Glycosylation of IgG regulates the effector function of this antibody in the immune response. Glycosylated IgG is a potent therapeutic used for both research and clinical purposes. While there is ample research on how different cell culture conditions affect IgG glycosylation, the data are missing on the stability of IgG glycome during long cell passaging, i.e., cell “aging”. To test this, we performed three independent time course experiments in FreeStyle 293-F cells, which secrete IgG with a human-like glycosylation pattern and are frequently used to generate defined IgG glycoforms. During long-term cell culturing, IgG glycome stayed fairly stable except for galactosylation, which appeared extremely variable. Cell transcriptome analysis revealed no correlation in galactosyltransferase B4GALT1 expression with galactosylation change, but with expression of EEF1A1 and SLC38A10, genes previously associated with IgG galactosylation through GWAS. The FreeStyle 293-F cell-based system for IgG production is a good model for studies of mechanisms underlying IgG glycosylation, but results from the present study point to the utmost importance of the need to control IgG galactosylation in both in vitro and in vivo systems. This is especially important for improving the production of precisely glycosylated IgG for therapeutic purposes, since IgG galactosylation affects the inflammatory potential of IgG.

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

Aging, a complex biological process, involves the progressive decline of physiological functions across various systems, leading to increased susceptibility to neurodegenerative diseases. In society, demographic aging imposes significant economic and social burdens due to these conditions. This review specifically examines the association of protein glycosylation with aging and neurodegenerative diseases. Glycosylation, a critical post-translational modification, influences numerous aspects of protein function that are pivotal in aging and the pathophysiology of diseases such as Alzheimer's disease, Parkinson's disease, and other neurodegenerative conditions. We highlight the alterations in glycosylation patterns observed during aging, their implications in the onset and progression of neurodegenerative diseases, and the potential of glycosylation profiles as biomarkers for early detection, prognosis, and monitoring of these age-associated conditions, and delve into the mechanisms of glycosylation. Furthermore, this review explores their role in regulating protein function and mediating critical biological interactions in these diseases. By examining the changes in glycosylation profiles associated with each part, this review underscores the potential of glycosylation research as a tool to enhance our understanding of aging and its related diseases. 

Glycosylation in aging and neurodegenerative diseases

INTRODUCTION
Glycans attached to immunoglobulin G are an important regulator of chronic systemic inflammation, one of the key drivers of aging. As people age, glycans that suppress inflammation are being replaced with inflammation-promoting glycans, but the rate of this conversion is highly individual and is affected by genetic, epigenetic, and environmental factors.


AREAS COVERED
This review summarizes key studies of IgG glycosylation changes in aging and disease, effects of lifestyle and pharmacological interventions, and mechanisms that regulate IgG glycosylation.


EXPERT OPINION
IgG glycome is an important contributor to the process of aging that can be modulated by both lifestyle and pharmacological interventions. Small molecule drugs that would suppress chronic systemic inflammation by modulation of the IgG glycome are still not available, but since gene network regulating IgG glycosylation has been identified and a high-throughput in vitro screening system is available, it is likely that this highly innovative approach to manage chronic systemic inflammation will be developed soon.

Can we suppress chronic systemic inflammation and postpone age-related diseases by targeting the IgG glycome?

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the 10 candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the-art clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9) molecular tool for the downregulation of the HNF1A, HNF4A, and FOXA2 genes in HepG2 cells—a human liver cancer cell line. The results show that the downregulation of all three genes individually and in pairs affects the transcriptional activity of many glyco-genes, although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures. The effect is better seen as an overall change in the total HepG2 N-glycome, primarily due to the extension of biantennary glycans. We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure. We also propose a model showing feedback loops with the mutual activation of HNF1A–FOXA2 and HNF4A–FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells. 

Transcription Factors HNF1A, HNF4A, and FOXA2 Regulate Hepatic Cell Protein N-Glycosylation

Summary All four subclasses of immunoglobulin G (IgG) antibodies have glycan structures attached to the protein part of the IgG molecules. Glycans linked to the Fc portion of IgG are found in all IgG antibodies, while about one‐fifth of IgG antibodies in plasma also have glycans attached to the Fab portion of IgG. The IgG3 subclass is characterized by more complex glycosylation compared to other IgG subclasses. In this review, we discuss the significant influence that glycans exert on the structural and functional properties of IgG. We provide a comprehensive overview of how the composition of these glycans can affect IgG's effector functions by modulating its interactions with Fcγ receptors and other molecules such as the C1q component of complement, which in turn influence various immune responses triggered by IgG, including antibody‐dependent cell‐mediated cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC). In addition, the importance of glycans for the efficacy of therapeutics like monoclonal antibodies and intravenous immunoglobulin (IVIg) therapy is discussed. Moreover, we offer insights into IgG glycosylation characteristics and roles derived from general population, disease‐specific, and interventional studies. These studies indicate that IgG glycans are important biomarkers and functional effectors in health and disease. 

The importance of <scp>IgG</scp> glycosylation—What did we learn after analyzing over 100,000 individuals

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 the in vitro IgG expression system. Up- and downregulation of three genes, EEF1A1, MANBA and TNFRSF13B, changed the IgG glycome composition, which confirmed that these three genes are involved in IgG galactosylation in this in vitro expression system.

Mapping of the gene network that regulates glycan clock of ageing

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Mapping of the gene network that regulates glycan clock of ageing