Matsumura K, Chiba A, Yamada H, Fukuta-Ohi H, Fujita S, Endo T, Kobata A, Anderson L V, Kanazawa I, Campbell K P, Shimizu T
Department of Neurology and Neuroscience, Teikyo University School of Medicine, Tokyo 173, Japan.
J Biol Chem. 1997 May 23;272(21):13904-10. doi: 10.1074/jbc.272.21.13904.
Dystroglycan is encoded by a single gene and cleaved into two proteins alpha- and beta-dystroglycan by posttranslational processing. Recently, alpha-dystroglycan was demonstrated to be an extracellular laminin-binding protein anchored to the cell membrane by a transmembrane protein beta-dystroglycan in striated muscle and Schwann cells. However, the biological functions of the dystroglycan-laminin interaction remain obscure, and in particular, it is still unclear if dystroglycan plays a role in cell adhesion. In the present study, we characterized the role of dystroglycan in the adhesion of schwannoma cells to laminin-1. Immunochemical analysis demonstrated that the dystroglycan complex, comprised of alpha- and beta-dystroglycan, was a major laminin-binding protein complex in the surface membrane of rat schwannoma cell line RT4. It also demonstrated the presence of alpha-dystroglycan, but not beta-dystroglycan, in the culture medium, suggesting secretion of alpha-dystroglycan by RT4 cells. RT4 cells cultured on dishes coated with laminin-1 became spindle in shape and adhered to the bottom surface tightly. Monoclonal antibody IIH6 against alpha-dystroglycan was shown previously to inhibit the binding of laminin-1 to alpha-dystroglycan. In the presence of IIH6, but not several other control antibodies in the culture medium, RT4 cells remained round in shape and did not adhere to the bottom surface. The adhesion of RT4 cells to dishes coated with fibronectin was not affected by IIH6. The known inhibitors of the interaction of alpha-dystroglycan with laminin-1, including EDTA, sulfatide, fucoidan, dextran sulfate, heparin, and sialic acid, also perturbed the adhesion of RT4 cells to laminin-1, whereas the reagents which do not inhibit the interaction, including dextran, chondroitin sulfate, dermatan sulfate, and GlcNAc, did not. Altogether, these results support a role for dystroglycan as a major cell adhesion molecule in the surface membrane of RT4 cells.
肌营养不良聚糖由单个基因编码，并通过翻译后加工裂解为两种蛋白质，即α-和β-肌营养不良聚糖。最近，α-肌营养不良聚糖被证明是一种细胞外层粘连蛋白结合蛋白，在横纹肌和施万细胞中通过跨膜蛋白β-肌营养不良聚糖锚定在细胞膜上。然而，肌营养不良聚糖与层粘连蛋白相互作用的生物学功能仍不清楚，特别是，肌营养不良聚糖是否在细胞粘附中起作用仍不清楚。在本研究中，我们表征了肌营养不良聚糖在施万细胞瘤细胞与层粘连蛋白-1粘附中的作用。免疫化学分析表明，由α-和β-肌营养不良聚糖组成的肌营养不良聚糖复合物是大鼠施万细胞瘤细胞系RT4表面膜中的主要层粘连蛋白结合蛋白复合物。它还证明了培养基中存在α-肌营养不良聚糖，但不存在β-肌营养不良聚糖，这表明RT4细胞分泌α-肌营养不良聚糖。在涂有层粘连蛋白-1的培养皿上培养的RT4细胞变成纺锤形并紧密粘附在底面。先前已证明，针对α-肌营养不良聚糖的单克隆抗体IIH6可抑制层粘连蛋白-1与α-肌营养不良聚糖的结合。在培养基中存在IIH6而非其他几种对照抗体的情况下，RT4细胞保持圆形，不粘附在底面。RT4细胞与涂有纤连蛋白的培养皿的粘附不受IIH6影响。已知的α-肌营养不良聚糖与层粘连蛋白-1相互作用的抑制剂，包括乙二胺四乙酸（EDTA）、硫苷脂、岩藻依聚糖、硫酸葡聚糖、肝素和唾液酸，也扰乱了RT4细胞与层粘连蛋白-1的粘附，而不抑制相互作用的试剂，包括葡聚糖、硫酸软骨素、硫酸皮肤素和N-乙酰葡糖胺（GlcNAc），则没有。总之，这些结果支持肌营养不良聚糖作为RT4细胞表面膜中主要细胞粘附分子的作用。
