Affinity of galectin-8 and its carbohydrate recognition domains for ligands in solution and at the cell surface

Abstract

Galectin-8 has two different carbohydrate recognition domains (CRDs), the N-terminal Gal-8N and the C-terminal Gal-8C linked by a peptide, and has various effects on cell adhesion and signaling. To understand the mechanism for these effects further, we compared the binding activities of galectin-8 in solution with its binding and activation of cells. We used glycan array analysis to broaden the specificity profile of the two galectin-8 CRDs, as well as intact galectin-8s (short and long linker), confirming the unique preference for sulfated and sialylated glycans of Gal-8N. Using a fluorescence anisotropy assay, we examined the solution affinities for a subset of these glycans, the highest being 50 nM for NeuAcalpha2,3Lac by Gal-8N. Thus, carbohydrate-protein interactions can be of high affinity without requiring multivalency. More importantly, using fluorescence polarization, we also gained information on how the affinity is built by multiple weak interactions between different fragments of the glycan and its carrier molecule and the galectin CRD subsites (A-E). In intact galectin-8 proteins, the two domains act independently of each other in solution, whereas at a surface they act together. Ligands with moderate or weak affinity for the isolated CRDs on the array are bound strongly by intact galectin-8s. Also galectin-8 binding and signaling at cell surfaces can be explained by combined binding of the two CRDs to low or medium affinity ligands, and their highest affinity ligands, such as sialylated galactosides, are not required.

Structural basis for recognition of autophagic receptor NDP52 by the sugar receptor galectin-8

“…Previous in-depth studies of GAL8 showed that its N-CRD is a really unique domain for recognizing sulphated and sialylated sugars 19,28,29 . In our structure, the diphosphate moiety attached to the pentose ring in NAD binds to the sugar-binding site of N-CRD specifically.…”

Different fractions of human serum glycoproteins bind galectin-1 or galectin-8, and their ratio may provide a refined biomarker for pathophysiological conditions in cancer and inflammatory disease

“…haptoglobin so far, but this does not rule out Carlsson et al, page 14 that they exist in a fraction of it. Alternatively, galectin-8N binds NeuAcα2-3Galβ1-3GlcNAc, which it also prefers over the galectin-1 binding NeuAcα2-3Galβ1-4GlcNAc [20], and which may occur in N-glycans. Hence, galectin-1 and -8N may be detecting the balance between two different glycosylation features, which in turn would be determined by the activity of glycosyltransferases typical for the cell making the glycoprotein and further regulated by surrounding pathophysiological conditions.…”

“…In another study we found that the N-terminal carbohydrate recognition domain (CRD) 1 of galectin-8 (galectin-8N) binds significantly more glycoproteins, including haptoglobin, in sera of patients with IgA-nephritis (IgAN) compared to patients with other forms of kidney disease and healthy subjects [18]. Galectin-8N has a strong preference for 2-3 sialylated galactosides [20][21], and its binding of serum glycoproteins requires the presence of 2-3 sialic acid [18], in contrast to the case of galectin-1, suggesting a different binding site.…”

Galectin-8 binds to LFA-1, blocks its interaction with ICAM-1 and is counteracted by anti-Gal-8 autoantibodies isolated from lupus patients

“…The CRDs of diff erent galectins and even the N-terminal or C-terminal CRDs of tandem repeat galectins display variations which serve to engage different subsets of functionally distinct glycoproteins (Hirabayashi et al, 2002;Ideo et al, 2003;Patnaik et al, 2006;Stillman et al, 2006;Carlsson et al, 2007;Stowell et al, 2008;Ideo et al, 2011). Thus, understanding the functions of a particular galectin requires defi ning the range of its interacting elements in diff erent cellular contexts.…”