Anti-LMNA monoclonal antibody [Alexa Fluor® 594]

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 beta-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' (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. We conclude that residues deleted in progeria are required for substrate recognition and/or modification by OGT in vitro. Interestingly, deletion Delta 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.

Laminopathies are a family of monogenic multi-system diseases resulting from mutations in theLMNAgene which include a wide range of neuromuscular disorders. Although lamins are expressed in most types of differentiated cells,LMNAmutations selectively affect only specific tissues by mechanisms that remain largely unknown. We have employed the combination of functional in vitro experiments and transcriptome analysis in order to determine how twoLMNAmutations associated with different phenotypes affect skeletal muscle development and metabolism. We used a muscle differentiation model based on C2C12 mouse myoblasts genetically modified with lentivirus constructs bearing wild-type humanLMNA(WT-LMNA) or R482L-LMNA/G232E-LMNAmutations, linked to familial partial lipodystrophy of the Dunnigan type and muscular dystrophy phenotype accordingly. We have shown that both G232E/R482L-LMNAmutations cause dysregulation in coordination of pathways that control cell cycle dynamics and muscle differentiation. We have also found that R482/G232E-LMNAmutations induce mitochondrial uncoupling and a decrease in glycolytic activity in differentiated myotubes. Both types of alterations may contribute to mutation-induced muscle tissue pathology.