X-ray structures of human galectin-9 C-terminal domain in complexes with a biantennary oligosaccharide and sialyllactose

Abstract

Galectin-9, a tandem-repeat-type β-galactoside-specific animal lectin with two carbohydrate recognition domains (CRDs) at the N- and C-terminal ends, is involved in chemoattraction, apoptosis, and the regulation of cell differentiation and has anti-allergic effects. Its ability to recognize carbohydrates is essential for its biological functions. Human galectin-9 (hG9) has high affinity for branched N-glycan-type oligosaccharides (dissociation constants of 0.16-0.70 μM) and linear β1-3-linked poly-N-acetyllactosamines (0.09-8.3 μM) and significant affinity for the α2-3-sialylated oligosaccharides (17-34 μM). Further, its N-terminal CRD (hG9N) and C-terminal CRD (hG9C) differ in specificity. To elucidate this unique feature of hG9, x-ray structures of hG9C in the free form and in complexes with N-acetyllactosamine, the biantennary pyridylaminated oligosaccharide, and α2-3-sialyllactose were determined. They are the first x-ray structural analysis of C-terminal CRD of the tandem-repeat-type galectin. The results clearly revealed the mechanism by which branched and α2-3-sialylated oligosaccharides are recognized and explained the difference in specificity between hG9N and hG9C. Based on structural comparisons with other galectins, we propose that the wide entrance for ligand binding and the shallow binding site of hG9C are favorable for branched oligosaccharides and that Arg(221) is responsible for recognizing sialylated oligosaccharides.

FIGURE 1.

The chemical structures of LacNAc, BIPA, and SiaLac. The names of sugar units with glucoside bonds and subsite numbers are indicated.

FIGURE 2.

Crystal structure of hG9C. a, the overall structure of hG9C is shown with the bound LacNAc (blue), BIPA (yellow), and SiaLac (green). Secondary elements and subsite numbers are also indicated. b, a trimer of hG9C with the three molecules (pink, yellow, and green) in a crystal is shown with a three-fold axis and 6₃ screw axes. Ni²⁺ on the three-fold axis, three coordinated His residues, and the bound LacNAc are also shown. c, area of contact between two molecules (pink and green) with intermolecular hydrogen bonds and metal coordination shown by dotted lines.

FIGURE 3.

Stereo views of the carbohydrate-binding sites of hG9C and a structural comparison between hG9C and hG9N. Selected hydrogen bonds with BIPA (a) and SiaLac (b) are shown by dotted lines. Although two conformers of Arg²²¹ are found in hG9C-SiaLac, only conformer-2 is shown in b for clarity. c, superimposition of hG9C (pink) and hG9N (cyan) with the bound BIPA (yellow), SiaLac (green), and LN3 (blue). The loops with large deviations are indicated by dark colors.

FIGURE 4.

Amino acid alignment among hG9N, hG9C, bG1, hG1, and hG8N, with the linker peptide sequence of hG9 short isoform (National Center for Biotechnology Information (NCBI) reference: NP_002299). Secondary elements are shown by arrows (β-strands) and a rectangle (α-helix). The loops with large deviations found between hG9N and hG9C are indicated by boxes. The positions of Arg²²¹ and Asp²⁴¹ in hG9C and of Arg⁶⁶ in hG8N are highlighted in black.

FIGURE 5.

The cross-linking of bG1 (PDB code: 1SLA, green) (a) and hG9C (pink) (b) by the biantennary oligosaccharides. Subsite numbers and selected amino acid residues are given. In b, the modeled sugar units at subsites +4 and +5 are shown in blue.