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.
Highlights
O-GlcNAc is a regulatory post-translational modification of nucleocytoplasmic proteins that has been implicated in multiple biological processes, including transcription
An X-chromosome exome screen identified a missense mutation, which encodes an amino acid in the tetratricopeptide repeat, in O-GlcNAc transferase (OGT) (759G>T (p.L254F)) that segregates with X-linked intellectual disability (XLID) in an affected family
We have characterized a missense mutation in OGT (759GϾT (p.L254F)) that segregates with XLID in a family
Summary
Exome sequencing of multiple family members (family K9427), revealed a mutation (759GϾT (p.L254F)) encoding an amino acid in the seventh TPR of OGT perfectly segregating with XLID (Fig. 1A). The known structure of the OGT TPR repeats (PDB code 1W3B) was used to calculate ⌬⌬G for the L254F variant. The average predicted ⌬⌬G upon substitution is relatively small, indicating that the single amino acid replacement probably does not grossly affect overall protein stability. Consistent with this finding, we visualized the structures of wild-type and L254F variant OGT TPR domain with UCSF Chimera, and the substitution sites are marked in red (Fig. 1B). The substitution with Phe, which is a bulkier residue, would be difficult to accom-
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