accessible surface area of the
2nd CRD (2)
solvent-accessible surface area (%)
Distance between carbohydrate
recognition sitesa ()
Interface area (2)
(Mol-A) 6784
(Mol-A) 7077
(Mol-A) 6773
Side-to-side 581 6840
(5GM0) Back-to-back
Face-to-face 671 7287
(3WV6) Back-to-back
Back-to-back 417 7011
(C-CRD, Mol-A) 7297
Galectin-8Null/NDP52- peptide (4HAN)
Back-to-side 391 8636
(N-CRD, Mol-A) 7252
(N-CRD, Mol-B) 8575 10.4 —
a The distance is defined as the distance between the O4 atoms of Gal+1 (Man+1 for galectin-10/Man) of the bound ligand molecules at the two CRDs. In rat galectin-5 and galectin-8Null/NDP52-peptide (4HAN), there is no oligosaccharide to bind.
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the S4–S5 loop (shown in red), including His52 and Gly53, over- laps BIOS, creating a deep carbohydrate-binding site. His52 and Trp68 sandwich Gal+1 and GlcNAc+2 to fix their positions, and Gly53 efficiently forms van der Waals contacts with Man3+ and Man+4.
The structure of galectin-9_C-CRD/BIOS (3NV3) is shown in Fig. 3B (17). As the electron density of Man+4 and GlcNAc+5 was invisible due to high disorder, they were modeled. The S4–S5 loop, including Asp241 and Glu242, is not directed toward BIOS, creating a shallow carbohydrate-binding site. The entrance for the ligand-binding of galectin-9_C-CRD is widely opened, and Man+3 and Man+4 of BIOS are free from the protein without any direct interactions. Galectin-9_N-CRD also has a similar S4–S5 loop structure with galectin-9_C-CRD, creating a shallow carbohydrate- binding site.
Bovine galectin-1 with a deep carbohydrate-binding site can form stable protein–ligand complexes of low structural energy through many attractive interactions, compared with galectin-9 CRDs. However, the conformation of the bound oligosaccharide may be restricted by strong protein–ligand interactions. Indeed, the extended conformation of the bound oligosaccharides was only found in X-ray structures of bovine galectin-1/BIOS in three crys- tal forms (Fig. 3A, right). In the case of galectin-9 CRDs, protein– ligand interactions were limited to Gal+1 and GlcNAc+2, and other sugar units were free from the protein, meaning that galectin-9 CRDs recognizes the antennae of a branched oligossacharide in several conformations (Fig. 3B, right). In galectin-9 CRDs, the less structural energy in the formation of a protein–ligand complex is probably compensated for by the ability to accept branched oligos- sacharides in different conformations. This may be one of the rea- sons why galectin-9_N-CRD and C-CRD have high affinities for N-glycan-type branched oligossacharides (17).
The X-ray structure of galectin-7 in complex with synthetic galactose-based dendron with three arms (galectin-7/D2 (4UW5)) was reported (13). In this structure, each galactose-terminus of the three arms of D2 is recognized by one galectin-7 molecule (Fig. 3C). The prototype galectin-7 forms a homo-dimer. Thus, D2 links three molecules of galectin-7, and the dimer partner of these galec- tin-7 molecules binds to another D2, likely forming supramolecu- lar assemblies with a lattice structure. (Fig. 3C, right) (13, 24).
D. Spatial Arrangements of CRDs of Prototype Galectins In the X-ray structures of prototype galectins, three spatial
arrangements of CRDs were found, the side-to-side, the back-to- back, and the face-to-face orientations, to form a homo-dimer and a homo-tetramer. Figure 4 shows their structures with schematic diagrams. Geometrical parameters for spatial arrangements of
CRDs are listed in Table 1. Galectin-1 (1W6P) (8) and galectin-2 (1HLC) (9) form a
homo-dimer with a 2-fold symmetry, making contacts between the right sides of the CRDs (the side-to-side orientation) (Fig. 4A). On dimerization, pairs of the same β-sheets are connected to give two large antiparallel β-sheets with an interface area of 620 2. Two carbohydrate-binding sites are located on the same side of the homo-dimer and separated from each other by 41 .
Galectin-7 (2GAL) (12) forms a homo-dimer with a 2-fold symmetry, making contact between the β-sheets on the back (the back-to-back orientation) with an interface area of 768 2. Two carbohydrate-binding sites are located at both ends of the homo- dimer with a distance of 50 , facing opposite each other.
