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Functional validation of GWAS hits associated with IgG glycosylation using CRISPR/dCas9 transient expression system

Alternative glycosylation of immunoglobulin G (IgG) Fc region has a crucial role in defining pro- or anti-inflammatory effector function of the antibody. Gene network involved in regulation of IgG glycosylation is still poorly understood because glycoyltransferases and glycosydases are not the major players in this regulatory process. In this study we functionally validated gene loci associated with IgG glycosylation in previous genome-wide association studies (GWAS). We utilized established stably transfected cell line FreeStyle™ 293-F with CRISPR/ dCas9 fusions for direct regulation of genes targeted with specific sgRNAs. This cell system was designed to secrete IgG molecules so that glycans on IgG can be analysed following gene manipulations. We manipulated 22 GWAS hits, grouped according to glycosylation traits such as galactosylation, fucosylation, sialylation and bisecting GlcNAC. Following gene expression manipulations, IgG glycosylation was analysed. Out of seven genes associated with galactosylation, only MANBA, HIVEP2, TNFRSF13B and EEF1A1 showed change of agalactosylated structures when upregulated using dSaCas9-VPR. Out of six hits associated with fucosylation, only upregulation of TBX21 and TBKBP1 showed significant changes in galactosylation, but no change was observed for fucosylated glycan structures. These results suggest that a different cell model might be preferable to validate different GWA hits, such as Lymphoblastoid Cell Line (LCL) which is known to be rich in fucose glycan structures. Out of five loci associated with bisecting GlcNac, upregulation of KDELR2 resulted in 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 hyperglycosylation with concomitant increase in sialylated and galactosylated structures and decrease in agalactosylated glycans, despite the fact that all genes were successfully up- or downregulated. Overall, these results have proven the functional role of several GWA hits which are not glycosyltransferases but are associated with the IgG glycosylation pathway. Ongoing research on LCL cell line might unravel the exact role of these and other GWAS hits.

CRISPR/dCas9, IgG glycosylation, FreeStyle™ 293-F Cells, GWAS

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