Dystroglycan is a transmembrane glycoprotein that links the extracellular basement membrane to cytoplasmic dystrophin. Disruption of the extensive carbohydrate structure normally present on α-dystroglycan causes an array of congenital and limb girdle muscular dystrophies known as dystroglycanopathies. The essential role of dystroglycan in development has hampered elucidation of the mechanisms underlying dystroglycanopathies. Here, we developed a dystroglycanopathy mouse model using inducible or muscle-specific promoters to conditionally disrupt fukutin (Fktn), a gene required for dystroglycan processing. In conditional Fktn-KO mice, we observed a near absence of functionally glycosylated dystroglycan within 18 days of gene deletion. Twentyweek-old KO mice showed clear dystrophic histopathology and a defect in glycosylation near the dystroglycan O-mannose phosphate, whether onset of Fktn excision driven by muscle-specific promoters occurred at E8 or E17. However, the earlier gene deletion resulted in more severe phenotypes, with a faster onset of damage and weakness, reduced weight and viability, and regenerating fibers of smaller size. The dependence of phenotype severity on the developmental timing of muscle Fktn deletion supports a role for dystroglycan in muscle development or differentiation. Moreover, given that this conditional Fktn-KO mouse allows the generation of tissue-and timingspecific defects in dystroglycan glycosylation, avoids embryonic lethality, and produces a phenotype resembling patient pathology, it is a promising new model for the study of secondary dystroglycanopathy.

Mutations in fukutin-related protein (FKRP) give rise to mild and more severe forms of muscular dystrophy. FKRP patients have reduced glycosylation of the extracellular protein dystroglycan, and FKRP itself shows sequence similarity to glycosyltransferases, implicating FKRP in the processing of dystroglycan. However, FKRP localization is controversial, and no FKRP complexes are known, so any FKRP-dystroglycan link remains elusive. Here, we demonstrate a novel FKRP localization in vivo; in mouse, both endogenous and recombinant FKRP are present at the sarcolemma. Biochemical analyses revealed that mouse muscle FKRP and dystroglycan co-enrich and co-fractionate, indicating that FKRP coexists with dystroglycan in the native dystrophin-glycoprotein complex. Furthermore, FKRP sedimentation shifts with dystroglycan in disease models involving the dystrophin-glycoprotein complex, and sarcolemmal FKRP immunofluorescence mirrors that of dystroglycan in muscular dystrophy mice, suggesting that FKRP localization may be mediated by dystroglycan. These data offer the first evidence of an FKRP complex in muscle and suggest that FKRP may influence the glycosylation status of dystroglycan from within the sarcolemmal dystrophin-glycoprotein complex. Mutations in FKRP3 lead to the allelic muscular dystrophies, congenital muscular dystrophy 1C and limb girdle muscular dystrophy 2I, characterized by progressive muscle weakness with variable heart, respiratory, and brain involvement (1-3). Although the specific function of FKRP is unclear, FKRP and its closest known homolog fukutin share sequence homology with phosphoryl ligand transferases and contain DXD domains common to some glycosyltransferases (4, 5). In addition, FKRPassociated muscular dystrophies fall into a growing family of "dystroglycanopathies," which exhibit reduced glycosylation of membrane-associated ␣DG (6). Extracellular ␣DG and the transmembrane-spanning ␤DG bind to dystrophin, sarcoglycans, and other proteins to form the dystrophin-glycoprotein complex (DGC), which serves as a critical structural link between the cell cytoskeleton, the sarcolemma, and the extracellular basement membrane. ␣DG glycans, detected by antibody IIH6, mediate the interaction between the DGC and extracellular matrix proteins that contain laminin LG domains. Therefore, reduced ␣DG glycosylation may weaken the cell to matrix link, increasing structural instability and disease (6).Previous studies have examined the localization of FKRP in a spectrum of cultured cells (e.g. COS7, SH-SY5Y, C2C12). The variable results suggested that FKRP is a resident of the Golgi, the rough endoplasmic reticulum, or perinuclear regions (7-11). In this study, we have examined FKRP protein complexes and their location in skeletal muscle of wild-type and dystrophic mice. We report that FKRP is localized at the muscle sarcolemma and that it co-fractionates with the DGC. Furthermore, disruption of the DGC (by alkaline treatment or genetic deletion) revealed that FKRP sedimentation and localization are dependent on...