Abstract
IgG, the main serum immunoglobulin isotype, exists in four subclasses which selectively appear with distinctive glycosylation profiles. However, very little is known about the biological consequences mainly due to the difficulties in the generation of distinct IgG subtypes with targeted glycosylation. Here, we show a comprehensive expression and glycan modulation profiling of IgG variants in planta that are identical in their antigen binding domain but differ in their subclass appearance. While IgG1, 2, and 4 exhibit similar expression levels and purification yields, IgG3 is generated only at low levels due to the in planta degradation of the heavy chain. All IgG subtypes are produced with four distinct N-glycosylation profiles, differing in sugar residues previously shown to impact IgG activities, i.e., galactosylation, sialylation and core fucosylation. Affinity purified IgG variants are shown to be fully assembled to heterodimers but display different biochemical/physical features. All subtypes are equally well amenable to targeted glycosylation, except sialylated IgG4 which frequently accumulates substantial fractions of unusual oligo-mannosidic structures. IgG variants show significant differences in aggregate formation and endotoxin contamination which are eliminated by additional polishing steps (size exclusion chromatography, endotoxin removal treatments). Collectively we demonstrate the generation of 16 IgG variants at high purity and large glycan homogeneity which constitute an excellent toolbox to further study the biological impact of the two main Fc features, subclass and glycosylation.
Highlights
IgG is the main immunoglobulin class that is induced during an immune response against foreign antigens
All four IgG variants were transiently expressed in Nicotiana benthamiana glycosylation mutant XTFT, synthesizing human type bi-antennary complex N-glycans lacking plant-specific xylose and core fucose residues (Strasser et al, 2008)
Immunoblotting using anti IgG antibodies revealed the presence of two strong signals corresponding to an apparent molecular mass (MM) of 55 (65 kD for IgG3) and 25 kDa, as expected for HC and LC (Figure 1A)
Summary
IgG is the main immunoglobulin class that is induced during an immune response against foreign antigens. Comparable to the other antibody classes, IgGs are characterized by a bifunctional action mode. While Fab region precisely recognize an antigen, the Fc domain conveys a wide range of effector functions that modulate various aspects of innate and adaptive immunity. Fc mediated activities are mainly initiated upon interactions with the various types of Fc gamma receptors (FcγRs), a process that is largely determined by the structural heterogeneity of the IgG Fc domain. The two main features that generate a spectrum of Fc phenotypes are modulation of Fc-glycosylation along with differences in the amino acid sequence among the IgG subclasses. The 30–35 N-glycan structures usually detected on serum Fc-IgG are of biantennary complex type, most
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