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
Department of Cell Biology, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205, USA.
Department of Chemistry, University of Virginia, Charlottesville, VA 22904, USA.
Cells. 2018 May 17;7(5):44. doi: 10.3390/cells7050044.
The gene encodes lamins A and C with key roles in nuclear structure, signaling, gene regulation, and genome integrity. Mutations in 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--linked -acetylglucosamine--GlcNAc)-modified in human hepatoma (Huh7) cells and in mouse liver. In vitro assays with purified -GlcNAc transferase (OGT) enzyme showed robust -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 -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 -GlcNAc-modified at seven sites. By contrast, -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 -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.
该基因编码核纤层蛋白A和C，它们在核结构、信号传导、基因调控和基因组完整性方面发挥关键作用。该基因的突变会导致超过12种疾病（“核纤层蛋白病”）。核纤层蛋白A和C的前566个残基相同。然而，它们在体内形成独立的细丝，具有明显不同的作用。我们报告称，在人肝癌（Huh7）细胞和小鼠肝脏中，核纤层蛋白A被β-连接的N-乙酰葡糖胺（O-GlcNAc）修饰。用纯化的O-GlcNAc转移酶（OGT）进行的体外试验表明，重组成熟核纤层蛋白A尾部（残基385⁻646）有强烈的O-GlcNAc糖基化，而核纤层蛋白B1、核纤层蛋白C或“早老素”（Δ50）尾部未检测到修饰。通过质谱分析，我们在核纤层蛋白A特有的“甜蜜点”中鉴定出11个O-GlcNAc位点，每个肽段最多有7个糖。大多数位点是当前算法无法预测的。双突变（S612A/T643A）核纤层蛋白A尾部在7个位点仍有强烈的O-GlcNAc修饰。相比之下，在导致哈钦森-吉尔福德早衰综合征的缺失Δ50的尾部未检测到O-GlcNAc糖基化，而缺失Δ35使其大大减少。我们得出结论，早衰中缺失的残基是体外OGT进行底物识别和/或修饰所必需的。有趣的是，缺失Δ35虽然没有去除大多数已鉴定的O-GlcNAc位点，但确实去除了与小鼠Tet1中同源的潜在OGT结合基序（核纤层蛋白A残基622⁻625和639⁻645）。这些生化结果意义重大，因为它们确定了一条可能深刻影响核纤层蛋白A功能的新分子途径。核纤层蛋白A受OGT选择性调控的假说值得未来在体内进行验证，同时还有两个预测：基因变异可能通过干扰OGT依赖的调控导致疾病，营养或其他应激可能导致OGT对野生型核纤层蛋白A调控失调。
