Abstract
Limb-Girdle Muscular Dystrophy type 2I (LGMD2I) is an inheritable autosomal, recessive disorder caused by mutations in the FuKutin-Related Protein (FKRP) gene (FKRP) located on chromosome 19 (19q13.3). Mutations in FKRP are also associated with Congenital Muscular Dystrophy (MDC1C), Walker-Warburg Syndrome (WWS) and Muscle Eye Brain disease (MEB). These four disorders share in common an incomplete/aberrant O-glycosylation of the membrane/extracellular matrix (ECM) protein α-dystroglycan. However, further knowledge on the FKRP structure and biological function is lacking, and its intracellular location is controversial. Based on immunogold electron microscopy of human skeletal muscle sections we demonstrate that FKRP co-localises with the middle-to-trans-Golgi marker MG160, between the myofibrils in human rectus femoris muscle fibres. Chemical cross-linking experiments followed by pairwise yeast 2-hybrid experiments, and co-immune precipitation, demonstrate that FKRP can exist as homodimers as well as in large multimeric protein complexes when expressed in cell culture. The FKRP homodimer is kept together by a disulfide bridge provided by the most N-terminal cysteine, Cys6. FKRP contains N-glycan of high mannose and/or hybrid type; however, FKRP N-glycosylation is not required for FKRP homodimer or multimer formation. We propose a model for FKRP which is consistent with that of a Golgi resident type II transmembrane protein.
Highlights
Defects of a-dystroglycan (a-DG) O-glycosylation are associated with several forms of inheritable muscular dystrophies (Limb Girdle Muscular Dystrophy type 2I; Limb-Girdle Muscular Dystrophy type 2I (LGMD2I)), of which some are congenital (Congenital Muscular Dystrophy type 1C; MDC1C), and some are associated with brain (Fukyama Congenital Muscular Dystrophy; FCMD, Walker-Warburg Syndrome; WWS) and eye abnormalities (Muscle Eye Brain disease; MEB) [1]. a-DG is a component of the dystrophinglycoprotein complex (DGC) and contains multiple sites for Olinked glycosylation [2,3]
Proper O-glycosylation of a-DG is crucial for its interaction with the extracellular laminin-a2 and agrin in muscle, and neurexin in brain [4,5,6]. a-DG hypoglycosylation precludes these interactions and the disruption of the link between these extracellular components and the actin cytoskeleton, is thought to be part of the molecular pathogenesis of the muscular dystrophy phenotype in LGMD2I and the additional brain involvement seen in WWS and MEB [7]
To investigate the in vivo localisation of endogenous FuKutin-Related Protein (FKRP) in muscle cells, separate double immunogold labelling experiments were performed on human rectus femoris longitudinal sections, using antibodies against FKRP and protein markers specifying either the sarcolemma, Golgi or endoplasmic reticulum (ER)
Summary
Defects of a-dystroglycan (a-DG) O-glycosylation are associated with several forms of inheritable muscular dystrophies (Limb Girdle Muscular Dystrophy type 2I; LGMD2I), of which some are congenital (Congenital Muscular Dystrophy type 1C; MDC1C), and some are associated with brain (Fukyama Congenital Muscular Dystrophy; FCMD, Walker-Warburg Syndrome; WWS) and eye abnormalities (Muscle Eye Brain disease; MEB) [1]. a-DG is a component of the dystrophinglycoprotein complex (DGC) and contains multiple sites for Olinked glycosylation [2,3]. A-DG hypoglycosylation precludes these interactions and the disruption of the link between these extracellular components and the actin cytoskeleton, is thought to be part of the molecular pathogenesis of the muscular dystrophy phenotype in LGMD2I and the additional brain involvement seen in WWS and MEB [7]. These disorders are collectively known as dystroglycanopathies, and they can be caused by mutations in any one of six different genes encoding known or putative glycosyltransferases or phosphotransferases. This family of proteins share sequence similarity with phosphoryl ligand transferases [16,23]
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