Croatian Science Foundation Project (HRZZ project)
Epigenetic regulation of immunoglobulin G glycosylation (EpiGlycoIgG)
Changes in protein glycosylation are reported in different inflammatory and autoimmune diseases. Immunoglobulin G (IgG) is an excellent model to study importance of glycosylation for protein function since it has only one glycosylation site on each heavy chain (Figure 1). The glycans attached to IgG are essential structural components of the Fc region and a minute change in glycan composition can significantly change the conformation of the Fc region with dramatic consequences for IgG effector function. Fab is determined early in the B cell development, however effector function of an antibody is defined only when the antibody comes in contact with an antigen. Our preliminary results show that different clones of hybridomas (mouse model for B cells) undergo distinct IgG glycosylation (Figure 2) suggesting that, once it established, the pattern of glycosylation stays stable. We believe that distinct IgG glycosylation patterns in different B cell clones are fixed by epigenetic mechanisms, most probably DNA methylation (Figure 3).
Figure 1: Immunoglobulin G (IgG) has only one glycosylation site on each heavy chain: a glycan part is attached to a highly conserved Asn residue of the Fc region of an IgG antibody.
We aim to explore the importance of the GWAS hits for IgG glycosylation by studying promoter methylation and expression of these genes and correlating the data with N-glycans on IgG secreted from hybridoma and/or lymphoblastoma cells (human model for B cells). The main goal is to understand how patterns of IgG glycosylation are established during development and maturation of B cells and their differentiation to plasma cells. We will apply CRISPR/Cas9-based methodology for genome editing (gene knockouts) as well as for epigenome editing (modulation of CpG methylation). Also, epigenetic regulation of candidate genes will be examined by study of epigenetic modifications such as CpG methylation (using bisulphite pyrosequencing) and histone modifications (using ChIP-qPCR and ChIP-seq methods). Besides answering to fundamental biological questions, the results are expected to shed light on mechanisms involved in development and progression of complex diseases such as inflammatory bowel diseases, HNF1A-MODY subtype of diabetes and cancer.
Figure 2: IgG glycosylation patterns are distinct for different clones of hybridomas but stay stable during time: different colours present different glycans on Fc region of IgG. Each hybridoma clone is presented at three time points (first three lines).
Type of funding scheme: Research projects
Host institution: Faculty of Science University of Zagreb
Project leader: Prof Vlatka Zoldoš, PhD, Faculty Science University of Zagreb
HRZZ Funding: HRK 1.000.000,00