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的严重肌营养不良伴脑畸形，在日本流行。本研究的目的是制备针对FCMD基因产物fuklavin的特异性抗体，分析fuklavin的定位和功能，并建立该疾病的小鼠模型。虽然获得了一些检测哺乳动物细胞中过表达的fuklavin的抗体，但这些抗体不能检测内源性fuklavin。由于根据我们最近的研究，假定fuklavin是一种糖基转移酶，并且与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fuklavin，因此认为不能通过抗体进行内源性fuklavin的检测。也就是说，结果表明，使用荧光抗体进行分析是困难的。此外，免疫组化分析表明，在高尔基体中存在着fuketamine。 ...更多信息 在FCMD患者中进行了突变分析，并且新发现了一些额外的突变。通过使用重组fuketin的亲和柱层析和质谱分析，尝试鉴定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，为了鉴定作为可能的糖基转移酶的fuklase的靶蛋白和可能的fuklase复合物的伴侣蛋白，使用二维电泳、免疫沉淀和双杂交方法进行另一分析。肌眼脑疾病（Muscle-eye-brain disease，MEB）与FCMD有着惊人的相似性，我们鉴定了MEB的基因，该基因编码POMGnT1糖基转移酶。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