Profiling the N-Glycan Composition of IgG with Lectins and Capillary Nanogel Electrophoresis

Abstract

Glycosylated human IgG contains fucosylated biantennary N-glycans with different modifications including N-acetylglucosamine, which bisects the mannose core. Although only a limited number of IgG N-glycan structures are possible, human IgG N-glycans are predominantly biantennary and fucosylated and contain varying levels of α2–6-linked sialic acid, galactose, and bisected N-acetylglucosamine. Monitoring the relative abundance of bisecting N-acetylglucosamine is relevant to physiological processes. A rapid, inexpensive, and automated method is used to successfully profile N-linked IgG glycans and is suitable to distinguish differences in bisection, galactosylation, and sialylation in N-glycans derived from different sources of human IgG. The separation is facilitated with self-assembled nanogels that also contain a single stationary zone of lectin. When the lectin specificity matches the N-glycan, the peak disappears from the electropherogram, identifying the N-glycan structure. The nanogel electrophoresis generates separation efficiencies of 500 000 plates and resolves the positional isomers of monogalactosylated biantennary N-glycan and the monogalactosylated bisected N-glycan. Aleuria aurantia lectin, Erythrina cristagalli lectin (ECL), Sambucus nigra lectin, and Phaseolus vulgaris Erythroagglutinin (PHA-E) are used to identify fucose, galactose, α2–6-linked sialic acid, and bisected N-acetylglucosamine, respectively. Although PHA-E lectin has a strong binding affinity for bisected N-glycans that also contain a terminal galactose on the α1–6-linked mannose branch, this lectin has lower affinity for N-glycans containing terminal galactose and for agalactosylated bisected biantennary N-glycans. The lower affinity to these motifs is observed in the electropherograms as a change in peak width, which when used in conjunction with the results from the ECL lectin authenticates the composition of the agalactosylated bisected biantennary N-glycan. For runs performed at 17 °C, the precision in migration time and peak area was less than or equal to 0.08 and 4% relative standard deviation, respectively. The method is compatible with electrokinetic and hydrodynamic injections, with detection limits of 70 and 300 pM, respectively.

The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto’s Thyroiditis: An in Vitro Model of Thyroid Autoimmunity

“…The whole pool of serum IgG isolated from HT patients and healthy donors was desialylated by Neu with broad specificity to α2-3,6,8-linked sialic acid. The efficiency of enzymatic desialylation was verified by lectin blotting with SNA specific for α2,6-linked SA, because α2,6-sialylated N-glycans are present on IgG much more frequently than are oligosaccharides with α2,3-SA [20]. The absence of an SNA staining signal in Neu-treated IgG from both HT and control samples confirmed the efficiency of IgG desialylation (Figure 3).…”

Clinical and pharmaceutical applications of affinity ligands in capillary electrophoresis: A review

“…Lectins are a third group of binding agents that have been used in CE [44][45][46][47][48][49][50][51][52][53][54][55]. These are carbohydrate-binding proteins that are not produced by the immune system and that can be used to interact with compounds that contain glycans, such as glycopeptides, glycoproteins, and glycolipids [45].…”

“…These are carbohydrate-binding proteins that are not produced by the immune system and that can be used to interact with compounds that contain glycans, such as glycopeptides, glycoproteins, and glycolipids [45]. The combined use of lectins with CE can produce an analytical method that is selective for a given type of glycan-containing target, as may be used to characterize the binding between carbohydrates and lectins, to identify glycans in a mixture, or to resolve some glycoforms of the target [45][46][47][48][49][50][51][52][53][54][55].…”

“…A partial filling technique has been used along with glycosidases to characterize N-glycans of the therapeutic antibody rituximab [50]. Other work has used thermally-reversible nanogels with ACE to trap and immobilize a lectin within a plug of the nanogel [51,52]. This method has been used to profile the N-glycan composition of IgG [52].…”

“…Other work has used thermally-reversible nanogels with ACE to trap and immobilize a lectin within a plug of the nanogel [51,52]. This method has been used to profile the N-glycan composition of IgG [52]. Lectins have been used in ACE for the characterization of glycoproteins other than antibodies.…”