Department of Molecular Physiology and Biophysics, Department of Neurology, Howard Hughes Medical Institute, University of Iowa Roy J. and Lucille A. Carver College of Medicine, 4283 Carver Biomedical Research Building, 285 Newton Road, Iowa City, IA, 52242-1101, USA.
Children's Cancer Therapy Development Institute, 12655 SW Beaverdam Road W, Beaverton, OR, 97005, USA.
Skelet Muscle. 2019 May 4;9(1):11. doi: 10.1186/s13395-019-0195-0.
α-Dystroglycan is the highly glycosylated component of the dystrophin-glycoprotein complex (DGC) that binds with high-affinity to extracellular matrix (ECM) proteins containing laminin-G-like (LG) domains via a unique heteropolysaccharide [-GlcA-beta1,3-Xyl-alpha1,3-] called matriglycan. Changes in expression of components of the DGC or in the O-glycosylation of α-dystroglycan result in muscular dystrophy but are also observed in certain cancers. In mice, the loss of either of two DGC proteins, dystrophin or α-sarcoglycan, is associated with a high incidence of rhabdomyosarcoma (RMS). In addition, glycosylation of α-dystroglycan is aberrant in a small cohort of human patients with RMS. Since both the glycosylation of α-dystroglycan and its function as an ECM receptor require over 18 post-translational processing enzymes, we hypothesized that understanding its role in the pathogenesis of RMS requires a complete analysis of the expression of dystroglycan-modifying enzymes and the characterization of α-dystroglycan glycosylation in the context of RMS.
A series of cell lines and biopsy samples from human and mouse RMS were analyzed for the glycosylation status of α-dystroglycan and for expression of the genes encoding the responsible enzymes, in particular those required for the addition of matriglycan. Furthermore, the glycosyltransferase LARGE1 was ectopically expressed in RMS cells to determine its effects on matriglycan modifications and the ability of α-dystroglycan to function as a laminin receptor.
Immunohistochemistry and immunoblotting of a collection of primary RMS tumors show that although α-dystroglycan is consistently expressed and glycosylated in these tumors, α-dystroglycan lacks matriglycan and the ability to bind laminin. Similarly, in a series of cell lines derived from human and mouse RMS, α-dystroglycan lacks matriglycan modification and the ability to bind laminin. RNAseq data from RMS cell lines was analyzed for expression of the genes known to be involved in α-dystroglycan glycosylation, which revealed that, for most cell lines, the lack of matriglycan can be attributed to the downregulation of the dystroglycan-modifying enzyme LARGE1. Ectopic expression of LARGE1 in these cell cultures restored matriglycan to levels comparable to those in muscle and restored high-affinity laminin binding to α-dystroglycan.
Collectively, our findings demonstrate that a lack of matriglycan on α-dystroglycan is a common feature in RMS due to the downregulation of LARGE1, and that ectopic expression of LARGE1 can restore matriglycan modifications and the ability of α-dystroglycan to function as an ECM receptor.
α- 肌聚糖是营养不良蛋白聚糖复合物（DGC）的高度糖基化成分，通过一种独特的杂多糖[-GlcA-β1,3-Xyl-α1,3-]称为基质聚糖，与富含层粘连蛋白-G 样（LG）结构域的细胞外基质（ECM）蛋白高亲和力结合。DGC 成分的表达变化或 α- 肌聚糖的 O- 糖基化变化导致肌肉营养不良，但也在某些癌症中观察到。在小鼠中，两种 DGC 蛋白（肌营养不良蛋白或 α- 横纹肌聚糖）之一的缺失与横纹肌肉瘤（RMS）的高发生率相关。此外，在一小部分患有 RMS 的人类患者中，α- 肌聚糖的糖基化异常。由于 α- 肌聚糖的糖基化及其作为 ECM 受体的功能需要超过 18 种翻译后加工酶，因此我们假设，要了解其在 RMS 发病机制中的作用，需要对肌聚糖修饰酶的表达进行全面分析，并在 RMS 的背景下对 α- 肌聚糖糖基化进行表征。
分析了一系列来自人类和小鼠 RMS 的细胞系和活检样本，以研究 α- 肌聚糖的糖基化状态以及负责该过程的基因的表达情况，特别是那些负责添加基质聚糖的基因。此外，在 RMS 细胞中异位表达糖基转移酶 LARGE1，以确定其对基质聚糖修饰和 α- 肌聚糖作为层粘连蛋白受体功能的影响。
对一组原发性 RMS 肿瘤的免疫组织化学和免疫印迹分析表明，尽管这些肿瘤中 α- 肌聚糖始终表达并发生糖基化，但 α- 肌聚糖缺乏基质聚糖和与层粘连蛋白结合的能力。同样，在一系列源自人类和小鼠 RMS 的细胞系中，α- 肌聚糖缺乏基质聚糖修饰和与层粘连蛋白结合的能力。对 RMS 细胞系的 RNAseq 数据进行分析，以研究已知参与 α- 肌聚糖糖基化的基因的表达情况，结果表明，对于大多数细胞系，缺乏基质聚糖可归因于肌聚糖修饰酶 LARGE1 的下调。在这些细胞培养物中异位表达 LARGE1，可将基质聚糖恢复到与肌肉相当的水平，并恢复 α- 肌聚糖与层粘连蛋白的高亲和力结合。
总之，我们的研究结果表明，由于 LARGE1 的下调，缺乏基质聚糖是 RMS 的一个常见特征，而异位表达 LARGE1 可以恢复基质聚糖修饰和 α- 肌聚糖作为 ECM 受体的功能。
