Effect of deglycosylation on the conformation of an IgG1 antibody
Recombinant monoclonal antibodies (rmAb) are an important class of biopharmaceutical proteins. Antibodies are used to treat
a wide array of medical conditions from autoimmune diseases to cancer to various other diseases (22). Today, more antibodies
are in biopharmaceutical development pipelines and in clinical programs then are any other class of protein (23). The most
common therapeutic rmAb is the immunoglobulin gamma 1 (IgG1). The IgG1 is made up of two identical subunits, a so–called homodimer.
It is also a glycosylated protein with an apparent molecular weight approaching 150 kDa. An IgG1 is composed of two identical
heavy chains and two identical light chains that are covalently connected by disulfide bonds. On each heavy chain (typically
on residue Asn298), there is a single N–linked biantennary type oligosaccharide (6, 24). For IgG1 antibodies, glycosylation
is a major source of heterogeneity, as most antibody expression systems (such as Chinese Hamster Ovary cells (25)) are incapable
of producing an antibody containing a glycan with a uniform structural composition. Changes in IgG1 glycosylation (i.e., different
levels of sialic acid, galactose, fucose, high mannose structures) have been shown to induce thermal destabilization, influence
effector function, drug clearance, and may also provoke an immunogenic response (3, 26). Therefore, understanding the impact
that glycans have on the conformation of an IgG1 (or any other glycosylated protein) is very important.
Figure 3: A summary of the H/DX-MS results (24, 27) obtained when comparing different IgG1 glycovariants. (Top) Representative
structure of an IgG1 antibody [PDB: 1HZH] (36) and common glycans. (Bottom) The left-hand panel shows the structural changes
observed by H/DX-MS when the oligosaccharides were removed from the IgG1, while the right-hand panel shows the structural
changes observed by H/DX-MS when only galactose was removed from the IgG1 oligosaccharide.
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Recently, H/DX–MS was used to study the effect of glycans on the conformation of an intact IgG1 (24–27). It was demonstrated
that complete removal of the IgG1 glycan resulted in differences in deuterium incorporation throughout the IgG1 Fc region
(see Figure 3). Some regions were shown to incorporate more deuterium as a result of glycan removal, while other regions incorporated
less deuterium. These data correlated nicely with previous structural studies on deglycosylated IgG1–Fc (26, 28). Since IgG1
glycans were shown by X–ray crystallography to make several contacts with many residues within the IgG1 Fc region (29), it
is not surprising that removing the glycan results in a change in the IgG1 Fc conformation. This change in conformation ultimately
resulted in thermal instability of the IgG1 Fc (30) and complete loss of effector function (27), relative to the native IgG1.
Because glycans are thought to anchor the IgG1 Fc in a stable conformation, which promotes favorable interactions with effector
function proteins. This and other results (31) suggest that removal of the glycans weakens the structural rigidity of the
IgG1 Fc. As a result, glycan removal leads to a significant change in the conformation of the IgG1 Fc. The change observed
by H/DX clearly indicates that structural perturbation may extend well beyond the protein regions directly in contact with
the oligosaccharide. This type of change is indicative of allosteric effects, where perturbations in one region influence
conformation at a distance.
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