Abstract
Glycosylation of immunoglobulin G (IgG) influences IgG effector function by modulating binding to Fc receptors. To identify genetic loci associated with IgG glycosylation, we quantitated N-linked IgG glycans using two approaches. After isolating IgG from human plasma, we performed 77 quantitative measurements of N-glycosylation using ultra-performance liquid chromatography (UPLC) in 2,247 individuals from four European discovery populations. In parallel, we measured IgG N-glycans using MALDI-TOF mass spectrometry (MS) in a replication cohort of 1,848 Europeans. Meta-analysis of genome-wide association study (GWAS) results identified 9 genome-wide significant loci (P<2.27×10−9) in the discovery analysis and two of the same loci (B4GALT1 and MGAT3) in the replication cohort. Four loci contained genes encoding glycosyltransferases (ST6GAL1, B4GALT1, FUT8, and MGAT3), while the remaining 5 contained genes that have not been previously implicated in protein glycosylation (IKZF1, IL6ST-ANKRD55, ABCF2-SMARCD3, SUV420H1, and SMARCB1-DERL3). However, most of them have been strongly associated with autoimmune and inflammatory conditions (e.g., systemic lupus erythematosus, rheumatoid arthritis, ulcerative colitis, Crohn's disease, diabetes type 1, multiple sclerosis, Graves' disease, celiac disease, nodular sclerosis) and/or haematological cancers (acute lymphoblastic leukaemia, Hodgkin lymphoma, and multiple myeloma). Follow-up functional experiments in haplodeficient Ikzf1 knock-out mice showed the same general pattern of changes in IgG glycosylation as identified in the meta-analysis. As IKZF1 was associated with multiple IgG N-glycan traits, we explored biomarker potential of affected N-glycans in 101 cases with SLE and 183 matched controls and demonstrated substantial discriminative power in a ROC-curve analysis (area under the curve = 0.842). Our study shows that it is possible to identify new loci that control glycosylation of a single plasma protein using GWAS. The results may also provide an explanation for the reported pleiotropy and antagonistic effects of loci involved in autoimmune diseases and haematological cancer.
Highlights
Glycosylation is a ubiquitous post-translational protein modification that modulates the structure and function of polypeptide components of glycoproteins [1,2]
Results from our study suggest that immunoglobulin G (IgG) N-glycome composition is regulated through a complex interplay between loci affecting an overlapping spectrum of glycome measurements, and through interaction of genes directly involved in glycosylation and those that presumably have a ‘‘higher-level’’ regulatory function
The suggested higher-level regulation is apparent from the differences in IgG Fab and Fc glycosylation, observed in human IgG [78,79] and different myeloma cell lines [80], and further supported by recent observation that various external factors exhibit specific effects on glycosylation of IgG produced in cultured B lymphocytes [81]
Summary
Glycosylation is a ubiquitous post-translational protein modification that modulates the structure and function of polypeptide components of glycoproteins [1,2]. Variation in protein glycosylation has physiological significance, with immunoglobulin G (IgG) being a well-documented example. Each heavy chain of IgG carries a single covalently attached bi-antennary N-glycan at the highly conserved asparagine 297 residue in each of the CH2 domains of the Fc region of the molecule. The attached oligosaccharides are structurally important for the stability of the antibody and its effector functions [5]. Some 15–20% of normal IgG molecules have complex bi-antennary oligosaccharides in the variable regions of light or heavy chains [6,7]. 36 different glycans (Figure 1) can be attached to the conserved Asn297 of the IgG heavy chain [8,9], leading to hundreds of different IgG isomers that can be generated from this single glycosylation site Some 15–20% of normal IgG molecules have complex bi-antennary oligosaccharides in the variable regions of light or heavy chains [6,7]. 36 different glycans (Figure 1) can be attached to the conserved Asn297 of the IgG heavy chain [8,9], leading to hundreds of different IgG isomers that can be generated from this single glycosylation site
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