In the X-ray structure of galectin-10 (1QKQ) (18), CRDs re- lated by a crystallographic 2-fold symmetry are associated in the face-to-face orientation with an interface area of 821 2 (Fig. 4C). The distance between the two carbohydrate-binding sites is 17 . However, it is still unclear whether galectin-10 forms a homo- dimer in the face-to-face orientation in solution. The X-ray struc- ture of the rat galectin-5 (5JPG), recently released on PDB, was a homo-dimer in the face-to-face orientation.
The Xenopus laevis skin galectin Va (3WUC) (25) and the marine sponge (Cinachyrella sp.) galectin (4AGR) (26) form homo-tetramers in which two homo-dimers in the side-to-side ori- entation are associated in the back-to-back orientation (Fig. 4D). As four carbohydrate-binding sites are located on the solvent-ac- cessible surface of the homo-tetramer, these galectins are expected to be tetravalent in glycan binding.
E. Spatial Arrangements of CRDs of Tandem-Repeat Type Galectins
In the X-ray structures of tandem-repeat type galectins having two CRDs, three spatial arrangements of CRDs were found, the back-to-back, the face-to-face, and the back-to-side orientations, as shown in Fig. 5.
In the X-ray structure of the protease-resistant mutant form of galectin-9 with a short linker of 19 amino acid residues and the replacement of Arg221 by Ser (galectin-9Null_R221S (3WV6)) (22), the two CRDs are associated, making contact between the β-sheets on the back with many hydrophobic interactions (the back-to-back orientation) (Fig. 5A). Compared with the homo- dimer of galectin-7 in the back-to-back orientation, the two CRDs of galectin-9Null_R221S are distorted from the 2-fold symmetry, giving a small interface area of 632 2 and a distance between two carbohydrate-binding sites of 47 (Table 1). The back surfaces of the galectin-9 CRDs exhibited high hydrophobicity with low solubility (27). These hydrophobic residues are buried between
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the two CRDs in the back-to-back orientation, giving a favorable structure to the protein in solution. The tandem-repeat type Toxas- caris leonina galectin (5GM0) (19) with 34% amino acid sequence similarity with galectin-9 also adopts the back-to-back orientation with hydrophobic interactions as found in galectin-9Null_R221S.
The Porcine Adenovirus Type 4 has a fiber protein contain- ing a tandem-repeat type galectin domain. In the X-ray structure of this galectin domain (2WSV) (28), the face-to-face orientation was observed (Fig. 5B). Compared with the homo-dimer of ga- lectin-10 in the face-to-face orientation, the two CRDs are largely distorted from the 2-fold symmetry with a small interface area of 671 2, to form the deep groove for ligand-binding between the two CRDs. Two carbohydrate-binding sites approach the distance of 12 , exposing the solvent-accessible surface. As a long oligo- lactosamine (LN3) was found to bind the groove between the two CRDs (2WT2), the face-to-face orientation of the two CRDs was proposed to allow both CRDs to interact with the same oligosac- charide in the recognition of complex sugars (28).
In the X-ray structure of the protease-resistant mutant form of galectin-8 with a short linker of 7 amino acid residues, in which the N-CRD recognized SiaLac and the C-CRD recognized Lac, (galectin-8Null/SiaLac/Lac (3VKM)) (21), the two CRDs are as- sociated in the back-to-side orientation (Fig. 5C). The N-CRD of G8Null has two additional β-strands at the N-terminal site, F01N and F02N, and F01N interacts with S1C, to participate in a β-sheet on the face of C-CRD, giving the back-to-side orientation. The car- bohydrate-binding sites make a right angle with each other at a dis- tance of 55 . In crystal, two molecules of galectin-8Null possibly form a dimer, in which the β-sheets on the back of the C-CRDs face each other to form an interface (the back-to-back orienta- tion) (Fig. 5D). Four carbohydrate-binding sites are located on the solvent-accessible surface of the dimer. The formation of dimeric species of galectin-8 was reported to be related with its biological function (29). The back-to-side orientation found in galectin-8Null may be favorable for forming a dimeric structure.
Galectin-8 was reported to activate antibacterial autophagy by interacting with the autophagic receptor NDP52 (30), and the X-ray structure of galectin-8Null in complex with the peptide frag- ment of NDP52 (galectin-8Null/NDP52-peptide (4HAN)) was reported (31). These results clearly demonstrate the roles of galec- tin-8 in the cytosol. In the galectin-8Null/NDP52-peptide, the two CRDs are associated in the back-to-side orientation, and an addi- tional β-strand, F0N, interacts with F1C, to participate in a β-sheet on the back of C-CRD (Fig. 5E). The additional β-strand F0N also interacts with that of another molecule to form a dimer, making contact between the β-sheets on the back of the N-CRDs (the back- to-back orientation) (Fig. 5F). The two C-CRDs recognize the
NDP52-peptides on the back side. Unexpectedly, an NAD from the crystallization solution was located at one of the carbohydrate- binding sites (N-CRD of Mol-A).
