Table 1.

Challenges to the structure-function understanding of glycans.

Key challenge	Features	Impact on study of glycans
Glycan biosynthesis	Nontemplate-driven process, unlike DNA/RNA and protein	Replication- or translation-like ‘rules’ cannot be easily applied; no direct methods to amplify glycans, unlike DNA (PCR) and protein (recombinant expression)
	Limited availability of glycans from natural sources (e.g., cells, tissues)	Without amplification tools, analytical and functional methods often require high sensitivity
	Tissue-, developmental-, and metabolic-dependent expression of glycan biosynthetic machinery (glycosyltransferases and glycosidases)	Glycan structure is sensitive to cellular conditions, tissue type, and developmental stages
	Lack of proofreading in glycan biosynthetic process	Increases structural diversity of glycans to be analyzed
Glycan structural complexity and heterogeneity	Presence of isomers and different anomeric configurations	Properties generally not present in DNA and proteins; challenges structural characterization by single method
	Microheterogeneity – a range of glycan structures (length, composition, branching) found at any given glycosylation site on a glycoprotein	Highly similar physicochemical properties of glycan microheterogeneities challenges their characterization
	Branching	Unambiguous designation of branches and their locations challenged by analytical approaches
	Presence of multiple modifications (sulfation, acetylation, methylation) and high diversity of linkages (location of linkages and anomericity)	Chemical synthesis is difficult and limited to small oligosaccharides due to the need of complex protecting and deprotecting strategies
	Site of attachment to protein/lipid	Requires glycan-protein and/or glycan-lipid characterization in addition to glycan structure
Glycan presentation and interactions	Presentation of an ensemble of different (often related) structures within a biological system or interaction	Studies must account for a population of glycans with similar structures, rather than an ‘average’ single structure
	Glycan-protein interactions often achieve high affinity and specificity by multivalency	Correct presentation of glycan and glycan-binding protein/domain(s) is critical for experimental design
	Glycan-protein interactions modulate biology in an analog-like nature	Functional readouts must be characterized in terms of gradation of effects (not binary “on/off” effects)
	High torsional flexibility of glycans mediates presentation of a range of conformations for a particular glycan	Sequence of glycan is often not sufficient to characterize glycan-protein interactions; analysis of conformations and topologies should be considered