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

Mathias Ingemann Nielsen,John Stegmayr,Oliver C Grant,Zhang Yang,Ulf J Nilsson,Irene Boos,Michael C Carlsson,Robert J Woods,Carlo Unverzagt,Hakon Leffler,Hans H Wandall

doi:10.1074/jbc.ra118.004636

Abstract

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for β-galactose-containing glycans. Moreover, galectins gain higher affinity and fine-tune specificity by glycan interactions at sites adjacent to their β-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.

Highlights

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for ␤-galactose– containing glycans
Galectins were conjugated to fluorescein, with cell binding measured by flow cytometry as described under “Materials and methods.”
This work demonstrates the use of a genetically glycoengineered cell platform to dissect the binding affinities of 10 different galectins to specific O- and N-linked glycans presented at the cell surface or on soluble glycoproteins

Summary

Introduction

Galectins compose a protein family defined by a conserved sequence motif conferring affinity for ␤-galactose– containing glycans. Shared specificity for ␤-galactose residues that are widely distributed on glycoproteins and glycosphingolipids, individual galectins require additional distinct structures for high-affinity binding. These specificities have been extensively analyzed by a variety of biochemical methods using panels of purified, naturally occurring glycans or fragments of them (16 –21). It has been suggested that the unique specificity of the N-terminal CRD of galectin-8 enables high-affinity binding to O-glycans [22, 30] These investigations have contributed greatly to our current understanding of galectin specificity, but most are limited by their evaluation of synthetic glycans in the absence of any appropriate protein or cellular context. The specificity of glycan binding in a more physiologically relevant setting has yet to be addressed in a systematic manner

Methods

Results

Conclusion