Galectin-8 may form two types of dimeric structures by mak- ing contact between N-CRDs or between C-CRDs. In both forms, four carbohydrate-binding sites are located on the solvent-accessi- ble surface of the dimer, to be tetravalent in glycan binding.
F. Conclusion Galectin CRDs recognize the β-galactoside moiety through
protein–ligand interactions by the conserved galectin signature amino acids. Each galectin CRD exhibits particular preference of oligosaccharide-binding by unique amino acid residues on each galectin CRD such as Arg59 of galectin-8_N-CRD and Ala46 of galectin-9_N-CRD (Fig. 2B). The structural comparison between bovine galectin-1/BIOS and galectin-9_C-CRD/BIOS suggested that the shallow carbohydrate-binding site of galectin-9 CRDs is related with the high affinity for N-glycan-type branched oligossa- charides (Figs. 3A, B).
The spatial arrangements of the prototype galectins are sym- metric (Fig. 4), whereas those of the tandem-repeat type galectins are distorted from the symmetry with less interactions between CRDs (Fig. 5 and Table 1). Tandem-repeat type galectins with a linker may flexibly change the spatial arrangements of their two CDRs depending on their biological roles (Fig. 3B, right).
The structure of galectin-7/D2 suggested that galectin-7 and the ligand with three arms form supramolecular assemblies with a lattice structure (Fig. 3C). The Xenopus laevis skin galectin Va (Fig. 4D) and galectin-8 (Figs. 5D, F) may be tetravalent in glycan binding. Furthermore, the back of galectin-8_C-CRD targets the peptide ligand (Figs. 5E, F). It will be very interesting to elucidate the unknown molecular mechanisms underlying the biological functions of galectins.
Acknowledgments The author thanks Dr. Hiromi Yoshida, Dr. Nozomu Nishi,
Dr. Shin-ichi Nakakita, and Dr. Yasuhiro Nonaka for their useful discussions and critical reading of the manuscript. The research performed by the author et al. was supported in part by Grants-in- Aid for Scientific Research (23370054) from the Japan Society for the Promotion of Science (JSPS), and the fund for Characteristic Prior Research from Kagawa University.
Abbreviations CRD — carbohydrate recognition domain N-CRD — N-terminal CRD C-CRD — C-terminal CRD
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galectin-8_N-CRD — N-CRD of galectin-8 galectin-9_N-CRD — N-CRD of galectin-9 galectin-9_C-CRD — C-CRD of galectin-9 Lac — lactose LacNAc — N-acetyllactosamine
SiaLac — α (2-3)-sialyllactose SiaLacNAc — α (2-3)-N-acetylsialyllactosamine LN3 — tri(N-acetyllactosamine) BIOS — biantennary oligosaccharide D2 — synthetic galactose-based dendron
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Shigehiro Kamitori: Graduated from Osaka City University in 1984, and received a Ph.D. in chemistry from the graduate school of Osaka City University in 1989. He worked as a research associate at Kyowa Hakko Kogyo Co., Ltd. (1989–1991), as a postdoctoral fellow at the University of Kansas (1991–1994), and as an associate professor at the Tokyo University of Agriculture and Technology (1994–2004), and became a professor at the Life Science Research Center and Faculty of Medicine, Kagawa University since 2004. His current research interests are the structure–function relationship of carbohydrate-binding proteins and the catalytic reaction mechanisms of sugar isomerases, as deduced from X-ray structures.
Information of the Authors
Trends in Glycoscience and Glycotechnology Vol. 30 No. 172 (January–May 2018) pp. SE41–SE50
Three-Dimensional Structures of Galectins
Shigehiro Kamitori Life Science Research Center and Faculty of Medicine, Kagawa University, 1750–1, Ikenobe, Miki-cho, Kita-gun, Kagawa 761–0793, Japan
FAX: +81–87–891–2421, E-mail: [email protected]
(Received on August 18, 2017, accepted on October 5, 2017)
Key Words: carbohydrate recognition domain, oligosaccharide, prototype galectin, tandem-repeat type galectin, X-ray crystal structure
Abstract The galectins are a family of β-galactoside-specific animal lectins that contain a conserved carbohydrate recognition domain
(CRD) with approximately 140 amino acid residues. There are 14 members in the mammalian galectin family (galectin-1–10, and 11– 15), and they have different specificities for oligosaccharides. X-ray structures of the galectin CRD in complexes with oligosaccha- rides have provided important clues about the oligosaccharide-recognition mechanisms of galectins giving the different specificities. Galectin is divalent in glycan binding due to the association of two CRDs that crosslink with oligosaccharides. The spatial arrange- ment of the two CRDs is very important for elucidating the biological functions of galectins. Several different spatial arrangements of CRDs are found in X-ray structures of galectins. I herein examined the three-dimensional structures of galectins relevant for biological functions, based on the protein–ligand interactions related with oligosaccharide-specificity, the cross-linking structure by galectin and oligosaccharides, and the spatial arrangements of CRDs.
