Molecular analysis of Fukuyama muscular dystrophy and functional analysis of the gene product fukutin.

福山型先天性肌营养不良症(FCMD)是一种常染色体隐性遗传性重度肌营养不良症，伴有脑畸形，流行于日本。本研究的目的是制备针对FCMD基因产物Fukutin的抗体，分析Fukutin的定位和功能，并建立该病的小鼠模型。虽然已经获得了一些抗体可以检测到哺乳动物细胞中高表达的芦丁，但这些抗体并不能检测到内源性的芦丁。由于我们最近的研究假设Fukutin是一种糖基转移酶，并且像许多已知的糖基转移酶一样，细胞中存在非常少量的内源性Fukutin，因此认为不能用抗体检测内源性Fukutin。也就是说，事实证明，使用河豚毒素抗体进行分析很困难。此外，免疫组织化学Ana…结果表明，芦丁存在于高尔基体中。在FCMD患者中进行了突变分析，并新发现了一些新发现的突变。用重组Fukutin亲和柱层析和质谱分析鉴定了Fukutin结合蛋白。虽然获得了一些蛋白质，但我们正在检查这些蛋白质是否是真正的Fukutin结合蛋白。此外，我们还利用双向电泳、免疫沉淀和双杂交方法对可能是糖基转移酶的目的蛋白和可能的复合体蛋白进行了分析。为了建立在3‘-非翻译区插入反转录转座子的转基因小鼠，构建了敲入载体并将其导入胚胎干细胞。肌肉-眼-脑疾病(MEB)与FCMD有惊人的相似性，我们鉴定了编码POMGNT1糖基转移酶的MEB基因。较少

Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive severe muscular dystrophy accompanied by brain malformation, prevalent in Japan. This research was performed for the purpose of making the antibodies specific for the FCMD gene product fukutin, analyzing localization and function of fukutin, and creating the mouse model of this disease. Then the following things were clarified.Although some antibodies were obtained which detect overexpressed fukutin in mammalian cells, these could not detect endogenous fukutin. Since it is supposed that fukutin is a glycosyltransferase from our recent researches and that very small quantity of endogenous fukutin exists in cells like many of known glycosyltransferases, it was thought that detection of the endogenous fukutin by the antibodies cannot be made. That is, it turned out that the analysis is difficult using the fukutin antibodies. Moreover, it was shown that fukutin exists in a Golgi body by the immunohistochemical ana … More lysis of mammalian cells overexpressing fukutin, and that is not contradictory to the possibility of being a glycosyltransferase.Mutational analysis was performed in the FCMD patients and some additional mutations were newly discovered.Identification of fukutin-binding proteins is tried by affinity column chromatography using recombinant fukutin and by mass spectrometric analysis. Although some proteins were obtained, we are checking whether these are the actual fukutin-binding proteins. Moreover, another analysis is performed using 2-dimensional electrophoresis, immunoprecipitation, and two-hybrid methods in order to identify the target protein of fukutin as a possible glycosyltransferase and the partner protein of the possible fukutin complex.To create the knock-in mice which carry the retrotransposon insertion in 3'-untranslated region of the fukutin gene, the knock-in vector was constructed and introduced to embryonic stem cells.Muscle-eye-brain disease (MEB) bears a striking resemblance to FCMD, We identified the gene responsible for MEB which encodes POMGnT1 glycosyltransferase. Less