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

Abstract

Age and sex dependence of subclass specific immunoglobulin G (IgG) Fc N-glycosylation was evaluated for 1709 individuals from two isolated human populations. IgGs were obtained from plasma by affinity purification using 96-well protein G monolithic plates and digested with trypsin. Fc Nglycopeptides were purified and analyzed by negative-mode MALDI-TOF-MS with 4-chloro-α-cyanocinnamic acid (Cl-CCA) matrix. Age-associated glycosylation changes were more pronounced in younger individuals (<57 years) than in older individuals (>57 years) and in females than in males. Galactosylation and sialylation decreased with increasing age and showed significant sex dependence. Interestingly, the most prominent drop in the levels of galactosylated and sialylated glycoforms in females was observed around the age of 45 to 60 years when females usually enter menopause. The incidence of bisecting N-acetylglucosamine increased in younger individuals and reached a plateau at older age. Furthermore, we compared the results to the total IgG N-glycosylation of the same populations recently analyzed by hydrophilic interaction liquid chromatography (HILIC). Significant differences were observed in the levels of galactosylation, bisecting N-acetylglucosamine and particularly sialylation, which were shown to be higher in HILIC analysis. Age and sex association of glycosylation features was, to a large extent, comparable between MALDI-TOF-MS and HILIC IgG glycosylation profiling.

A prominent lack of IgG1-Fc fucosylation of platelet alloantibodies in pregnancy

“…The main difference between the patient groups was observed for core fucosylation, with the majority of the FNAIT patients with anti-HPA-1a-specific antibodies showing a markedly lowered IgG1-Fc fucosylation in pregnancy (levels down to 10% fucosylation), a very atypical form of IgG-Fc glycosylation (normal values being .90%). 13,37 To our knowledge, a lowered fucosylation has only been found for HIV-specific antibodies in elite controllers of HIV infection, but unlike in our study, these also displayed agalactosylated and asialylated glycans. 38 We further observed normal high levels of core fucosylation for anti-HLA class I antibodies (RT patients), of which 12 of the 13 patients were women who we cannot exclude to have been immunized already during a previous pregnancy.…”

Estrogens regulate glycosylation of IgG in women and men

“…It is therefore possible that, by providing more galactose substrate, estrogens could indirectly promote greater IgG Fc sialylation. Consistent with this possibility, women but not men manifest a decrease in IgG Fc sialylation around the transition between the fifth and sixth decade of life (9). Further study is required to assess whether gonadal hormones affect sialylation, fucosylation, and other glycan features independently of galactosylation.…”

“…Gender differences have been noted to extend to these features as well (9,12). Galactosylation is required for attachment of sialic acid (see Figure 1), and galactosylation and sialylation frequently exhibit marked colinearity (2,12).…”

“…Epidemiological data suggest that endocrine factors may play a role. Population studies reveal an increase in G0 glycans in midlife in women but not in men (9)(10)(11)(12). During pregnancy, a marked…”

Elevated core-fucosylated IgG is a new marker for hepatitis B virus-related hepatocellular carcinoma

“…6 Structural details of the attached glycans are of great physiological significance and are associated with many pathological conditions. 6,7 For example, levels of galactose-deficient IgG are increased in liver fibrosis, cirrhosis, and ovarian and lung cancer [8][9][10] and increased core fucosylated IgG was described as a diagnostic marker in ovarian cancer. 11 Additionally, individual variation in IgG glycosylation changes during acute systemic inflammation was associated with increased mortality risk.…”