A. Introduction The galectins are a family of β-galactoside-specific animal
lectins that contain a conserved carbohydrate recognition domain (CRD) with approximately 140 amino acid residues, and have at- tracted much attention as novel regulators of the immune system (1, 2). There are 14 members in the galectin family (galectin-1–10, and 12–15), which are classified into three subtypes based on structure. The prototypes (galectin-1, 2, 5, 7, 10, 13, 14 and 15) have a single CRD. The chimera type (galectin-3) has a single CRD and a non-lectin N-terminal domain. The tandem-repeat types (galectin-4, 6, 8, 9, and 12) have two different CRDs in the N- and C-terminal regions (N-CRD and C-CRD) that are joined by a linker peptide (Fig. 1A). The prototype galectins mostly form homo-dimers, and the chimera type galectin is expected to form an oligomer based on its non-lectin N-terminal domain. Thus, ga- lectins are divalent and/or multivalent in glycan binding. The most well-characterized role of galectins is crosslinking with oligosac- charides in the extracellular space, which is involved in cell–cell and cell–matrix interactions. Recently, additional roles of galectins in the cytosol have attracted interest (3, 4).
Galectin CRDs have different specificities for oligosaccha- rides. The N-CRD of galectin-8 (galectin-8_N-CRD) exhibits a strong affinity for α(2-3)-sialylated oligosaccharides, but the C- CRD does not. The N-CRD of galectin-9 (galectin-9_N-CRD) has high affinity for oligolactosamines with a linear structure, but the C-CRD does not. Both the galectin-9_N-CRD and C-CRD have high affinities for N-glycan-type branched oligosaccharides (bi- anntenary oligosaccharides) (5). Many X-ray structures of galectin CRDs in complexes with oligosaccharides have been deposited into PDB (6), and have provided important clues about the oligo-
saccharide-recognition mechanisms of galectins giving the differ- ent specificities (7–18). Selected oligosaccharides and a synthetic ligand used in structural studies of galectin CRDs are listed in Fig. 1B with their abbreviations.
Most of the reported X-ray structures of galectins contained only a single type of CRD. The homo-dimer structures of the prototype galectins were found to show the spatial arrangements of CRDs. However, there was no information about the spatial ar- rangement of the CRDs of chimera type and tandem-repeat type galectins. Tandem-repeat type galectins are inherently divalent in glycan binding with different specificities, and structural informa- tion about the spatial arrangement of the two CRDs is very impor- tant for elucidating their biological functions. Tandem-repeat type galectins are sensitive to proteases due to the long linker. To carry out X-ray crystal structure determinations of tandem-repeat type galectins with two CRDs, the galectins with a short linker(19) and/ or the protease-resistant mutant forms with a modified linker pep- tide were used (20–22).
In this review, I examined three-dimensional structures of galectins based on the protein–ligand interactions related with oligosaccharide-specificity, the cross-linking structure by galectin and oligosaccharides, and the spatial arrangements of CRDs of the prototype and tandem-repeat type galectins. Galectin-1, 2, 7, 8, 9, 10 stand for human galectin-1, 2, 7, 8, 9, 10, respectively (unless stated otherwise). Figures 2, 3, 4, 5 were drawn with the program PyMOL(23).
B. Three-Dimensional Structure of Galectin CRD The overall structure of galectin-9_C-CRD in complex with
LacNAc (galectin-9_C-CRD/LacNAc (PDB ID: 3NV2)) (17) is
MINIREVIEW doi: 10.4052/tigg.1731.1SE(Article for special issue on Galectins)
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Fig. 1. Three subtypes of galectins and ligands used in structural studies of galectins. (A) Schematic diagrams showing the three subtypes of galectins are illustrated. (B) Chemical structures of oligosaccharides and a synthetic ligand are illustrated with their abbreviations.
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shown in Fig. 2A. The galectin-9_C-CRD adopts a β-sandwich structure formed by two anti-parallel β-sheets consisting of six (S1–S6) and five (F1–F5) β-strands, respectively. The strand S6 is divided into two strands (S6a and S6b). A short helix (H1) exists between F5 and S2. The carbohydrate-binding sites are exposed to the solvent-accessible surfaces of the molecule, and a LacNAc binds to the concave surfaces formed by S3, S4, S5 and S6, via non-reducing and reducing ends located at S3 to S6. The overall structure is well conserved among galectin CRDs, and can be represented by a tetragonal prism. For clarity, the face, back, top, bottom and sides of the CRD are defined as in Fig. 2A (right). The
carbohydrate-binding site is on the face of the CRD, and the oppo- site side is the back. The short helix (H1) is on the bottom and the opposite side is the top. The β-strand of S1 is on the right side and S6 is on the left side.
