Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, California 90095, USA.
J Biol Chem. 2012 Jun 29;287(27):22759-70. doi: 10.1074/jbc.M111.334581. Epub 2012 May 8.
Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.
杜氏肌营养不良症是一种 X 连锁疾病，其特征是肌营养不良蛋白的缺失，肌营养不良蛋白是一种细胞骨架蛋白，将骨骼肌细胞中的肌动蛋白细胞骨架连接到细胞外基质。肌营养不良蛋白与跨膜糖蛋白β-肌营养不良蛋白（β-DG）的细胞质结构域结合，β-DG 与细胞表面α-肌营养不良蛋白（α-DG）结合，α-DG 与细胞外基质中的层粘连蛋白结合。β-DG 还可以与 utrophin 结合，这种差异结合与α-DG 上特定的糖基化变化相关。α-DG 糖基化的遗传修饰可以促进 utrophin 结合并挽救小鼠肌营养不良模型中的肌营养不良表型。我们使用植物凝集素 Wisteria floribunda agglutinin (WFA) 进行高通量筛选，以鉴定改变肌肉细胞膜表面糖基化的化合物，目的是找到增加α-DG 及其相关肌膜糖蛋白丰度、增加 utrophin 利用和增加层粘连蛋白结合的化合物。我们从美国食品和药物管理局批准的 Prestwick 化合物库中鉴定出一种化合物 lobeline，符合这些标准，增加了 WFA 与 C2C12 细胞和野生型和 mdx 小鼠的原代肌肉细胞的结合。WFA 结合和 lobeline 增强需要复杂的 N-聚糖，但不需要与层粘连蛋白结合的 O-甘露糖聚糖。然而，抑制复杂的 N-聚糖加工减少了层粘连蛋白与肌肉细胞糖蛋白的结合，尽管 O-甘露糖化是完整的。糖基化分析表明，用 lobeline 处理的细胞中 N-聚糖普遍增加，而不是细胞膜表面糖基化的特定改变，这与多种肌膜糖蛋白丰度的增加一致。这证明了使用植物凝集素进行高通量筛选以鉴定改变肌肉细胞糖基化的化合物的可行性，并确定了 N-聚糖在调节肌肉细胞功能中的新作用。
