Terminal sugars of Fc glycans influence antibody effector functions of IgGs
Introduction
Immunoglobulins (Igs) are soluble serum glycoproteins involved in protecting vertebrates against foreign substances [1, 2]. IgG molecules consist of two Fab and one Fc fragment, which are linked through a flexible hinge region [1, 2, 3]. The Fc fragment is involved in defining antibody effector functions and pharmacokinetic properties by its interaction with Fc receptors, C1q, and FcRn [2, 3]. The Fab fragment of IgG seems relatively resistant to proteases, whereas the Fc and hinge regions are more sensitive to proteolysis [4•, 5••, 6•].
IgG molecules are N-glycosylated in the CH2 domain of the Fc fragment and about 30% of circulating human IgG is also glycosylated in the Fab region [3, 7]. In human IgG, the majority of the Fc glycans are complex biantennary structures with a high degree of heterogeneity, on account of the presence or absence of different terminal sugars [2, 3, 7]. Fc glycosylation is required for the induction of antibody-mediated effector functions including ADCC and CDC [3, 8]. This review discusses the structural heterogeneity of Fc glycans and their role in determining antibody effector functions.
Section snippets
Structural heterogeneity of Fc glycans
The N-glycans present in the Fc portion of IgG molecules contain a common core region of two N-acetylglucosamine residues (GlcNAc) linked to asparagine 297 (Asn297) via an amide bond and three mannose (Man) residues (Figure 1) [2, 3]. This core structure is present in high mannose, hybrid or complex structures and may contain additional terminal-sugar residues such as Man, GlcNAc, galactose (Gal), core fucose (Fuc), bisecting GlcNAc, and sialic acid (Sia or NANA for N-acetylneuraminic acid)
Terminal Gal residues affect CDC activity of IgG molecules
Unlike serum glycoproteins that contain surface-exposed glycans that are 60–95% sialylated, only about 10% of Fc glycans of human serum IgG are sialylated and <5% of Fc glycans of recombinant IgGs (rIgGs) produced in Chinese hamster ovary (CHO) cells are sialylated [16]. As a result, Fc glycans may contain 0, 1, or 2 terminal Gal residues (G0, G1, or G2) in their antennae (Figure 1). The terminal Gal content of IgG affects CDC, but not ADCC activity [17]. For Rituxan, for example, an increase
Effect of terminal GlcNAc residues on IgG functions
Glycoproteins containing terminal GlcNAc residues have been shown to bind to the mannose receptor and thereby exhibit reduced serum half-life [18•]. However, many rIgGs containing significant amounts of terminal GlcNAc residues in the form of G0 and G1 structures show considerably longer serum half-lives compared to glycoproteins containing such terminal GlcNAc residues [2, 3, 18•]. Therefore, terminal GlcNAc residues of Fc glycans may not affect pharmacokinetic properties of IgG.
IgG molecules
Absence of core fucose results in enhanced ADCC activity of IgG
Fc glycans contain a core Fuc residue in α1,6-position linked to the core GlcNAc residue (Figure 1) [10]. Biosynthesis of core-fucosylated glycans is the result of a transfer of a Fuc residue from GDP-Fuc-mediated by α1,6-fucosyltransferase in the trans-Golgi [20]. The absence of core Fuc residues in the Fc glycans substantially increases the ADCC activity of IgG as nonfucosylated antibodies bind to the FcγRIIIa receptor with significantly increased affinity [21••, 22]. However, >80% of the Fc
Terminal sialylation affects IgG functions
The N-glycans of serum glycoproteins are often terminated with sialic acid residues [32]. Increased terminal sialylation can increase the serum half-life of many glycoproteins [2, 3]. Fc glycans of IgGs contain variable amounts of sialylated glycans [10]. Increased sialylation of Fc glycans results in decreased ADCC activity of rIgGs as terminal sialylation negatively affects antibody binding to the FcγRIIIa receptor [33••, 34••]. In addition, increased sialylation may result in decreased
Effect of high mannose structures on ADCC activity of IgGs
Human IgG molecules may contain small amounts of Man5GlcNAc2 structure [9, 10, 11]. Other high mannose type glycans such as Man6GlcNAc2, Man7GlcNAc2, Man8GlcNAc2, Man9GlcNAc2, and Glc3Man9GlcNAc2 structures that are present in chicken IgGs were either not present or only at very low levels in human IgG [9]. In rIgG, the high mannose content varies with cell lines and batches [5••, 6•, 36].
Glycoproteins containing high mannose structures bind the mannose receptor and therefore exhibit a reduced
Hydrophobic and hydrophilic interactions between terminal-sugar residues and amino acid residues in the Fc
The impact of terminal-sugar residues on antibody effector functions may be because of hydrophobic and hydrophilic interactions between amino acid and sugar residues, as observed in structural analyses of human IgG Fc fragments using X-ray crystallography, NMR spectroscopy, and differential scanning microcalorimetry [40, 41••, 42•, 43, 44•].
Fc fragments of G2, G0, and G-2 glycoforms of IgG1 (see Figure 3 for glycan structures) were crystallized in complex with miniZ peptide, which represents an
Conclusions
The presence or absence of various terminal sugars of Fc glycans increases microheterogeneity, which affects not only antibody effector functions but also antibody stability and binding to certain cell surface antigens. Structural studies of intact antibodies and Fc fragments suggest that hydrophobic and hydrophilic interactions between sugar residues and amino acid residues in the CH2 domain of the Fc fragment affect the fine structural conformation of IgG molecules. Terminal-sugar residues
References and recommended reading
Papers of particular interest, published within the period of the review, have been highlighted as:
• of special interest
•• of outstanding interest
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