Three structures of galectin CRDs in complexes with oligo- saccharides are shown in Fig. 2B. Sugar units of the bound oligo- saccharides are numbered −1, +1, and +2, from the non-reducing end to reducing end (Fig. 1B). Gal+1 occupies the same position in each complex.
In galectin-9_C-CRD/LacNAc (3NV2) (17), the galactose moiety (Gal+1) forms stacking interactions with Trp255, and forms
Fig. 2. X-ray structures of galectin CRDs with the bound oligosaccharides. (A) Overall structure of galectin-9_C-CRD/LacNAc (3NV2) is illustrated as viewed from the face (left) and the left side (middle). The β-sheet on the back of the CRD is shown in a dark color, and a short helix (H1) is shown in red. A schematic diagram of tetragonal prisms showing the CRD is illustrated (right). The back, bottom and right sides of tetragonal prisms are shown in gray. (B) Galectin-9_C-CRD/LacNAc (3NV2) (left), Galectin-8_N-CRD/SiaLacNAc (3VKO) (middle) and Galectin-9_N-CRD/ LN3 (2ZHM) (right) are illustrated with the protein–ligand interactions.
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six hydrogen bonds with the protein: O4-His235, O4-Asn237, O4-Arg239, O5-Arg239, O6-Asn248 and O6-Glu258. The axial conformation of the O4 of Gal+1 is strictly recognized by three hy-
drogen bonds from His235, Asn237 and Arg239. The glucosamine moiety (GlcNAc+2) forms hydrogen bonds with the protein by O3: O3-Arg239, O3-Glu258, and O3-Arg260. As Arg239 and Glu258
Fig. 3. Cross-linking structures by galectins and oligosaccharides. (A) Bovine galectin-1/BIOS (1SLA) is illustrated. The S4–S5 loop is shown in red. A schematic diagram showing the cross-linked structure is illustrated (right). (B) Galectin-9_C-CRD/BIOS (3NV3) is illustrated. The modeled GlcNAc+5 and Man+4 are shown in blue. (C) Galectin-7/D2 (4UW5) is illustrated.
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form bifurcated hydrogen bonds with both Gal+1 and GlcNAc+2, they efficiently recognize the β (1-4) glycoside bond of LacNAc. The protein–ligand interactions found in galectin-9_C-CRD/ LacNAc are conserved in galectin CRD/oligosaccharide complex structures.
In galectin-8_N-CRD/SiaLacNAc (3VKO) (21), in addition to the conserved protein–ligand interactions, Arg59 forms ef- ficient salt–bridge interactions with the carboxyl group of Sia−1, and Gln47 and Trp86 hold the carboxyl group from both sides via hydrogen bonds. Arg59 is unique to galectin-8_N-CRD, and may be responsible for the strong affinity for α(2-3)-sialylated oligosac- charides.
In galectin-9_N-CRD/LN3 (2ZHM) (16), the bound LN3 has a linear structure which enables GlcNAc−1 to form a hydrogen bond with Asn48. Furthermore, Ala46, which is unique to galec- tin-9_N-CRD, is proposed to be one of the residues responsible for the high affinity for oligolactosamines (13). This is because an amino acid residue with bulky side chain group at the position of Ala46 (His223 in galectin-9_C-CRD and/or Gln47 in galectin-8_ N-CRD) causes steric hindrance with GlcNAc−1 of LN3.
C. Cross-Linking Structure by Galectin and Oligosac- charides
Galectin crosslinks with oligosaccharides, and the N-glycan-
Fig. 4. Spatial arrangements of CRDs of prototype galectins. (A) The homo-dimer of galectin-1/LacNAc (1W6P) is illustrated with a sche- matic diagram showing the spatial arrangement of the two CRDs. The β-sheet on the back of the CRD is shown in a dark color. (B) The homo-dimer of galectin-7/Gal (2GAL) is illustrated. (C) The homo-dimer of galectin-10/Man (1QKQ) is illustrated. (D) The homo-tetramer of Xenopus laevis skin galectin Va (3WUC) is illustrated.
