OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A
AbstractThe 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 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’ (Δ50) 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 Δ50, which causes Hutchinson–Gilford progeria syndrome, and greatly reduced by deletion Δ35. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Δ35, 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. View Full-Text
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Simon, D.N.; Wriston, A.; Fan, Q.; Shabanowitz, J.; Florwick, A.; Dharmaraj, T.; Peterson, S.B.; Gruenbaum, Y.; Carlson, C.R.; Grønning-Wang, L.M.; Hunt, D.F.; Wilson, K.L. OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A. Cells 2018, 7, 44.
Simon DN, Wriston A, Fan Q, Shabanowitz J, Florwick A, Dharmaraj T, Peterson SB, Gruenbaum Y, Carlson CR, Grønning-Wang LM, Hunt DF, Wilson KL. OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A. Cells. 2018; 7(5):44.Chicago/Turabian Style
Simon, Dan N.; Wriston, Amanda; Fan, Qiong; Shabanowitz, Jeffrey; Florwick, Alyssa; Dharmaraj, Tejas; Peterson, Sherket B.; Gruenbaum, Yosef; Carlson, Cathrine R.; Grønning-Wang, Line M.; Hunt, Donald F.; Wilson, Katherine L. 2018. "OGT (O-GlcNAc Transferase) Selectively Modifies Multiple Residues Unique to Lamin A." Cells 7, no. 5: 44.