Identification of a Post-translational Modification with Ribitol-Phosphate and Its Defect in Muscular Dystrophy

Abstract

Glycosylation is an essential post-translational modification that underlies many biological processes and diseases. α-dystroglycan (α-DG) is a receptor for matrix and synaptic proteins that causes muscular dystrophy and lissencephaly upon its abnormal glycosylation (α-dystroglycanopathies). Here we identify the glycan unit ribitol 5-phosphate (Rbo5P), a phosphoric ester of pentose alcohol, in α-DG. Rbo5P forms a tandem repeat and functions as a scaffold for the formation of the ligand-binding moiety. We show that enzyme activities of three major α-dystroglycanopathy-causing proteins are involved in the synthesis of tandem Rbo5P. Isoprenoid synthase domain-containing (ISPD) is cytidine diphosphate ribitol (CDP-Rbo) synthase. Fukutin and fukutin-related protein are sequentially acting Rbo5P transferases that use CDP-Rbo. Consequently, Rbo5P glycosylation is defective in α-dystroglycanopathy models. Supplementation of CDP-Rbo to ISPD-deficient cells restored α-DG glycosylation. These findings establish the molecular basis of mammalian Rbo5P glycosylation and provide insight into pathogenesis and therapeutic strategies in α-DG-associated diseases.

B4GALNT2 (GALGT2) Gene Therapy Reduces Skeletal Muscle Pathology in the FKRP P448L Mouse Model of Limb Girdle Muscular Dystrophy 2I

“…15,16,19 Abnormal glycosylation includes the reduced expression of O-mannose-phosphateelinked glycans needed for ECM protein binding, 16,19 secondary to reductions in ribitol 5-phosphate synthesized on a dystroglycan by FKRP and fukutin. 24 Here, we have used the FKRP P448Lneo À mouse model made by Lu and colleagues 25 to test a surrogate gene therapy, rAAVrh74.MCK.GALGT2, that is known to inhibit muscle pathology in the mdx mouse model for Duchenne MD, the dy W mouse model for congenital MD 1A, and the Sgca À/À mouse model for LGMD2D. 36e39 Because GALGT2 overexpression normally induces the glycosylation of a dystroglycan in skeletal muscle 29,36 and because the full therapeutic potential of GALGT2 requires the expression of Figure 9 Protein and mRNA expression of dystrophin and laminin a2 surrogates after rAAVrh74.MCK.GALGT2 treatment of FKRP P448Lneo À muscles.…”

“…24 Merged images of WFA staining and laminin a2 are shown in Figure 2 to show regions of coincident staining. Cumulatively, these data show that rAAVrh74.MCK.GALGT2 treatment of FKRP P448Lneo À muscles led to increased expression of vgs, GALGT2 mRNA, and functional GALGT2 production of the CT glycan in all treated muscles; however, some expression was lost for all three measures as time after treatment increased.…”

“…49 Here, we observed a slight increase in WFA precipitated material even in untreated FKRP P448Lneo À muscles, but this material contained little or no CT glycan, suggesting it may be recognizing incompletely glycosylated structures of a dystroglycan such as GalNAcb1,3GlcNAcb1,4Man(6P)-O that would normally be modified by FKRP but that are not the CT glycan. 24 Treated FKRP P448Lneo À muscles showed a slight increase in WFA precipitated material, with a dystroglycan identified when 0.5 mg of protein was used for the precipitation Figure 6 Immunostaining for natively glycosylated a-dystroglycan and for embryonic myosin in GALGT2-overexpressing FKRP P448Lneo À muscles. Data shown are from rAAVrh74.MCK.GALGT2-injected (treated) or PBS-injected (untreated) gastrocnemius muscles of FKRP P448Lneo À mice at 3 months after injection.…”

“…22,23 Recently, both FKRP and fukutin have been shown to possess ribitol 5-phosphate transferase activity, and a tandem ribitol 5-phosphate moiety was shown to be present on a dystroglycan on which laminin binding glycans could be synthesized and which are themselves absent in FKRP-, fukutin-, and isoprenoid synthase domain containingedeficient cells. 24 Lu and colleagues 25 have designed a series of mouse knockin models of disease-relevant human FKRP mutations that give rise to varying degrees of muscle disease pathology and dysfunction. Introduction of the FKRP P448L mutation, in the presence of a neomycin gene cassette, gives rise to severe muscle pathology consistent with WWS, 26 whereas deletion of the neo cassette from the P448L knockin allele gives rise to a milder disease spectrum more analogous to LGMD2I, with adult onset muscle necrosis and evidence of muscle damage and regeneration, coupled with inflammation, fibrosis, and muscle wasting.…”

Gènes impliqués dans les alpha-dystroglycanopathies

“…Ainsi, à côté de la structure initialement décrite M1, Yoshida-Moriguchi et al [7] ont proposé en 2013, l'existence de deux autres structures glycaniques (=core), M2 et M3 ( Figures 1A, 1B, 1C). Très récemment, la structure M3 a pu ainsi être complétée avec la description d'une séquence répétée de deux sucres (résidus xylosyles et glucuronyles) nommée « matriglycan » ( Figure 1E) [8,9]. Ces découvertes ont permis de compléter le lien entre l'ensemble des gènes identifiés à ce jour et les -DGpathies.…”