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Fig. 5. Spatial arrangements of CRDs of tandem-repeat type galectins. (A) Galectin-9Null_R221S/Lac (3WV6) is illustrated. The N-CRD, C-CRD and linker are shown in blue, pink and yellow, respectively, and the β-sheet on the back of the CRD is shown in a dark color. (B) Porcine Ad- enovirus Type 4 galectin domain/Lac (2WSV) is illustrated. The C-CRD is shown in salmon pink. A LN3 molecule binding to the groove (2WT2) is superimposed. (C) Galectin-8Null/SiaLac/Lac (3VKM) is illustrated. The C-CRD is shown in green. (D) The homo-dimer of galectin-8Null/SiaLac/ Lac is illustrated. (E) Galectin-8Null/NDP52-peptide (4HAN) is illustrated. (F) The homo-dimer of galectin-8Null/NDP52-peptide is illustrated.
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type branched oligosaccharide is also able to crosslink with galec- tins.
The X-ray structure of bovine galectin-1 in complex with
BIOS was reported with three crystal forms, hexagonal, trigonal and monoclinic, and the structure in the hexagonal form (bovine galectin-1/BIOS (1SLA)) is shown in Fig. 3A (7). In this structure,
Table 1. Geometrical parameters for spatial arrangements of the two CRDs.
CRD orientation Solvent-accessible
Solvent-accessible surface area of the
2nd CRD (2)
solvent-accessible surface area (%)
Distance between carbohydrate
recognition sitesa ()
Interface area (2)
(Mol-A) 6784
(Mol-A) 7077
(Mol-A) 6773
Side-to-side 581 6840
(5GM0) Back-to-back
Face-to-face 671 7287
(3WV6) Back-to-back
Back-to-back 417 7011
(C-CRD, Mol-A) 7297
Galectin-8Null/NDP52- peptide (4HAN)
Back-to-side 391 8636
(N-CRD, Mol-A) 7252
(N-CRD, Mol-B) 8575 10.4 —
a The distance is defined as the distance between the O4 atoms of Gal+1 (Man+1 for galectin-10/Man) of the bound ligand molecules at the two CRDs. In rat galectin-5 and galectin-8Null/NDP52-peptide (4HAN), there is no oligosaccharide to bind.
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the S4–S5 loop (shown in red), including His52 and Gly53, over- laps BIOS, creating a deep carbohydrate-binding site. His52 and Trp68 sandwich Gal+1 and GlcNAc+2 to fix their positions, and Gly53 efficiently forms van der Waals contacts with Man3+ and Man+4.
The structure of galectin-9_C-CRD/BIOS (3NV3) is shown in Fig. 3B (17). As the electron density of Man+4 and GlcNAc+5 was invisible due to high disorder, they were modeled. The S4–S5 loop, including Asp241 and Glu242, is not directed toward BIOS, creating a shallow carbohydrate-binding site. The entrance for the ligand-binding of galectin-9_C-CRD is widely opened, and Man+3 and Man+4 of BIOS are free from the protein without any direct interactions. Galectin-9_N-CRD also has a similar S4–S5 loop structure with galectin-9_C-CRD, creating a shallow carbohydrate- binding site.
Bovine galectin-1 with a deep carbohydrate-binding site can form stable protein–ligand complexes of low structural energy through many attractive interactions, compared with galectin-9 CRDs. However, the conformation of the bound oligosaccharide may be restricted by strong protein–ligand interactions. Indeed, the extended conformation of the bound oligosaccharides was only found in X-ray structures of bovine galectin-1/BIOS in three crys- tal forms (Fig. 3A, right). In the case of galectin-9 CRDs, protein– ligand interactions were limited to Gal+1 and GlcNAc+2, and other sugar units were free from the protein, meaning that galectin-9 CRDs recognizes the antennae of a branched oligossacharide in several conformations (Fig. 3B, right). In galectin-9 CRDs, the less structural energy in the formation of a protein–ligand complex is probably compensated for by the ability to accept branched oligos- sacharides in different conformations. This may be one of the rea- sons why galectin-9_N-CRD and C-CRD have high affinities for N-glycan-type branched oligossacharides (17).
The X-ray structure of galectin-7 in complex with synthetic galactose-based dendron with three arms (galectin-7/D2 (4UW5)) was reported (13). In this structure, each galactose-terminus of the three arms of D2 is recognized by one galectin-7 molecule (Fig. 3C). The prototype galectin-7 forms a homo-dimer. Thus, D2 links three molecules of galectin-7, and the dimer partner of these galec- tin-7 molecules binds to another D2, likely forming supramolecu- lar assemblies with a lattice structure. (Fig. 3C, right) (13, 24).
