The immunoglobulin G (IgG) fragment crystallizable (Fc) domain contains a single, highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain, which is buried within the hydrophobic core of each of the two heavy chains. The biantennary core glycan structure, composed of 2 N-acetylglucosamine (GlcNAc) and 3 mannose residues, can be further decorated with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid. Presence or absence of distinct residues can alter IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Here, we provide a protocol for the generation of IgG-Fc de-galactosylated, galactosylated, de-sialylated and sialylated IgG antibodies using recombinant glycosidases and glycosyltransferases.
[Background] The use of glycosyltransferases for antibody glycan modification allows the attachment of sugar substrates to pre-existing glycan residues. Immunoglobulin G carries a single, highly conserved N-glycosylation site in each of its CH2 domains (Arnold et al., 2007) (Figure 1) allowing site-specific glycan modification with glycosyltransferases. Antibodies may carry additional N-glycans if their Fab domains contain Asn-X-Ser/Thr (X ≠ Pro) sequences (Mellquist et al., 1998). Careful selection of a monoclonal antibody lacking Fab glycosylation is therefore important for Fc-specific glycan modification. The protocol described herein was developed based on the following publications (Kingston, 2003; Kaneko et al., 2006; Anthony et al., 2008; Barb et al., 2009; Quast et al., 2015).
Figure 1. The IgG-Fc N-glycan. Schematic depiction of IgG with two fully processed IgG-Fc N-glycans (left) and composition of the glycan (right).
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