Galectin binding to cells and glycoproteins with genetically modified glycosylation reveals galectin–glycan specificities in a natural context

• Galectins compose a protein family defined by a conserved sequence motif conferring affinity for -galactose– containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their -galactoside– binding site, as revealed by extensive testing against panels of purified glycans. However, in cells, galectins bind glycans on glycoproteins and glycolipids in the context of other cellular components, such as at the cell surface. Because of difficulties in characterizing natural cellular environments, we currently lack a detailed understanding of galectin-binding specificities in the cellular context. To address this challenge, we used a panel of genetically stable glycosylation mutated CHO cells that express defined glycans to evaluate the binding affinities of 10 different carbohydrate-recognition domains in galectins to N-glycans and mucin-type O-glycans. Using flow cytometry, we measured the cell-surface binding of the galectins. Moreover, we used fluorescence anisotropy to determine the galectin affinities to recombinant erythropoietin used as a reporter glycoprotein produced by the glycoengineered cells and to synthetic N-glycans with defined branch structures. We found that all galectins, apart from galectin-8N, require complex N-glycans for high-affinity binding. Galectin-8N targeted both N- and O-linked glycans with high affinity, preferring 2,3-sialylated N-acetyllactosamine (LacNAc) structures. Furthermore, we found that 2,3-sialylation suppresses high-affinity binding of select galectins, including galectin-2, -3, -4N, and -7. Structural modeling provided a basis for interpreting the observed binding preferences. These results underscore the power of a glycoengineered platform to dissect the glycan-binding specificities of carbohydrate-binding proteins.

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NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

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