High Throughput Isolation and Glycosylation Analysis of IgG–Variability and Heritability of the IgG Glycome in Three Isolated Human Populations

Abstract

All immunoglobulin G molecules carry N-glycans, which modulate their biological activity. Changes in N-glycosylation of IgG associate with various diseases and affect the activity of therapeutic antibodies and intravenous immunoglobulins. We have developed a novel 96-well protein G monolithic plate and used it to rapidly isolate IgG from plasma of 2298 individuals from three isolated human populations. N-glycans were released by PNGase F, labeled with 2-aminobenzamide and analyzed by hydrophilic interaction chromatography with fluorescence detection. The majority of the structural features of the IgG glycome were consistent with previous studies, but sialylation was somewhat higher than reported previously. Sialylation was particularly prominent in core fucosylated glycans containing two galactose residues and bisecting GlcNAc where median sialylation level was nearly 80%. Very high variability between individuals was observed, approximately three times higher than in the total plasma glycome. For example, neutral IgG glycans without core fucose varied between 1.3 and 19%, a difference that significantly affects the effector functions of natural antibodies, predisposing or protecting individuals from particular diseases. Heritability of IgG glycans was generally between 30 and 50%. The individual's age was associated with a significant decrease in galactose and increase of bisecting GlcNAc, whereas other functional elements of IgG glycosylation did not change much with age. Gender was not an important predictor for any IgG glycan. An important observation is that competition between glycosyltransferases, which occurs in vitro, did not appear to be relevant in vivo, indicating that the final glycan structures are not a simple result of competing enzymatic activities, but a carefully regulated outcome designed to meet the prevailing physiological needs.

Classical galactosaemia: novel insights in IgG N-glycosylation and N-glycan biosynthesis

“…The assignment of glycan structures by HILIC-UPLC in serum was performed by comparison with an updated GlycoBase 3.2 (http://glycobase.nibrt.ie/), as well as published assignments. 13,32 Assignment of the IgG glycans in each peak was based on the analysis in Pucic et al 34 Glycan feature statistical analysis…”

Effects of temporary low-dose galactose supplements in children aged 5–12 y with classical galactosemia: a pilot study

“…Galactosylation of IgG is one of the most comprehensively studied glycosylation features, and galactose ratios (G-ratios) have been identified as highly informative indicators of the level of galactose incorporation (8). We determined peak % areas of agalactosylated (G0), mono-(G1) and digalactosylated (G2) structures to provide a quantitative measurement of galactose incorporation into released undigested IgG N-glycans.…”

“…Glycosylation is a widespread post-translational modification of proteins and deregulation of glycosylation is associated with a wide range of diseases (8). Along these lines, we have previously described galactose incorporation ratios as a method of studying the presence of N-glycan processing defects in children with galactosemia (7).…”

The “less-is-more” in therapeutic antibodies: Afucosylated anti-cancer antibodies with enhanced antibody-dependent cellular cytotoxicity

“…13-15 It is now widely recognized that removal of the core fucose from Fc N -glycans represents the most effective approach to enhance ADCC activity 14 15 A high-throughput study of the IgG glycome of three isolated human populations showed that most of the human plasma IgG antibodies are core fucosylated with levels of afucosylated IgG ranging from 1.3% to 19.3%, underlying the difference in ADCC efficacy of naturally occurring antibodies to protect against diseases 16 . Dramatic shifts in IgG glycan profile towards reduced galactosylation and fucosylation have been observed in human immunodeficiency virus (HIV)-specific antibodies and are associated with improved antiviral activity and HIV control 17 …”