G.P.2.09 Glycosylation of α-dystroglycan in cultured cells and its restoration by glycosyltransferase

Abstract

Dystroglycan (DG) is a central component of dystrophin–glycoprotein complex that links extracellular matrix and cytoskeleton. α-Dystroglycanopathy is a disorder characterized by muscular dystrophy often associated with brain anomaly, mental retardation and eye abnormalities. Mutations of known or putative glycosyltransferases, POMGnT1, POMT1, POMT2, fukutin, FKRP and LARGE, were identified and defective glycosylation of α-DG has been implicated in the pathogenesis of this disorder. Recently, it was reported that overexpression of LARGE recover a function of α-dystroglycan and bypasses the defective glycosylation of α-DG in cultured cells deficient in fukutin, POMGnT1 or POM1. In this study, we characterized glycosylation status of α-DG in several cultured cell lines and examined the restorative effect of glycosyltransferases on the defective glycosylation of α-DG. We demonstrated that benign tumor or embryonic cell lines such as RT4, HEK293 or C2C12 express α-DG detectable by an antibody against sugar chain of α-DG, whereas malignant carcinoma cell lines such as HeLa or MCF7 did not. Using an antibody against core protein, α-DG with molecular mass of 75 kDa was detected in these carcinoma cells, suggesting that functional α-dystroglycan is lost in carcinoma cells. Next, we examined if the function of α-DG is restored in these cells by gene transfer of glycosyltransferases, mutation of which leads to α-dystroglycanopathy. Transfection of the cells with LARGE greatly restored the laminin binding activity of α-DG, whereas other glycosyltransferases including POMGnT1, POMT1, POMT2, fukutin or FKRP did not. These results demonstrate that the restoration of functional α-DG by LARGE is a possible molecular target when developing therapeutic strategies for α-dystroglycanopathy.