Abstract
Concanavalin A (ConA) chromatography has been extensively used to separate asymmetric Immunoglobulin G (IgG), which possesses oligosaccharide attached to one of the two F(ab')2 arms, from symmetric IgG with no glycan attached to Fab fragments. In this study, applying affinity chromatography, silver stain, Western blot and lectin stain techniques, N- linked oligosaccharide attached to Fab fragment was demonstrated to be exposed on the surface of the protein and be accessible by ConA. In contrast, N- linked oligosaccharide attached to asparagine (Asn) 297 of IgG Fc was located in the inside of the natural protein and was inaccessible by ConA. In addition to asymmetric IgG, there are also detectable level of IgG with both F(ab')2 arms glycosylated that has not been reported previously. The discoveries of new basic molecular structure of IgG would have implications in understanding the function and properties of this important immune molecule with clinical applications.
Highlights
Immunoglobulin G (IgG) has long been known as a glycoprotein with two highly conserved glycosylation sites located at Asn 297 in the CH2 domains of the Fc region
N- linked oligosaccharide attached to asparagine (Asn) 297 of IgG Fc was located in the inside of the natural protein and was inaccessible by Concanavalin A (ConA)
Human IgG was purified from serum with Protein G column and fractionated with ConA Sepharose 4B
Summary
IgG has long been known as a glycoprotein with two highly conserved glycosylation sites located at Asn 297 in the CH2 domains of the Fc region. Further oligosaccharides may be found attached to the variable regions of IgG molecules [1]. The Fc glycosylation has been extensively studied to illustrate glycans’ structure, influence on antibodies’ function and glycoform changes associated with diseases. The common characteristic of IgG Fc oligosaccharides is of the complex type with a constant heptasaccharide core. Additional sugar residues such as core fucose (Fuc), bisecting N-acetylglucosamine (GlcNAc), galactose(s) (Gal), and N-aetylneuraminic acid(s) (NeuAc) may attached to the “core” [2] (Figure 1B)
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