Reading the glyco-code: New approaches to studying protein-carbohydrate interactions

Abstract

The surface of all living cells is decorated with carbohydrate molecules. Hundreds of functional proteins bind to these glycosylated ligands; such binding events subsequently modulate many aspects of protein and cell function. Identifying ligands for glycan-binding proteins (GBPs) is a defining challenge of glycoscience research. Here, we review recent advances that are allowing protein-carbohydrate interactions to be dissected with an unprecedented level of precision. We specifically highlight how cell-based glycan arrays and glyco-genomic profiling are being used to define the structural determinants of glycan-protein interactions in living cells. Going forward, these methods create exciting new opportunities for the study of glycans in physiology and disease.

Figure 1

The cell surface glyco-code. Scaffolds are the proteins or lipids to which carbohydrate chains are attached. These molecules play a key role in the presentation of glycans on the cell surface. Writers are enzymes (principally glycosyltransferases) that build complex carbohydrate chains. These enzymes can directly link a glycan to a protein/lipid. They can also extend glycans by covalently linking new monosaccharide building blocks to existing oligosaccharide chains. Readers are glycan-binding proteins that bind to specific glycan-containing molecular motifs, which are termed ligands. Often, binding between a reader and its ligand will initiate transduction of downstream biological signaling pathways. UDP = Uridine diphosphate, CMP = Cytidine-5-monophosphate.

Figure 2

Affinity capture of GBP-binding ligands. In one approach, cells are incubated with a synthetic monosaccharide modified with a photocrosslinking group. After incorporation of this sugar into cell surface glycans, cells are incubated with a recombinant GBP. Exposure of cells to light induces the formation of covalent cross-links between the GBP and its ligands. Proteins can then be affinity purified and subjected to glyocoproteomic analysis to identify glycoproteins bound by the GBP.

Figure 3

Synthetic and biological glycan arrays. Synthetic arrays may contain glycans that form components of a physiological ligand for a GBP, but these glycans may not be presented in the correct valency and orientation to mediate high-affinity binding events. Synthetic glycans may also be anchored to a solid support via flexible linkers that don’t recapitulate physiological interactions with a protein scaffold. Engineering of cell surface glycans by genetic or chemical approaches, conversely, allows for dissection of glycan-binding interactions in their correct biochemical context.

Figure 4

Glyco-genomic profiling of GBP-ligand interactions. A genetically heterogenous cell population is either generated through CRISPR genome-wide mutagenesis or isolated from physiological sources. Cells exhibiting altered (either diminished or elevated) binding to a glycan-binding protein are isolated (via FACS) and genetically characterized by high-throughput sequencing. This method allows unbiased identification of genes required for expression of ligands for the given glycan-binding protein, information that can be used to infer structural characteristics of the ligand.