Background/Purpose: Systemic Lupus Erythematosus (SLE) is an autoimmune inflammatory disease characterized by altered T cell signaling. SLE is characterized by epigenetic mechanisms that cause hypomethylation of the inactive X-chromosome predisposing females to the disease, and a global T cell DNA hypomethylation, which causes overexpression of immune-related genes.

OGT (O-linked N-acetylglucosamine transferase) is an X-linked gene overexpressed in female lupus T cells. OGT causes posttranslational modifications to proteins by adding β-N-acetyl-glucosamine (O-GlcNAc) to Ser or Thr residues in competition with phosphorylation. OGT mRNA and protein levels and O-GlcNAc-modified proteins are increased in CD4+ experimentally demethylated T cells and in T cells from active women with lupus.

Recent evidence shows that OGT plays a critical role in chromatin structure through O-GlcNAcylation of histones in vivo and in vitro, and GlcNAc is considered now part of the histone code.

Hypothesis:  OGT overexpression in female lupus alters T cell signaling affecting posttranslational modifications of histones, thereby regulating chromatin remodeling and modifying gene expression.

Methods: PBMC from healthy donors were PHA-stimulated and treated with 5-azaC for 72 h. Then CD4+ T cells were isolated by negative selection and stimulated with PMA/Ionomycin for 6 h. T cells from lupus patients were activated with PMA/Ionomycin for 6 h. Cell fractions were obtained by using a nuclear extract kit (Active Motif) and the chromatin-bound proteins were isolated from the insoluble nuclear fraction. Subcellular fractions were subjected to SDS-PAGE gel electrophoresis followed by immunoblot. To detect glycosylated proteins we used CTD110.6 antibody (Abcam) and histone glycosylation was specifically detected by using anti-Histone H2B (glcnac S112) antibody (Abcam). T cells were nucleofected with 2µg of OGT siRNA using a nucleofector device (Amaxa).  

Results: OGT and GlcNAcylated protein levels were increased in the nuclear fraction which correlates with translocation of OGT to the nucleus after lymphocyte activation, and the increment was higher in experimentally demethylated T cells, (OGT: p=0.047 control vs 5-AzaC T cells; GlcNAcylated proteins: p=0.047 control vs 5-AzaC T cells). OGT levels in T cells from males did not show any significant difference regardless of the treatment and/or cell fraction.  We also found OGT bound to chromatin-bound proteins. H2B was GlcNAcylated at Ser112 in T cells and increased in women patients with active lupus with respect to healthy female donors (mean ± SEM: 2.85 ± 0.95; p<0.05 vs healthy donors; n=3). Histone glycosylation was inhibited in T cells transfected with OGT siRNA by 81 ± 11%, indicating the specificity of the effect.

Conclusion: These results support our hypothesis that OGT overexpression in lupus T cells causes modifications in histone glycosylation that may result in altered gene expression.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/female-specific-increase-in-t-cell-glycosylation-in-lupus/