To orchestrate a successful pregnancy, the maternal body induces adaptations in many biological processes, both locally and systemically. In collaboration with the Erasmus MC, we have been studying pregnancy-associated systemic serum glycome changes. The serum samples provided by the Erasmus MC – collected before, during and after pregnancy – have proven to be an ideal biological toolbox to show the applicability of our high-throughput methods.
At the targeted glycoprotein level, we have shown changes on the constant region of immunoglobulin G(IgG) associated with a lower inflammatory state.1 The variable region, while occupied by a largely different glycan population, shows similar pregnancy-associated changes.2 Interestingly, on another class of immunoglobulins, IgA, only few and minor glycosylation changes were found.3 Furthermore, we have been able to monitor total serum glycome changes, although the direct link to function is yet to be determined.4,5
Currently, together with our collaborators at the Erasmus MC, but also within the LUMC and with others, we are trying to better understand how the glycomics changes are induced, e.g. by hormones and/or cytokines, with a focus on immunoglobulin G.
Protein glycosylation is dependent on the physiological state of an organism and can vary with age and various metabolic diseases. This makes glycosylation a potential marker for health and longevity. The Leiden Longevity cohort set up by the Molecular Epidemiology (Molepi) department of the LUMC provides a unique opportunity to investigate the biological processes behind aging. The cohort consists of people with a hereditary predisposition towards longevity, as well as their age-matched partners as controls.
Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), we previously found a significant correlation between longevity and the incidence of a bisecting N-acetylglucosamine on immunoglobulin G (IgG) N-glycans.6 Furthermore, high performance liquid chromatography (HPLC) analysis of the total profile of released plasma N-glycans revealed two afucosylated N-glycans which exhibited association with longevity.7 A high-throughput method involving affinity-capturing of proteins and capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) of released N-glycans was developed to evaluate the glycosylation of single proteins.8 Using this method, it was reported that the glycosylation of immunoglobulin A (IgA) and alpha1-antitrypsin (AAT) displays age- and sex-dependence, but a correlation with longevity was not observed.8
Research into protein glycosylation within the Leiden Longevity cohort is still ongoing. Recently developed methodologies will be used in order to gain a better understanding of the role of glycosylation in aging.
Next to the glycosidic changes during disease, we focus on exploring protein glycosylation in healthy volunteers, especially of the antibody immunoglobulin G (IgG). The glycosylation found in the constant region (Fc) of the antibody is well studied, for example how it changes during normal aging and pregnancy.2,9 To this knowledge base, we recently added information about the IgG Fc-glycosylation in healthy newborns and young children.10 This revealed that during childhood the IgG Fc-glycosylation behaves differently than in adults. For example, no difference in glycosylation between the sexes was observed in children, while this is the case for adults. Furthermore, as opposed to the decreasing adult IgG Fc-galactosylation with age, the galactosylation in children between 3 months and 17 years old stayed relatively constant.10
We anticipate to use our knowledge about IgG Fc-glycosylation in healthy children to study the changes of this glycosylation in diseased situations. Concrete examples are our studies into pediatric meningitis infections and stem cell transplantations. With these studies, we hope to obtain more knowledge about the regulation of IgG glycosylation and to find markers to predict disease outcome.
- Selman, M. H. J. et al. Fc specific IgG glycosylation profiling by robust nano-reverse phase HPLC-MS using a sheath-flow ESI sprayer interface. J. Proteomics 75, 1318-1329 (2012).
- Bondt, A. et al. Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes. Mol. Cell. Proteomics 13, 3029-3039 (2014).
- Bondt, A. et al. Longitudinal monitoring of immunoglobulin A glycosylation during pregnancy by simultaneous MALDI-FTICR-MS analysis of N- and O-glycopeptides. Sci. Rep. 6, 27955 (2016).
- Ruhaak, L. R., Uh, H.-W., Deelder, A. M., Dolhain, R. E. J. M. & Wuhrer, M. Total Plasma N-Glycome Changes during Pregnancy. J. Proteome Res. 13, 1657-1668 (2014).
- Jansen, B. C. et al. Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS. Sci. Rep. 6, 23296 (2016).
- Ruhaak, L. R. et al. Decreased levels of bisecting GlcNAc glycoforms of IgG are associated with human longevity. PLoS One 5, e12566 (2010).
- Ruhaak, L. R. et al. Plasma protein N-glycan profiles are associated with calendar age, familial longevity and health. J. Proteome Res. 10, 1667-1674 (2011).
- Ruhaak, L. R. et al. Targeted biomarker discovery by high throughput glycosylation profiling of human plasma alpha1-antitrypsin and immunoglobulin A. PLoS One 8, e73082 (2013).
- Bakovic, M. P. et al. High-throughput IgG Fc N-glycosylation profiling by mass spectrometry of glycopeptides. J. Proteome Res. 12, 821-831 (2013).
- de Haan, N., Reiding, K. R., Driessen, G., van der Burg, M. & Wuhrer, M. Changes in Healthy Human IgG Fc-Glycosylation after Birth and during Early Childhood. J. Proteome Res. 15, 1853-1861 (2016).