Research on molecular pathogenesis of congenital muscular dystrophy -Characterization of the intra-/extra-cellular interaction of dystroglycan-

为了阐明先天性肌营养不良的分子机制，我对营养不良鸡和fukutin缺陷嵌合小鼠的骨骼肌、神经肌肉接头和周围神经进行了表征，这是先天性肌营养不良动物模型。首先，通过对营养不良鸡的分析发现：1)营养不良鸡骨骼肌中α-糖歧义蛋白的糖基化存在缺陷，其层粘连蛋白结合活性降低；2)鸡体内存在两种不同的α-糖歧义蛋白分子种，较小的分子种不具有层粘连蛋白结合活性；3)与对照组相比，营养不良鸡α-营养不良聚糖上的Galβ1-3GalNAc片段量增加，而Siaa2-3Gal片段量减少。4)骨骼肌和心脏中β1整合子表达上调。其次，对fukutin缺陷嵌合小鼠进行的实验表明：1)嵌合小鼠的神经肌肉连接处外观小且碎片化；2)嵌合神经肌肉连接处的agrin结合活性及其表达降低；3)嵌合小鼠周围神经轴突径向分选机制缺陷，有髓纤维减少；4)嵌合神经中α-三磷酸腺苷异常糖基化，层粘连蛋白结合活性降低。此外，通过酵母双杂交筛选人类cDNA文库，我克隆了质膜Ca^<2+> atp酶作为β-三聚糖酐的潜在结合蛋白。然而，这些蛋白的分子相互作用并没有在蛋白水平上通过共免疫沉淀实验或拉下实验得到证实，这表明相互作用非常弱，这些方法无法检测到结合。

To clarify the molecular mechanisms underlying congenital muscular dystrophy, I characterized the skeletal muscle, neuromuscular junction and peripheral nerve of dystrophic chicken and fukutin deficient chimeric mouse, animal models of congenital muscular dystrophy. First, analysis using dystrophic chicken revealed that 1) glycosylation of α-dystroglycan is defective and its laminin binding activity is reduced in the skeletal muscle of dystrophic chicken, 2) two distinct molecular species of α-dystroglycan exists in the chicken and the smaller species doesn't possess the laminin binding activity, 3) amount of Galβ1-3GalNAc moiety is increased whereas Siaa2-3Gal is reduced on α-dystroglycan of the dystrophic chicken as compared to control 4) expression of β1-integron is up-regulated in the skeletal muscle and heart of the chicken. Second, experiments with fukutin deficient chimeric mice demonstrated that 1) neuromuscular junction of the chimeric mice is small and fragmented in appearance, 2) agrin binding activity and its expression is reduced in the chimeric neuromuscular junction, 3) the radial sorting mechanism of peripheral nerve axon is defective and myelinated fiber is decreased in the chimeric mice, 4) α-dystroglycan is aberrantly glycosylated and its laminin binding activity is decreased in the chimeric nerve. In addition, by yeast two-hybrid screening of human cDNA library, I cloned plasma membrane Ca^<2+> ATPase as a potential binding protein of β-dystroglycan. However, the molecular interaction of these proteins was not confirmed at protein level with co-immunoprecipitation experiments or pull-down assays, suggesting that the interaction is so weak that these methodology couldn't detect the binding.