D. Spatial Arrangements of CRDs of Prototype Galectins In the X-ray structures of prototype galectins, three spatial
arrangements of CRDs were found, the side-to-side, the back-to- back, and the face-to-face orientations, to form a homo-dimer and a homo-tetramer. Figure 4 shows their structures with schematic diagrams. Geometrical parameters for spatial arrangements of
CRDs are listed in Table 1. Galectin-1 (1W6P) (8) and galectin-2 (1HLC) (9) form a
homo-dimer with a 2-fold symmetry, making contacts between the right sides of the CRDs (the side-to-side orientation) (Fig. 4A). On dimerization, pairs of the same β-sheets are connected to give two large antiparallel β-sheets with an interface area of 620 2. Two carbohydrate-binding sites are located on the same side of the homo-dimer and separated from each other by 41 .
Galectin-7 (2GAL) (12) forms a homo-dimer with a 2-fold symmetry, making contact between the β-sheets on the back (the back-to-back orientation) with an interface area of 768 2. Two carbohydrate-binding sites are located at both ends of the homo- dimer with a distance of 50 , facing opposite each other.
In the X-ray structure of galectin-10 (1QKQ) (18), CRDs re- lated by a crystallographic 2-fold symmetry are associated in the face-to-face orientation with an interface area of 821 2 (Fig. 4C). The distance between the two carbohydrate-binding sites is 17 . However, it is still unclear whether galectin-10 forms a homo- dimer in the face-to-face orientation in solution. The X-ray struc- ture of the rat galectin-5 (5JPG), recently released on PDB, was a homo-dimer in the face-to-face orientation.
The Xenopus laevis skin galectin Va (3WUC) (25) and the marine sponge (Cinachyrella sp.) galectin (4AGR) (26) form homo-tetramers in which two homo-dimers in the side-to-side ori- entation are associated in the back-to-back orientation (Fig. 4D). As four carbohydrate-binding sites are located on the solvent-ac- cessible surface of the homo-tetramer, these galectins are expected to be tetravalent in glycan binding.
E. Spatial Arrangements of CRDs of Tandem-Repeat Type Galectins
In the X-ray structures of tandem-repeat type galectins having two CRDs, three spatial arrangements of CRDs were found, the back-to-back, the face-to-face, and the back-to-side orientations, as shown in Fig. 5.
In the X-ray structure of the protease-resistant mutant form of galectin-9 with a short linker of 19 amino acid residues and the replacement of Arg221 by Ser (galectin-9Null_R221S (3WV6)) (22), the two CRDs are associated, making contact between the β-sheets on the back with many hydrophobic interactions (the back-to-back orientation) (Fig. 5A). Compared with the homo- dimer of galectin-7 in the back-to-back orientation, the two CRDs of galectin-9Null_R221S are distorted from the 2-fold symmetry, giving a small interface area of 632 2 and a distance between two carbohydrate-binding sites of 47 (Table 1). The back surfaces of the galectin-9 CRDs exhibited high hydrophobicity with low solubility (27). These hydrophobic residues are buried between
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the two CRDs in the back-to-back orientation, giving a favorable structure to the protein in solution. The tandem-repeat type Toxas- caris leonina galectin (5GM0) (19) with 34% amino acid sequence similarity with galectin-9 also adopts the back-to-back orientation with hydrophobic interactions as found in galectin-9Null_R221S.
The Porcine Adenovirus Type 4 has a fiber protein contain- ing a tandem-repeat type galectin domain. In the X-ray structure of this galectin domain (2WSV) (28), the face-to-face orientation was observed (Fig. 5B). Compared with the homo-dimer of ga- lectin-10 in the face-to-face orientation, the two CRDs are largely distorted from the 2-fold symmetry with a small interface area of 671 2, to form the deep groove for ligand-binding between the two CRDs. Two carbohydrate-binding sites approach the distance of 12 , exposing the solvent-accessible surface. As a long oligo- lactosamine (LN3) was found to bind the groove between the two CRDs (2WT2), the face-to-face orientation of the two CRDs was proposed to allow both CRDs to interact with the same oligosac- charide in the recognition of complex sugars (28).
In the X-ray structure of the protease-resistant mutant form of galectin-8 with a short linker of 7 amino acid residues, in which the N-CRD recognized SiaLac and the C-CRD recognized Lac, (galectin-8Null/SiaLac/Lac (3VKM)) (21), the two CRDs are as- sociated in the back-to-side orientation (Fig. 5C). The N-CRD of G8Null has two additional β-strands at the N-terminal site, F01N and F02N, and F01N interacts with S1C, to participate in a β-sheet on the face of C-CRD, giving the back-to-side orientation. The car- bohydrate-binding sites make a right angle with each other at a dis- tance of 55 . In crystal, two molecules of galectin-8Null possibly form a dimer, in which the β-sheets on the back of the C-CRDs face each other to form an interface (the back-to-back orienta- tion) (Fig. 5D). Four carbohydrate-binding sites are located on the solvent-accessible surface of the dimer. The formation of dimeric species of galectin-8 was reported to be related with its biological function (29). The back-to-side orientation found in galectin-8Null may be favorable for forming a dimeric structure.
