Characterization of missense mutations in the tetratricopeptide region of O-GlcNAc transferase found in patients with X-linked intellectual disability
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE110616 | GEO | 2018/04/30
REPOSITORIES: GEO
Similar Datasets
Project description:An X-chromosome exome sequencing analysis identified a mutation in O-GlcNAc transferase (OGT) (pL254F) in a family with X-linked intellectual disability (XLID). Affected patient lymohoblastoids exhibit decreased steady state OGT levels owing to an unstable protein compared to the unaffected, related male controls. Suprisingly, global O-GlcNAc levels remained remained unaltered. This prompted us to check the both protein and mRNA levels of the other cycling enzyme, O-GlcNAcase (OGA) which was also lowered. This implies that a compensation mechanism exists, however imperfect, owing to the disease state of the individuals. We performed glabal transcriptome analysis to assess any other changes in message between patients and controls. Our study highlights small differences in the global transcriptome of patient lymhoblastoids that have the L254F mutation in O-GlcNAc transferase when compared to familial controls using Illumina sequencing of total RNA
2016-02-25 | E-GEOD-74263 | biostudies-arrayexpress
Project description:An X-chromosome exome sequencing analysis identified a mutation in O-GlcNAc transferase (OGT) (pL254F) in a family with X-linked intellectual disability (XLID). Affected patient lymohoblastoids exhibit decreased steady state OGT levels owing to an unstable protein compared to the unaffected, related male controls. Suprisingly, global O-GlcNAc levels remained remained unaltered. This prompted us to check the both protein and mRNA levels of the other cycling enzyme, O-GlcNAcase (OGA) which was also lowered. This implies that a compensation mechanism exists, however imperfect, owing to the disease state of the individuals. We performed glabal transcriptome analysis to assess any other changes in message between patients and controls.
2016-02-25 | GSE74263 | GEO
Project description:O-linked ?-N-acetyl-D-glucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential post-translational modification that is abundant in the brain. Recently, OGT mutations have been associated with intellectual disability, although it is not understood how they affect OGT structure and function. Using a multi-disciplinary approach we show that the L254F OGT mutation leads to conformational changes of the tetratricopeptide repeats and reduced activity, revealing the molecular mechanisms contributing to pathogenesis.
| S-EPMC5967971 | biostudies-literature
Project description:Intellectual disability (ID) is a neurodevelopmental condition that affects ~1% of the world population. In total 5-10% of ID cases are due to variants in genes located on the X chromosome. Recently, variants in OGT have been shown to co-segregate with X-linked intellectual disability (XLID) in multiple families. OGT encodes O-GlcNAc transferase (OGT), an essential enzyme that catalyses O-linked glycosylation with β-N-acetylglucosamine (O-GlcNAc) on serine/threonine residues of thousands of nuclear and cytosolic proteins. In this review, we compile the work from the last few years that clearly delineates a new syndromic form of ID, which we propose to classify as a novel Congenital Disorder of Glycosylation (OGT-CDG). We discuss potential hypotheses for the underpinning molecular mechanism(s) that provide impetus for future research studies geared towards informed interventions.
| S-EPMC7253464 | biostudies-literature
Project description:O-GlcNAc transferase (OGT) is an X-linked gene product that is essential for normal development of the vertebrate embryo. It catalyses the O-GlcNAc posttranslational modification of nucleocytoplasmic proteins and proteolytic maturation of the transcriptional coregulator Host cell factor 1 (HCF1). Recent studies have suggested that conservative missense mutations distal to the OGT catalytic domain lead to X-linked intellectual disability in boys, but it is not clear if this is through changes in the O-GlcNAc proteome, loss of protein-protein interactions, or misprocessing of HCF1. Here, we report an OGT catalytic domain missense mutation in monozygotic female twins (c. X:70779215 T > A, p. N567K) with intellectual disability that allows dissection of these effects. The patients show limited IQ with developmental delay and skewed X-inactivation. Molecular analyses revealed decreased OGT stability and disruption of the substrate binding site, resulting in loss of catalytic activity. Editing this mutation into the Drosophila genome results in global changes in the O-GlcNAc proteome, while in mouse embryonic stem cells it leads to loss of O-GlcNAcase and delayed differentiation down the neuronal lineage. These data imply that catalytic deficiency of OGT could contribute to X-linked intellectual disability.
| S-EPMC6660750 | biostudies-literature
Project description:It is estimated that ∼1% of the world's population has intellectual disability, with males affected more often than females. OGT is an X-linked gene encoding for the enzyme O-GlcNAc transferase (OGT), which carries out the reversible addition of N-acetylglucosamine (GlcNAc) to Ser/Thr residues of its intracellular substrates. Three missense mutations in the tetratricopeptide (TPR) repeats of OGT have recently been reported to cause X-linked intellectual disability (XLID). Here, we report the discovery of two additional novel missense mutations (c.775 G>A, p.A259T, and c.1016 A>G, p.E339G) in the TPR domain of OGT that segregate with XLID in affected families. Characterization of all five of these XLID missense variants of OGT demonstrates modest declines in thermodynamic stability and/or activities of the variants. We engineered each of the mutations into a male human embryonic stem cell line using CRISPR/Cas9. Investigation of the global O-GlcNAc profile as well as OGT and O-GlcNAc hydrolase levels by Western blotting showed no gross changes in steady-state levels in the engineered lines. However, analyses of the differential transcriptomes of the OGT variant-expressing stem cells revealed shared deregulation of genes involved in cell fate determination and liver X receptor/retinoid X receptor signaling, which has been implicated in neuronal development. Thus, here we reveal two additional mutations encoding residues in the TPR regions of OGT that appear causal for XLID and provide evidence that the relatively stable and active TPR variants may share a common, unelucidated mechanism of altering gene expression profiles in human embryonic stem cells.
