OGT (O-GlcNAc Transferase) selectively modifies multiple residues unique to lamin A

Simon, D. N.; Wriston, A.; Fan, Q.; Shabanowitz, J.; Florwick, A.; Dharmaraj, T.; Peterson, S. B.; Gruenbaum, Y.; Carlson, C. R.; Gronning-Wang, L. M.; Hunt, D. F.; Wilson, K. L.

doi:10.1101/206458

bioRxiv · 10.1101/206458

OGT (O-GlcNAc Transferase) selectively modifies multiple residues unique to lamin A

Simon, D. N.·Wriston, A.·Fan, Q.·Shabanowitz, J.·Florwick, A.·Dharmaraj, T.·Peterson, S. B.·Gruenbaum, Y.·Carlson, C. R.·Gronning-Wang, L. M.·Hunt, D. F.·Wilson, K. L.

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Abstract

The LMNA gene encodes lamins A and C with key roles in nuclear structure, signaling, chromatin organization, and genome integrity. Mutations in LMNA cause >12 diseases, termed laminopathies. Lamins A and C are identical for their first 566 residues. However, they form distinct filaments in vivo with apparently distinct roles. We report that lamin A is O-GlcNAc modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified 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 ({Delta}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 {Delta}50, which causes Hutchinson-Gilford progeria syndrome, and greatly reduced by deletion {Delta}35, suggesting this region is required for substrate recognition or modification by OGT in vitro. These results suggest OGT, an essential protein and master regulator, regulates partners or function(s) unique to lamin A that are lost in progeria.

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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., Gronning-Wang, L. M., Hunt, D. F., Wilson, K. L.. 2017-10-26. OGT (O-GlcNAc Transferase) selectively modifies multiple residues unique to lamin A. https://doi.org/10.1101/206458

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