Investigating the Influence of Glycosylation on Protein Conformation and Dynamics

The authors review and discuss the influence of glycans on the conformation of a representative IgG1 biopharmceutical using H/DX-MS as an analytical tool.
Oct 01, 2010

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.
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.