| S-EPMC6036218 | biostudies-literature
Project description:X-linked intellectual disabilities (XLID) are common developmental disorders. The enzyme O-GlcNAc transferase encoded by OGT, a recently discovered XLID gene, attaches O-GlcNAc to nuclear and cytoplasmic proteins. As few missense mutations have been described, it is unclear what the aetiology of the patient phenotypes is. Here, we report the discovery of a missense mutation in the catalytic domain of OGT in an XLID patient. X-ray crystallography reveals that this variant leads to structural rearrangements in the catalytic domain. The mutation reduces in vitro OGT activity on substrate peptides/protein. Mouse embryonic stem cells carrying the mutation reveal reduced O-GlcNAcase (OGA) and global O-GlcNAc levels. These data suggest a direct link between changes in the O-GlcNAcome and intellectual disability observed in patients carrying OGT mutations.
| S-EPMC7042088 | biostudies-literature
Project description:N-Acetylglucosamine (O-GlcNAc) transferase (OGT) regulates protein O-GlcNAcylation, an essential and dynamic post-translational modification. The O-GlcNAc modification is present on numerous nuclear and cytosolic proteins and has been implicated in essential cellular functions such as signaling and gene expression. Accordingly, altered levels of protein O-GlcNAcylation have been associated with developmental defects and neurodegeneration. However, mutations in the OGT gene have not yet been functionally confirmed in humans. Here, we report on two hemizygous mutations in OGT in individuals with X-linked intellectual disability (XLID) and dysmorphic features: one missense mutation (p.Arg284Pro) and one mutation leading to a splicing defect (c.463-6T>G). Both mutations reside in the tetratricopeptide repeats of OGT that are essential for substrate recognition. We observed slightly reduced levels of OGT protein and reduced levels of its opposing enzyme O-GlcNAcase in both patient-derived fibroblasts, but global O-GlcNAc levels appeared to be unaffected. Our data suggest that mutant cells attempt to maintain global O-GlcNAcylation by down-regulating O-GlcNAcase expression. We also found that the c.463-6T>G mutation leads to aberrant mRNA splicing, but no stable truncated protein was detected in the corresponding patient-derived fibroblasts. Recombinant OGT bearing the p.Arg284Pro mutation was prone to unfolding and exhibited reduced glycosylation activity against a complex array of glycosylation substrates and proteolytic processing of the transcription factor host cell factor 1, which is also encoded by an XLID-associated gene. We conclude that defects in O-GlcNAc homeostasis and host cell factor 1 proteolysis may play roles in mediation of XLID in individuals with OGT mutations.
| S-EPMC5535036 | biostudies-literature
Project description:O-GlcNAc transferase (OGT) is a nutrient-sensitive glycosyltransferase that is overexpressed in prostate cancer, the most common cancer in males. We recently developed specific and potent inhibitor targeting this enzyme, and here we report a synthetic lethality screen using this compound. Our screen identified pan-cyclin-dependent kinase (CDK) inhibitor AT7519 as lethal in combination with OGT inhibition. Follow-up chemical and genetic approaches identified CDK9 as the major target for synthetic lethality with OGT inhibition in prostate cancer cells. OGT expression is regulated through retention of the fourth intron in the gene and CDK9 inhibition blunted this regulatory mechanism. CDK9 phosphorylates carboxy-terminal domain (CTD) of RNA Polymerase II (RNA Pol II) to promote transcription elongation. We showed that OGT inhibition augments effects of CDK9 inhibitors on CTD phosphorylation and general transcription. Finally, the combined inhibition of both OGT and CDK9 blocked growth of organoids derived from metastatic prostate cancer patients but had minimal effects on normal prostate spheroids. We report a novel synthetic lethal interaction between inhibitors of OGT and CDK9 that specifically kills prostate cancer cells but not normal cells. Our study highlights the potential of combining OGT inhibitors with other treatments to exploit cancer specific vulnerabilities.
2020-08-18 | GSE116778 | GEO
Project description:Abstract: The essential mammalian enzyme O-GlcNAc Transferase (OGT) is uniquely responsible for transferring N-acetylglucosamine to over a thousand nuclear and cytoplasmic proteins, yet there is no known consensus sequence and it remains unclear how OGT recognizes its substrates. To address this question, we have developed a protein microarray assay that chemoenzymatically labels de novo sites of glycosylation with biotin, allowing us to simultaneously as-sess OGT activity across >6000 human proteins. We used this assay to examine the contribution of a conserved asparagine ladder within the lumen of OGT’s superhelical tetratri-copeptide repeat (TPR) domain to substrate selection. When these residues were mutated, OGT retained full activity against short peptides, but showed low to no activity against most of the OGT substrates on the microarray. O-GlcNAcylation of protein substrates in cell extracts was also greatly attenuated. We conclude that OGT recognizes a majority of its substrates by binding them to the asparagine ladder in the TPR lumen proximal to the catalytic domain. This series contains microarray data both comparing the new chemoenzymatic method to antibody-based detection as well as comparing arrays treated with wild-type OGT, 5N5A mutant OGT, or controls not treated with enzyme. Note: all CTD-stained arrays or control array raw files are contained in GSE107911_RAW.tar
2018-02-26 | GSE107911 | GEO