Site-specific glycosylation of human immunoglobulin G is altered in four rheumatoid arthritis patients

Abstract

Alterations in the glycosylation of human IgG have been shown to occur in rheumatoid arthritis (RA). However, the precise nature and location of these changes have not been fully established. Therefore we carried out a detailed analysis of the oligosaccharides chemically released from intact human serum IgG and fragments of the molecule. Serum samples were from three healthy ('normal') individuals, and from four patients with RA. Site-specific glycolsylation of the glycoprotein was shown to occur, which extended to sites even within the Fab fragment. These were differences in galactosylation, sialylation and the presence of a bisecting N-acetylglucosomide. Disease related alterations were also shown to be site-specific. In particular, an increase in the proportion of agalactosylated oligosaccharides occurred on the Fc fragment in RA (P=0.057), but, in contrast to previous reports there was an increase on the light chain in the proportion of fully galactosylated, bisected and core fucosylated oligosaccharides (from 13% of total in normal to between 18 and 35% in RA, P=0.057)). There was also an Fab-specific increase in oligosaccharides bearing a bisecting N-acetylglucosamine and a core fucose (P=0.075) The site-specific glycosylation changes described in this paper reveal the complexity of the regulatory mechanism, perhaps reflecting the many levels at which regulation can occur.

The Glycosylation and Structure of Human Serum IgA1, Fab, and Fc Regions and the Role of N-Glycosylation on Fcα Receptor Interactions

“…The proportion of bisected structures in pooled serum IgA1 (24%) was higher than that in IgG (14%; Ref. 59) although lower than that previously reported for IgA1 isolated from individuals (40%; Ref. 14).…”

“…In contrast, the C␥2 N-glycans of IgG1 extend from the N terminus of the domain into the interstitial region between the two heavy chains and interact with the protein surface (28,76). This limits C␥2 N-glycan processing such that 25-35% of glycans are not galactosylated and Ͻ10% are sialylated (31,59,68,77). On a molecular level, this suggests that the K m for the galactosyltransferase is higher for IgG than for IgA and that, in the case of IgG, the enzyme levels are insufficient to ensure that all of the glycans exposed on the CH2 domains are galactosylated.…”

High-Throughput IgG Fc N-Glycosylation Profiling by Mass Spectrometry of Glycopeptides

“…Increased levels of these glycosylation features observed in the HILIC analysis may largely be linked to the inclusion of Fab glycans, as Fab glycans are known to show an increased incidence of fully galactosylated, sialylated, and bisected structures compared to the Fc. 12,14,15,36 However, the analysis of sialylation levels by mass spectrometry is complicated by two phenomena: first, the charge introduced by the sialic acid will influence ionization. It may be assumed that negative-mode ionization of the sialylated species was more efficient than the ionization of glycopeptides with nonsialylated, neutral glycan chains.…”

Function characterization of a glyco-engineered anti-EGFR monoclonal antibody cetuximab in vitro

“…Considering the different oligosaccharide profiles of IgG Fab and Fc fragments revealed in some case-control studies [22,23] , we also analyzed the N-glycan profiles of antibody Fab and Fc fragments. Both the Fab and Fc fragments of the bisec-EGFR mAb showed an increased bisecting GlcNAc content compared to the wild type EGFR mAb, but the Fab and Fc fragments had different N-glycan profiles from each other.…”