engleski

Mapping the estradiol signalling network that regulates immunoglobulin G glycosylation using CRISPR/dCas9 based Freestyle293-F transient expression system

Immunoglobulin G (IgG) is a glycoprotein with a central role in adaptive immunity. Glycosylation of Fc domain defines IgG function and different studies link the change in IgG glycosylation with disease and aging. In healthy women, the most prominent change coincides with perimenopause, and a recent study has revealed that estradiol (E2) is involved in regulation of IgG glycosylation. Analysis of the Signaling Pathway Projects (SPP) web knowledgebase revealed that E2 affects expression of four genes with yet unknown role in IgG glycosylation, three of them being associated in previous genome-wide asLCKsociation studies (GWAS) with galactosylation (RUNX1, RUNX3, SPINK4) and one with sialylation (ELL2). To map downstream pathways linking E2 signaling and IgG glycosylation we utilized our FreeStyleTM293-F transient system, expressing IgG antibodies, for targeted manipulation of candidate loci. This system exploits stably integrated CRISPR dCas9-VPR or dCas9-KRAB expression cassettes for targeted activation or silencing of genes via transient transfection of cells with plasmids carrying specific gRNAs and recombinant IgG. Using this cell system we upregulated and downregulated RUNX1, RUNX3, SPINK4 and ELL2 loci but only upregulation of RUNX3 (Runt-related factor 3) and SPINK4 (Serine Peptidase Inhibitor Kazal Type 4) resulted in alternative IgG glycosylation. Upregulation of RUNX3 resulted in a significant decrease of galactosylated glycans accompanied with an increase of agalactosylated glycans. Upregulation of SPINK4 was also accompanied with a decrease of galactosylated glycans, but the ratio of agalactosylated glycans was unchanged. We hypothesized that RUNX3 acts as B4GALT1 repressor considering its role as a transcription factor that can either activate or suppress transcription. However, following RUNX3 upregulation, expression of B4GALT1 gene remained stable. To further investigate RUNX3 signaling, possibly involved in alternative IgG glycosylation, the total cell transcriptome was analyzed following RUNX3 overexpression. In sum, the results suggest a novel mechanism through which E2 could regulate IgG glycosylation, specifically galactosylation. Moreover, this was the first in vitro functional validation of RUNX3 and SPINK4, the GWAS hits associated with IgG glycosylation.

IgG glycosylation, estrogen, gene regulation, RUNX3, CRISPR/dCas9

nije evidentirano