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Functional validation of GWA hits for IgG glycosylation pleiotropic with inmflamatory diseases using CRISPR/dCas9 molecular tools

Pro- or anti-inflammatory activity of immunoglobulin G (IgG) depends largely on differential N- glycosylation of its Fc region. Alternative glycosylation affects binding efficiency of this antibody to antigens, ligands, or different complexes of the immune system, which modulates immune response. While previous genome-wide association studies (GWAS) succeeded to associate functional network of gene loci with IgG glycosylation pleiotropic with inflammatory diseases, it is still unclear whether these genes have a functional importance and if so, how they are involved in this process. For this purpose, we applied already established system based on FreeStyle™ 293-F cells, with stably integrated CRISPR/dCas9 fusions (dCas9-VPR or dCas9-KRAB) for direct regulation of the candidate genes using appropriate sgRNAs. The cell system is designed to secrete IgG molecules, so that glycans can be analyzed as the final phenotype following gene manipulations. The GWA hits were grouped with respect to different glycosylation traits such as galactosylation (KIF3C, NFKB1, MANBA, SLC38A10, TNFRSF13B, EEF1A1, HIVEP2), fucosylation (MYCBP, ACVR1C, KIF11, TBX21, TBKBP1, NFATC2), sialylation (IL6ST, GGA2, SLC17A9, SPPL3) and bisecting GlcNAC (TCF3, SMARCB1, DERL2, RRBP1, KDELR2). Out of seven genes associated with galactysylation only MANBA, NFKB1, TNFRSF13B and EEF1A1 showed change of agalactosylated structures when upregulated using dCas9-VPR. Out of six hits associated with fucosylation, only upregulation of TBX21 and TBKBP1 showed significant changes in galactosylation, but we did not detect any changes in fucosylation. This can suggest that a different cell model might be preferable to validate if these GWA hits are responsible for fucosylation, since glycans on IgG produced from FreeStyle™ 293-F cells possess only core fucose. Out of five loci associated with bisecting GlcNac, only the upregulation of KDELR2 produced an increase in biantennary glycan structures with bisecting GlcNac. The upregulation of DERL2 and RRBP1 resulted in an increase of digalactosylated biantennary glycans. Out of four GWA hits for sialylation, only downregulation of SPPL3 led to hyperglycoslation with the corresponding increase in sialylated and galactosylated structures and decrease in agalactosylated glycans, even though all the genes were successfully up - or downregulated. In conclusion, in our cell model, which produces human plasma-like IgG, we have proven the functional role of several GWA hits which are not glycosyltransferases but are associated with the IgG glycosylation pathway. Ongoing research will unravel the exact role of these genes in an integrated picture of molecular mechanism regulating IgG glycosylation.

GWAS, IgG, inflamatory diseases, CRISPR/dCas9

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