Impact of Fc N-glycan sialylation on IgG structure

Abstract

Human IgG antibodies containing terminal alpha 2,6-linked sialic acid on their Fc N-glycans have been shown to reduce antibody-dependent cell-mediated cytotoxicity and possess anti-inflammatory properties. Although terminal sialylation on complex N-glycans can happen via either an alpha 2,3-linkage or an alpha 2,6-linkage, sialic acids on human serum IgG Fc are almost exclusively alpha 2,6-linked. Recombinant IgGs expressed in Chinese hamster ovary (CHO) cells, however, have sialic acids through alpha 2,3-linkages because of the lack of the alpha 2,6-sialyltransferase gene. The impact of different sialylation linkages to the structure of IgG has not been determined. In this work, we investigated the impact of different types of sialylation to the conformational stability of IgG through hydrogen/deuterium exchange (HDX) and limited proteolysis experiments. When human-derived and CHO-expressed IgG1 were analyzed by HDX, sialic acid-containing glycans were found to destabilize the CH2 domain in CHO-expressed IgG, but not human-derived IgG. When structural isomers of sialylated glycans were chromatographically resolved and identified in the limited proteolysis experiment, we found that only alpha 2,3-linked sialic acid on the 6-arm (the major sialylated glycans in CHO-expressed IgG1) destabilizes the CH2 domain, presumably because of the steric effect that decreases the glycan-CH2 domain interaction. The alpha 2,6-linked sialic acid on the 3-arm (the major sialylated glycan in human-derived IgG), and the alpha 2,3-linked sialic acid on the 3-arm, do not have this destabilizing effect.

The Role of Fc Receptors on the Effectiveness of Therapeutic Monoclonal Antibodies

“…Additionally, for mAbs, hypersialysation was shown to reduce binding properties, and consequently ADCC and CDC activity [164,165,167]. Thereby, it is suggested that the added sialic acid might affect the structure of the hinge region, which impacts binding to FcγRs [168,169]. Furthermore, it has been demonstrated by Bas et al that hypersialylation of N297 is able to extend the half-life of IgG [170].…”

Glycoengineering Chinese hamster ovary cells: a short history

“…On the other hand, galactose is required for sialylation with N-acetylneuraminic acid (NeuAc), which in turn can improve the solubility, anti-inflammatory activity, thermal stability and serum half-life of mAbs [8,[23][24][25][26]. The type of sialic acid linkage also influences biotherapeutic properties, in fact when NeuAc is α2,3-linked (main sialylated species in CHO-derived glycoproteins) the conformational stability of the C H 2 domain of IgG can be reduced, most likely a result of a steric effect between the protein backbone and the α2,3-sialylated 6-arm [27,28]. In contrast, it is the α2,6-linked NeuAc (main sialic acid linkage in human IgG) that is solely required for the anti-inflammatory activity of IgG mediated by non-canonical IgG-Fc receptors [24].…”

Characterization of Signaling Pathways Associated with Pancreatic β-cell Adaptive Flexibility in Compensation of Obesity-linked Diabetes in db/db Mice

“…The significantly regulated SA glycopeptides were imported into STRING and the resulting interaction network is shown in Figure 6 (lower left panel). Here, the modulated SA glycopeptides could be associated with limited proteolysis (29,30), re-glycosylation with additional sugar residues such as SA in the ER and Golgi apparatus (31, 32), lysosomal function (33), renin-angiotensin system (34), cell adhesion molecules such as integrins and laminin (35)(36)(37), PI3K-Akt signaling (38), regulation of the actin cytoskeleton (39), focal adhesion (40), and extracellular matrix-receptor interaction (41). Moreover, ER by guest on July 8, 2020 lectin chaperone-like proteins (e.g., calnexin; CANX) and their associated co-factors (e.g., PDIA3) that recognize N-linked glycans are likely involved (42) were significantly regulated in D/N and restored towards normal after exposure to normal glucose (Table S1).…”