Abstract
A defect in O-mannosyl glycan is the cause of α-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant α-dystroglycan (α-DG) glycosylation. Recently, the entire structure of O-mannosyl glycan, [3GlcAβ1-3Xylα1]n-3GlcAβ1-4Xyl-Rbo5P-1Rbo5P-3GalNAcβ1-3GlcNAcβ1-4 (phospho-6)Manα1-, which is required for the binding of α-DG to extracellular matrix ligands, has been proposed. However, the linkage of the first Xyl residue to ribitol 5-phosphate (Rbo5P) is not clear. TMEM5 is a gene product responsible for α-dystroglycanopathy and was reported as a potential enzyme involved in this linkage formation, although the experimental evidence is still incomplete. Here, we report that TMEM5 is a xylosyltransferase that forms the Xylβ1-4Rbo5P linkage on O-mannosyl glycan. The anomeric configuration and linkage position of the product (β1,4 linkage) was determined by NMR analysis. The introduction of two missense mutations in TMEM5 found in α-dystroglycanopathy patients impaired xylosyltransferase activity. Furthermore, the disruption of the TMEM5 gene by CRISPR/Cas9 abrogated the elongation of the (-3GlcAβ1-3Xylα1-) unit on O-mannosyl glycan. Based on these results, we concluded that TMEM5 acts as a UDP-d-xylose:ribitol-5-phosphate β1,4-xylosyltransferase in the biosynthetic pathway of O-mannosyl glycan.
Highlights
A defect in O-mannosyl glycan is the cause of ␣-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant ␣-dystroglycan (␣-DG) glycosylation
TMEM5 Is a Xylosyltransferase—To address the possibility that TMEM5 acts as a xylose transferase to form Xyl-ribitol 5-phosphate (Rbo5P), we prepared a soluble form of TMEM5 and examined its activity using a potential glycopeptide substrate that contained the full glycan structure of FKRP products, based on our previous report (Fig. 1) [13]
Because the FKTN product has a Rbo5P residue on the non-reducing terminal of core M3 (Fig. 1), we examined whether the FKTN product served as an acceptor substrate for TMEM5. soluble form of TMEM5 (sTMEM5) was expressed in HEK293T cells and immunoprecipitated with anti-c-Myc antibody-agarose
Summary
A defect in O-mannosyl glycan is the cause of ␣-dystroglycanopathy, a group of congenital muscular dystrophies caused by aberrant ␣-dystroglycan (␣-DG) glycosylation. The entire structure of O-mannosyl glycan, [3GlcA1-3Xyl␣1]n3GlcA1-4Xyl-Rbo5P-1Rbo5P-3GalNAc1-3GlcNAc1-4 (phospho-6)Man␣1-, which is required for the binding of ␣-DG to extracellular matrix ligands, has been proposed. We report that TMEM5 is a xylosyltransferase that forms the Xyl1-4Rbo5P linkage on O-mannosyl glycan. Several groups, including ours, proposed the entire glycan structure on core M3, (3GlcA1-3Xyl␣1)n-3GlcA 1-4Xyl-Rbo5P-1Rbo5P-3GalNAc1-3GlcNAc1-4(phospho6)Man␣1-peptide/protein (Fig. 1) (8 –13). The first GlcA/Xyl unit is not formed by the action of LARGE [11, 12] Defects in this biosynthetic pathway seem to result in the loss of laminin binding and cause ␣-dystroglycanopathy. TMEM5 was thought to be a candidate enzyme involved in the LARGE-independent synthesis of the GlcA/Xyl unit, because the (-3GlcA1-3Xyl␣1-) repeat was absent in ␣-dystroglycanopathy patients with a mutation in the TMEM5 gene [16]. We proposed the complete glycan structure of functional ␣-DG
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