Abstract
N-linked glycosylation of the fragment crystallizable (Fc)-region of immunoglobulin G (IgG) is known to have a large influence on the activity of the antibody, an effect reported to be IgG subclass specific. This situation applies both to humans and mice. The mouse is often used as experimental animal model to study the effects of Fc-glycosylation on IgG effector functions, and results are not uncommonly translated back to the human situation. However, while human IgG Fc-glycosylation has been extensively characterized in both health and disease, this is not the case for mice. To characterize the glycosylation profile of murine IgG-Fc and in addition evaluate the systematic glycosylation differences between mouse strains, sexes, and IgG subclasses, we used nanoliquid chromatography mass spectrometry (nanoLC-MS(/MS)) to look at the subclass-specific IgG Fc-glycopeptides of male and female mice from the strains BALB/c, C57BL/6, CD-1, and Swiss Webster. The structural analysis revealed the presence of predominantly fucosylated, diantennary glycans, with varying amounts of galactosylation and α2,6-sialylation. In addition, we report glycosylation features not previously reported in an Fc-specific way on murine IgG, including monoantennary, hybrid, and high mannose structures, as well as diantennary structures without a core fucose, with a bisecting N-acetylglucosamine, or with α1,3-galactosylation. Pronounced differences were detected between strains and the IgG subclasses within each strain. Especially the large spread in galactosylation and sialylation levels found between both strains and subclasses may vastly influence IgG effector functions. Mouse strain-based and subclass-specific glycosylation differences should be taken into account when designing and interpreting immunological and glycobiological mouse studies involving IgG effector functions.
Highlights
Immunoglobulin G (IgG) is the most abundant antibody in human plasma and plays a crucial role in the humoral immune response [1]
Disodium hydrogen phosphate dihydrate (Na2HPO4⋅2H2O), potassium dihydrogen phosphate (KH2PO4), NaCl, sodium dodecyl sulfate (SDS), ethanol, glacial acetic acid, and trifluoroacetic acid were purchased from Merck (Darmstadt, Germany). 1-Hydroxybenzotriazole hydrate, dimethyl sulfoxide (DMSO), ammonium bicarbonate, formic acid, Nonidet P-40 substitute (NP-40), super-DHB, NaOH, and tosyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin from bovine pancreas were purchased from Sigma-Aldrich
The strain-specific IgG fragment crystallizable (Fc)-glycosylation was studied for four plasma pools of one male and one female mouse per strain (Figures 1 and 2A)
Summary
Immunoglobulin G (IgG) is the most abundant antibody in human plasma and plays a crucial role in the humoral immune response [1]. Various effector functions of IgG are affected by its fragment crystallizable (Fc)-glycosylation, which has shown to influence the binding of IgG-Fc to, e.g., Fcγ-receptors (FcγRs) and C-type lectins [2,3,4]. Galactosylation on total IgG is decreased in rheumatoid arthritis and active tuberculosis infections, and increases with pregnancy [5,6,7]. Fucosylation on the other hand, is decreased on alloantibodies against red blood cells and platelets as well as on gp120-specific antibodies in HIV-infected patients [8,9,10]. Modification of IgG Fc-glycosylation has shown to be a suitable measure to improve the efficacy of therapeutic monoclonal antibodies [11]
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