Galectin-8 was reported to activate antibacterial autophagy by interacting with the autophagic receptor NDP52 (30), and the X-ray structure of galectin-8Null in complex with the peptide frag- ment of NDP52 (galectin-8Null/NDP52-peptide (4HAN)) was reported (31). These results clearly demonstrate the roles of galec- tin-8 in the cytosol. In the galectin-8Null/NDP52-peptide, the two CRDs are associated in the back-to-side orientation, and an addi- tional β-strand, F0N, interacts with F1C, to participate in a β-sheet on the back of C-CRD (Fig. 5E). The additional β-strand F0N also interacts with that of another molecule to form a dimer, making contact between the β-sheets on the back of the N-CRDs (the back- to-back orientation) (Fig. 5F). The two C-CRDs recognize the
NDP52-peptides on the back side. Unexpectedly, an NAD from the crystallization solution was located at one of the carbohydrate- binding sites (N-CRD of Mol-A).
Galectin-8 may form two types of dimeric structures by mak- ing contact between N-CRDs or between C-CRDs. In both forms, four carbohydrate-binding sites are located on the solvent-accessi- ble surface of the dimer, to be tetravalent in glycan binding.
F. Conclusion Galectin CRDs recognize the β-galactoside moiety through
protein–ligand interactions by the conserved galectin signature amino acids. Each galectin CRD exhibits particular preference of oligosaccharide-binding by unique amino acid residues on each galectin CRD such as Arg59 of galectin-8_N-CRD and Ala46 of galectin-9_N-CRD (Fig. 2B). The structural comparison between bovine galectin-1/BIOS and galectin-9_C-CRD/BIOS suggested that the shallow carbohydrate-binding site of galectin-9 CRDs is related with the high affinity for N-glycan-type branched oligossa- charides (Figs. 3A, B).
The spatial arrangements of the prototype galectins are sym- metric (Fig. 4), whereas those of the tandem-repeat type galectins are distorted from the symmetry with less interactions between CRDs (Fig. 5 and Table 1). Tandem-repeat type galectins with a linker may flexibly change the spatial arrangements of their two CDRs depending on their biological roles (Fig. 3B, right).
The structure of galectin-7/D2 suggested that galectin-7 and the ligand with three arms form supramolecular assemblies with a lattice structure (Fig. 3C). The Xenopus laevis skin galectin Va (Fig. 4D) and galectin-8 (Figs. 5D, F) may be tetravalent in glycan binding. Furthermore, the back of galectin-8_C-CRD targets the peptide ligand (Figs. 5E, F). It will be very interesting to elucidate the unknown molecular mechanisms underlying the biological functions of galectins.
Acknowledgments The author thanks Dr. Hiromi Yoshida, Dr. Nozomu Nishi,
Dr. Shin-ichi Nakakita, and Dr. Yasuhiro Nonaka for their useful discussions and critical reading of the manuscript. The research performed by the author et al. was supported in part by Grants-in- Aid for Scientific Research (23370054) from the Japan Society for the Promotion of Science (JSPS), and the fund for Characteristic Prior Research from Kagawa University.
Abbreviations CRD — carbohydrate recognition domain N-CRD — N-terminal CRD C-CRD — C-terminal CRD
© 2018 FCCA (Forum: Carbohydrates Coming of Age) SE50
galectin-8_N-CRD — N-CRD of galectin-8 galectin-9_N-CRD — N-CRD of galectin-9 galectin-9_C-CRD — C-CRD of galectin-9 Lac — lactose LacNAc — N-acetyllactosamine
SiaLac — α (2-3)-sialyllactose SiaLacNAc — α (2-3)-N-acetylsialyllactosamine LN3 — tri(N-acetyllactosamine) BIOS — biantennary oligosaccharide D2 — synthetic galactose-based dendron
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Shigehiro Kamitori: Graduated from Osaka City University in 1984, and received a Ph.D. in chemistry from the graduate school of Osaka City University in 1989. He worked as a research associate at Kyowa Hakko Kogyo Co., Ltd. (1989–1991), as a postdoctoral fellow at the University of Kansas (1991–1994), and as an associate professor at the Tokyo University of Agriculture and Technology (1994–2004), and became a professor at the Life Science Research Center and Faculty of Medicine, Kagawa University since 2004. His current research interests are the structure–function relationship of carbohydrate-binding proteins and the catalytic reaction mechanisms of sugar isomerases, as deduced from X-ray structures.
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