Dataset Information

OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

ABSTRACT: The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is ?-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified O-GlcNAc transferase (OGT) enzyme showed robust O-GlcNAcylation of recombinant mature lamin A tails (residues 385?646), with no detectable modification of lamin B1, lamin C, or 'progerin' (?50) tails. Using mass spectrometry, we identified 11 O-GlcNAc sites in a 'sweet spot' unique to lamin A, with up to seven sugars per peptide. Most sites were unpredicted by current algorithms. Double-mutant (S612A/T643A) lamin A tails were still robustly O-GlcNAc-modified at seven sites. By contrast, O-GlcNAcylation was undetectable on tails bearing deletion ?50, which causes Hutchinson?Gilford progeria syndrome, and greatly reduced by deletion ?35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion ?35, which does not remove the majority of identified O-GlcNAc sites, does remove potential OGT-association motifs (lamin A residues 622?625 and 639?645) homologous to that in mouse Tet1. These biochemical results are significant because they identify a novel molecular pathway that may profoundly influence lamin A function. The hypothesis that lamin A is selectively regulated by OGT warrants future testing in vivo, along with two predictions: genetic variants may contribute to disease by perturbing OGT-dependent regulation, and nutrient or other stresses might cause OGT to misregulate wildtype lamin A.

SUBMITTER: Simon DN

PROVIDER: S-EPMC5981268 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

Simon Dan N DN Wriston Amanda A Fan Qiong Q Shabanowitz Jeffrey J Florwick Alyssa A Dharmaraj Tejas T Peterson Sherket B SB Gruenbaum Yosef Y Carlson Cathrine R CR Grønning-Wang Line M LM Hunt Donald F DF Wilson Katherine L KL

Cells 20180517 5

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in LMNA cause over 12 diseases ('laminopathies'). Lamins A and C are identical for their first 566 residues. However, they form separate filaments in vivo, with apparently distinct roles. We report that lamin A is β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays ...[more]

PMID: 29772801

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Project description:Neurodevelopmental disorders such as epilepsy and autism have been linked to an imbalance of excitation and inhibition (E/I) in the central nervous system. The simplicity and tractability of C. elegans allows our electroconvulsive seizure (ES) assay to be used as a behavioral readout of the locomotor circuit and neuronal function. C. elegans possess conserved nervous system features such as gamma-aminobutyric acid (GABA) and GABA receptors in inhibitory neurotransmission, and acetylcholine (Ach) and acetylcholine receptors in excitatory neurotransmission. Our previously published data has shown that decreasing inhibition in the motor circuit, via GABAergic manipulation, will extend the time of locomotor recovery following electroshock. Similarly, mutations in a HECT E3 ubiquitin ligase called EEL-1 leads to impaired GABAergic transmission, E/I imbalance and altered sensitivity to electroshock. Mutations in the human ortholog of EEL-1, called HUWE1, are associated with both syndromic and non-syndromic intellectual disability. Both EEL-1 and its previously established binding protein, OGT-1, are expressed in GABAergic motor neurons, localize to GABAergic presynaptic terminals, and function in parallel to regulate GABA neuron function. In this study, we tested behavioral responses to electroshock in wildtype, ogt-1, eel-1 and ogt-1; eel-1 double mutants. Both ogt-1 and eel-1 null mutants have decreased inhibitory GABAergic neuron function and increased electroshock sensitivity. Consistent with EEL-1 and OGT-1 functioning in parallel pathways, ogt-1; eel-1 double mutants showed enhanced electroshock susceptibility. Expression of OGT-1 in the C. elegans nervous system rescued enhanced electroshock defects in ogt-1; eel-1 double mutants. Application of a GABA agonist, Baclofen, decreased electroshock susceptibility in all animals. Our C. elegans electroconvulsive seizure assay was the first to model a human X-linked Intellectual Disability (XLID) associated with epilepsy and suggests a potential novel role for the OGT-1/EEL-1 complex in seizure susceptibility.

Dataset Information

OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

Publications

OGT (<i>O</i>-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A.

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