Fukutin-related Protein Associates with the Sarcolemmal Dystrophin-Glycoprotein Complex

Abstract

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...

Basal lamina strengthens cell membrane integrity via the laminin G domain-binding motif of α-dystroglycan

“…3E). This is in contrast to other muscular dystrophies involving the DGC, where one primary genetic defect leads to disruption of the entire DGC, as assessed by using the same assay (27,28). This finding indicates that it is not the loss of the entire DGC, but rather the disrupted linkage between the sarcolemma and the basal lamina (due to disrupted glycosylation of ␣-DG) (Fig.…”

Mouse fukutin deletion impairs dystroglycan processing and recapitulates muscular dystrophy

“…For secondary immunofluorescence, tissues were blocked with 5% donkey serum in PBS, incubated in primary antibody overnight, washed, incubated in Cy3 (Jackson ImmunoResearch Laboratories Inc.) or Alexa Fluor 488-conjugated (Molecular Probes, Invitrogen) secondary antibodies, washed, and coverslipped. With the exception of αDG glycosylation staining, tissue cryosections were processed for immunofluorescence as described previously (81). For αDG glyco-antibody IIH6, tissues were fixed in 2% PFA, washed, and denatured by incubations with 100 mM glycine at room temperature and 0.05% SDS at 50°C for 10 and 30 minutes, respectively (82).…”

“…PHOS-bead voids were buffer exchanged (same as desalting step) and concentrated, diluted 1:1 with phosphate buffer, and mixed with 5×LSB for SDS-PAGE. All protein samples were separated by 3%-15% SDS-PAGE and transferred to PVDF for Western blotting using standard protocols (81). Laminin overlays have been described previously (37).…”

Congenital muscular dystrophy. Part II: a review of pathogenesis and therapeutic perspectives

“…According to yamamoto et al 25 , although the functions of fukutin continue unclear, it seems to interact with alpha-DG during glycosylation but binding to the core area of alpha-DG instead of its sugar chain. Also concerning FKrP, it has recently been supposed that in normal and mutant mice it is detected in the sarcollema and coexists with DG in the native dystrophin-glycoprotein complex, perhaps having its localization mediated by DG 26 . It has not yet been demonstrated that lArGe is a glycosyltransferase itself but the Kanagawa et al 27 found that alpha-DG interacts with lArGe at two different domains, one involved with the glycosilation process itself and the other at intracellular level (N-terminal domain of α-DG) for defining a recognition motif necessary to initiate functional glycosylation.…”