Dystroglycan is a highly glycosylated peripheral membrane protein that functions as a cell surface receptor for proteins in the extracellular matrices and synapses. O-Mannosyl glycosylation is necessary for the ligand-binding activities of dystroglycan and a unique “post-phosphoryl moiety” modified via a phosphodiester linkage on the O-mannose likely forms the ligand-binding domain. Several proteins are involved in the process of this modification, the mechanism for which appears highly ordered. In various tissues, dystroglycan plays important physiological roles such as maintenance of muscle cell viability and structural development of the brain. Conversely, abnormal glycosylation causes a group of muscular dystrophy, collectively called “dystroglycanopathy,” which is often associated with brain abnormalities including type II lissencephaly and mental retardation. Here, we will be reviewing the structure, modification pathway, and physiological roles of dystroglycan glycosylation as well as their involvement in human diseases.

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Lipid rafts serve as a platform for important biological events such as signal transduction, cell adhesion, and protein trafficking. To elucidate the molecular mechanisms of these events, identification of interacting molecules in individual raft domains is required. We have developed a novel method termed enzyme-mediated activation of radical source (EMARS), by which molecules in the vicinity within 300 nm from horseradish peroxidase (HRP) set on the probed molecule are labeled. Recently, we have established a new version of the EMARS system, in which the EMARS reaction is catalyzed by HRP expressed by genetic engineering. In order to express HRP in lipid rafts, HRP was constructed as a GPI-anchored form (HRP-GPI). Two kinds of HRP-GPIs, in which GPI attachment signals of human decay accelerating factor and Thy-1 were separately connected to the C-terminus of HRP, were expressed in human HeLa S3 cells, and the EMARS reaction was catalyzed by these expressed HRP-GPIs under a living condition. As a result, these HRP-GPIs underwent different N-glycosylation and formed distinct molecular clusters. Thus, this novel approach is able to identify molecular clusters associated with particular GPI-anchored proteins, suggesting that it can segregate individual lipid raft domains.

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