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 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.

Analytical and Biochemical Perspectives of Protein O-GlcNAcylation

“…Even though such long peptides can be detected by certain types of mass spectrometry (e.g., by using ETD), their identification and site determination are often challenging with other commonly available instruments (e.g., CID mass spectrometry). Instead, less specific enzymes (e.g., chymotrypsin and elastase) and chemical cleavage (e.g., using CNBr) should generate peptides of a size amenable to MS analysis, ,, and in silico digestion will provide helpful information to guide the selection of appropriate enzymes.…”

Post-Translational Modification of Lamins: Mechanisms and Functions

“… Simon et al (2018) reported that lamin A could be β-O-linked N-acetylglucosamine-(O-GlcNAc)-modified in human hepatoma (Huh7) cells and in the mouse liver. O-linked glycosylation is the enzymatic addition of O-GlcNAc by the enzyme O-GlcNAc transferase (OGT) to protein Ser/Thr residues ( Hart et al, 2011 ).…”

“…The mechanisms underlying laminopathies have been extensively investigated. It is currently well recognized that lamins are functionally regulated by a myriad of PTMs, existing under certain biological or pathological conditions with specific functions, including farnesylation ( Farnsworth et al, 1989 ), phosphorylation ( Beausoleil et al, 2004 ; Olsen et al, 2006 ), acetylation ( Choudhary et al, 2009 ; Lundby et al, 2012 ; Weinert et al, 2018 ), SUMOylation ( Lumpkin et al, 2017 ), methylation ( Rao et al, 2019 ), ubiquitination ( Wagner et al, 2011 ; Povlsen et al, 2012 ), and O-GlcNAcylation ( Alfaro et al, 2012 ; Wang et al, 2012 ; Simon et al, 2018 ). PTMs of lamins might directly affect their structure, therefore affecting the functions or destructing LAD binding or its organization and gene expression ( Wong and Stewart, 2020 ), although the roles of different types and specific sites of PTMs remain to be further clarified.…”

Regulation of Lipid Metabolism by Lamin in Mutation-Related Diseases

“…Meanwhile, a growing number of studies have shown that the function of lamin not only at the cellular level but also in disease states is controlled by the PTMs of proteins, including phosphorylation (Machowska et al, 2015), SUMOylation (Moriuchi et al, 2016), glycosylation (Snider and Omary, 2014), farnesylation (Farnsworth et al, 1989), methylation (Rao et al, 2019), o-glcNAcylation (Alfaro et al, 2012;Wang et al, 2012;Simon et al, 2018), succinylation (Weinert et al, 2013), and ubiquitination (Wagner et al, 2011;Povlsen et al, 2012). Farnesylation is special among many PTMs in the regulation of prelamin A.…”