Structure and structural dynamics are important attributes that enable proteins to perform many different activities in vivo. Although most of the unique structural and functional properties of a protein are dictated by its amino-acid sequence, there
are many changes, called post–transitional modifications (PTMs) that can alter a protein as it is expressed, folds, and ages
(1). These alterations can significantly change and control the specificity and strength of protein interactions, as well
as influence the physico–chemical properties of the protein (e.g., stability, solubility, immunogenicity) (2). Probably one
of the most common and important of these PTMs is glycosylation—the attachment of oligosaccharides to proteins.
During recent years, it has become increasingly apparent that glycosylation is important in controlling the function and solution
behavior of proteins in solution (3, 4). More than half of all eukaryotic proteins are glycoproteins (5). The oligosaccharides,
also referred to as glycans or carbohydrates, commonly found on a protein exist as either N–linked or O–linked oligosaccharides
and typically consist of 2 to 14 monosaccharides chemically linked in a linear or branched configuration. N–linked oligosaccharides
are chemically linked to asparagines (and to a lesser extent arginine) within the consensus sequence –Asn–Xaa–Ser/Thr (6),
while O–linked oligosaccharides are chemically linked to any serine or threonine (7).
To date, most of the progress in characterizing the carbohydrates on glycoproteins has focused on studying their structure
and composition. These results have shown that a diversity of oligosaccharide structures can exist at any given glycosylation
site on a protein. This diversity is one of the major causes of the microheterogeneity observed in proteins and is one of
the major challenges biopharmaceutical companies face in demonstrating lot–to–lot comparability in manufacturing a glycosylated
protein biopharmaceutical. Differential glycosylation on proteins will also be an important factor in determining similarity
between innovator products and their follow–ons.
Figure 1: A representative glycosylated protein (PDB: 1AUC) with its glycan (shown as cyan sticks) in different hypothetical
orientations. A. Glycan not interacting with protein. B. Glycan interacting with the protein surface. C. Glycan interacting
with the protein interior.
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Progress has been made in developing analytical tools capable of characterizing oligosaccharides in great detail (8, 9) and
in associating their chemical differences with functional activities (10, 11). However, the physico–chemical basis for understanding
just how oligosaccharides alter the function and structural properties of a protein is significantly lacking. A major challenge
exists in understanding the details, at a molecular level, as to how oligosaccharides influence the structure and structural
dynamics of a protein. Do the oligosaccharides float freely about the protein linkage site, do they interact directly with
the protein, or are the oligosaccharides interacting with and influencing the protein surface or interior (see Figure 1)?
Although nuclear magnetic resonance (NMR) and X–ray crystallography have contributed greatly in providing some of the answers
to these and other questions, more orthogonal tools are needed. One tool that offers significant promise in helping to understand
carbohydrate–protein interactions is hydrogen/deuterium–exchange mass spectrometry (H/DX–MS). Although the application of
H/DX–MS to protein conformational studies is not new, recent developments now make H/DX–MS more capable, attractive, and informative
(12). The authors briefly highlight how H/DX–MS can enhance our knowledge of the molecular details driving carbohydrate–protein
interactions. In addition, we speculate on how these glycans may actually manipulate and control the biological function of
a protein. Such scientific understanding should facilitate the development of new and more useful biopharmaceuticals.
