Mapping of the gene network that regulates glycan clock of ageing

Abstract

Glycans are an essential structural component of Immunoglobulin G (IgG) that modulate its structure and function. However, regulatory mechanisms behind this complex posttranslational modification are not well known. Previous genome-wide association studies (GWAS) identified 29 genomic regions involved in regulation of IgG glycosylation, but only a few were functionally validated. One of the key functional features of IgG glycosylation is the addition of galactose (galactosylation). We performed GWAS of IgG galactosylation (N=13,705) and identified 16 significantly associated loci, indicating that IgG galactosylation is regulated by a complex network of genes that extends beyond the galactosyltransferase enzyme that adds galactose to IgG glycans. Gene prioritization identified 37 candidate genes. Using a recently developed CRISPR/dCas9 system we manipulated gene expression of candidate genes in thein vitroIgG expression system. Up- and downregulation of three genes,EEF1A1,MANBAandTNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

Long-Term Culturing of FreeStyle 293-F Cells Affects Immunoglobulin G Glycome Composition

“…Moreover, a key glycosyltransferase, B4GALT1 (β-1,4-galactosyltransferase 1), which catalyzes the addition of galactose, had a completely different pattern of expression in two analyzed experiments from the pattern of IgG galactosylation ( Figure 3 ). This was in line with previous genome-wide association studies (GWAS) of IgG glycosylation, which discovered that many genetic loci that associate with IgG glycosylation have no known role in the process of glycosylation [ 49 , 50 , 51 , 52 , 53 ]. So, we integrated the data from different GWA studies of IgG glycosylation and focused on changes of expression in IgG glycome GWAS hits in our two time course experiments.…”

“…At the same time, we found that the upregulation of another galactosylation-associated gene, SPINK4, led only to a decrease of G1 structures in the IgG glycome [65]. A different pattern of change for G1 and G2 glycans was also observed in consecutive studies performed in our lab [44,53,65]. Taken together, these results suggest that different regulatory mechanisms control IgG mono-and digalactosylation.…”

“…This finding is especially important in light of recent studies showing that galactose attached to IgG glycans can have a strong inflammatory potential, emphasizing the need to tightly control IgG galactosylation in both in vitro and in vivo systems [ 67 , 68 , 69 , 70 , 71 ]. On the other hand, previous studies demonstrated that the FreeStyle 293-F cell line is a good model with which to study the mechanisms underlying IgG glycosylation if appropriate controls are included in the experiments [ 44 , 53 , 65 ]. Considering that IgG galactosylation is the most variable trait in humans, the observation that the same is true in FreeStyle 293-F cells can only reinforce the notion that these cells can serve as an adequate model for specific studies of IgG glycosylation.…”

“…We compared the GWAS-prioritized genes with the transcriptome data from the time course experiments. Interestingly, the expression of two galactosylation-specific GWAS hits [ 53 ], EEF1A1 and SLC38A10 , changed significantly at the analyzed time points. At every time point when IgG galactosylation decreased, the expression of EEF1A1 increased and vice versa.…”

“…The opposite was true for SLC38A10, whose expression was positively correlated with IgG galactosylation changes. We recently manipulated the expression of both genes using CRISPR tools to functionally validate their role in IgG galactosylation in the FreeStyle 293-F cells [ 53 ]. dCas9-VPR mediated upregulation of SLC38A10 resulted in no change of IgG glycosylation, while the upregulation of EEF1A1 led to a decrease of galactosylated IgG glycans accompanied with an increase of agalactosylated IgG glycans, the same phenotype we observed in this study.…”