Identification and characterization of a missense mutation in the O-linked β- N-acetylglucosamine (O-GlcNAc) transferase gene that segregates with X-linked intellectual disability

Abstract

O-GlcNAc is a regulatory post-translational modification of nucleocytoplasmic proteins that has been implicated in multiple biological processes, including transcription. In humans, single genes encode enzymes for its attachment (O-GlcNAc transferase (OGT)) and removal (O-GlcNAcase (OGA)). An X-chromosome exome screen identified a missense mutation, which encodes an amino acid in the tetratricopeptide repeat, in OGT (759G>T (p.L254F)) that segregates with X-linked intellectual disability (XLID) in an affected family. A decrease in steady-state OGT protein levels was observed in isolated lymphoblastoid cell lines from affected individuals, consistent with molecular modeling experiments. Recombinant expression of L254F-OGT demonstrated that the enzyme is active as both a glycosyltransferase and an HCF-1 protease. Despite the reduction in OGT levels seen in the L254F-OGT individual cells, we observed that steady-state global O-GlcNAc levels remained grossly unaltered. Surprisingly, lymphoblastoids from affected individuals displayed a marked decrease in steady-state OGA protein and mRNA levels. We observed an enrichment of the OGT-containing transcriptional repressor complex mSin3A-HDAC1 at the proximal promoter region of OGA and correspondingly decreased OGA promoter activity in affected cells. Global transcriptome analysis of L254F-OGT lymphoblastoids compared with controls revealed a small subset of genes that are differentially expressed. Thus, we have begun to unravel the molecular consequences of the 759G>T (p.L254F) mutation in OGT that uncovered a compensation mechanism, albeit imperfect, given the phenotype of affected individuals, to maintain steady-state O-GlcNAc levels. Thus, a single amino acid substitution in the regulatory domain (the tetratricopeptide repeat domain) of OGT, which catalyzes the O-GlcNAc post-translational modification of nuclear and cytosolic proteins, appears causal for XLID.

Figure 1.

A mutation, predicted to destabilize the protein, in OGT segregates with XLID. A, partial pedigree of L254F family (K9427). Segregation of the mutation, c.759G>T, in OGT in family K9427. Genotype is given below the pedigree symbol. Red letters below symbols indicate control cell line (C1–C3), carrier female cell line (CA), or affected cell line (P1 and P2). Black squares indicate affected males, whereas a black dot inside the circle indicates a confirmed carrier female with skewed X-inactivation. B, modeling of the side chain conformation of the L254F variant in the OGT protein predicts loss in stability.

Figure 2.

L254F-OGT has reduced protein levels and half-life. A, immunoblotting (IB) of equal amounts of crude lysates displays a decrease in steady-state OGT levels in XLID lymphoblastoids (P1 and P2) when compared with a control female carrier (CA) using three independent antibodies to OGT. β-Actin immunoblotting was performed to confirm equal loading. B, immunoblotting of equal amounts of crude lysates from XLID lymphoblastoids (P1 and P2) displays less OGT protein when compared with control male relatives (C1–C3) or the control female carrier (CA) using a fourth α-OGT (11576-2-AP) antibody. α-Tubulin immunoblotting was performed to confirm equal loading. C, OGT has a reduced half-life in an XLID lymphoblastoid (P1) compared with control (C1) as measured by immunoblotting over time following blocking of translation with cycloheximide. For β-actin, which has a half-life in excess of 48 h, immunoblotting was performed to confirm equal loading. Blots shown are representative of results from three independent replicates.

Figure 3.

L254-OGT is an active glycosyltransferase and HCF1-protease. A, O-GlcNAc levels are elevated when WT or L254F-OGT is transiently overexpressed, compared with control (enhanced GFP (eGFP)), in HEK293F cells as measured by immunoblotting (IB) with a pan-O-GlcNAc antibody (110.6). B, purified WT and L254F-OGT are capable of glycoslyating purified CK2α. C, purified WT and L254F-OGT enzymes can glycosylate and cleave HCF1-rep1, whereas the catalytically compromised K852M-OGT can catalyze neither reaction. Blots shown are representative of results from at least three independent replicates.

Figure 4.

Global O-GlcNAc levels remain unaltered in XLID lymphoblastoids. Immunoblotting (IB) of equal amounts of crude lysates with pan-O-GlcNAc antibodies (mAb14 and 110.6) from carrier (CA)-affected L254F-OGT (P1 and P2) and unaffected (C1–C3) males detects no significant changes. Blots shown are representative of results from at least three independent replicates.

Figure 5.

OGA protein, mRNA, and promoter activity are reduced in XLID lymphoblastoids. A, immunoblotting (IB) of equal amounts of crude lysates displays a decrease in steady-state OGA levels in XLID lymphoblastoids (P1 and P2) when compared with a control female carrier (CA) using an α-OGA antibody (Whiteheart Ab). B, immunoblotting of equal amounts of crude lysates from XLID lymphoblastoids (P1 and P2) displays less OGA protein when compared with control related males (C1–C3) using an α-OGA antibody (14711-1-AP). Blots shown in A and B are representative of results from at least three independent replicates. C, steady-state OGA mRNA levels assayed by quantitative RT-PCR show decreased transcript levels in XLID lymphoblastoids (P1 and P2) compared with a control male, C1 (n = 3, p < 0.05). D, XLID lymphoblastoids (P1 and P2) displayed less luciferase activity compared with a control male (C1) when transfected with a plasmid containing 2 kb of the proximal promoter region of OGA driving luciferase expression (n = 3, p ≤ 0.001).

Figure 6.

OGT-mSin3A-HDAC1 complex is enriched at the OGA promoter in XLID lymphoblastoids. ChIP analyses of the OGA proximal promoter following enrichment with antibodies to OGT (A), O-GlcNAc (B), mSin3A (C), and HDAC1 (D) show enrichment in XLID lymphoblastoids (P1 and P2) compared with a control male (C1). All values were determined using qPCR relative to the percentage of input and are presented as -fold enrichment on the y axis with different replicates on the x axis.

Figure 7.

Gene expression is differentially regulated in XLID lymphoblastoids. A, Spearman correlation analysis shows segregation of RNA-seq data by disease (P1 and P2 expression data are more similar to one another than to either C, and this is also true for C1 and C2). B, the vast majority of transcripts change by < 3-fold (log₂ = 1.58). C, approximately 1% of quantifiable transcripts are altered 3-fold in XLID lymphoblastoids (P1 and P2) compared with control males (C1 and C2). D, number of quantifiable genes under differential -fold expression sets by disease versus natural variation.