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Effects of Estradiol on Immunoglobulin G Glycosylation: Mapping of the Downstream Signaling Mechanism

Glycosylation of immunoglobulin G (IgG) largely defines function of this antibody and regulates inflammation on multiple levels but the mechanisms and the genes involved in the process of IgG glycosylation are still vaguely understood. Genome wide association studies (GWAS) enabled identification of many genes potentially involved in IgG glycosylation but in the most cases functional validation is missing. Composition of the IgG glycome changes significantly with various diseases and ageing. In women, the most prominent change occurs during the perimenopausal period and a recent intervention study revealed that estrogen regulates IgG glycosylation. The most affected IgG glycome features are galactosylation, sialylation and structures with bisecting GlcNac. Analysis of the Signaling Pathway Projects (SPP) web knowledgebase revealed that E2 affects expression of four genes with yet unknown role in IgG glycosylation with three of them associated with galactosylation (RUNX1, RUNX3 and SPINK4) and one associated with sialylation (ELL2) through previous GWA studies. To map downstream signaling mechanisms responsible for the effect of estrogen on IgG glycome composition we utilized our recently developed in vitro IgG expression system HEK293-FS for targeted CRISPRa/CRISPRi manipulation of candidate loci. This system exploits stably integrated dCas9-VPR or dCas9-KRAB expression cassettes for targeted upregulation or downregulation of genes via transient transfection of FreeStyleTM 293-F cells with specific gRNAs. We successfully upregulated or downregulated RUNX1, RUNX3, SPINK4 and ELL2 but only upregulation of RUNX3 (Runt-related factor 3) and SPINK4 (Serine Peptidase Inhibitor Kazal Type 4) resulted with concomitant change in IgG glycome composition. Upregulation of RUNX3 resulted with significant decrease in galactosylated glycans accompanied with increase in agalactosylated glycans. Upregulation of SPINK4 was also accompanied with decrease in galactosylated glycans but the ratio of agalactosylated glycans remained stable. To further explore the role of SPINK4 and RUNX3 in IgG galactosylation we measured B4GALT1 (beta-1, 4- galactosyltransferase 1) expression after upregulation and observed no significant change in B4GALT1 transcript levels. This suggests that RUNX3 and SPINK4 do not suppress B4GALT1 on transcription level, but through some indirect pathway that should be further investigated. Taken together, the results of this study suggest the molecular mechanism through which E2 could regulate IgG glycosylation, specifically galactosylation.

estrogen, IgG, CRISPR/dCas9

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