Research Highlights

Congenital muscular dystrophy: Phospho-glycan connection

Dystroglycan glycosylation and its place in the dystrophin–glycoprotein complex. From Nat. Clin. Pract. Neurol. 2, 222-230 (2006) doi:10.1038/ncpneuro0155

To bind its extracellular matrix (ECM) ligands, including laminin, α-dystroglycan (α-DG) must be glycosylated. Mutations of several known or putative glycosyltransferase genes are implicated in cases of congenital muscular dystrophy (CMD), and – as we highlighted in Tumor suppression: Glycosyltransferases loom LARGE – in deficient laminin binding by α-DG in cancer cells. However, although an O-mannosyl glycan is thought to mediate the interaction, its identity is unknown. Kevin Campbell and colleagues now report in Science that laminin binding by α-DG requires a phosphorylated O-mannosyl modification, of a type only previously identified in yeast and fungi.

In many tissues the cytoskeleton is connected to the ECM by the dystrophin glycoprotein complex, of which α-DG is a subunit. In Duchenne muscular dystrophy, the protein component is defective, but several types of CMD are attributed to aberrant α-DG glycosylation. The most severe of these – fatal in early infancy – is Walker-Warburg syndrome (WWS). Some WWS cases have mutations in protein O-mannosyltransferase 1 (POMT1), which encodes a glycosyltransferase required for the initial O-mannosylation of α-DG. Other putative glycosyltransferases, including LARGE, are thought to modify the nascent glycan, and are mutated in some types of CMD. However, the precise synthesis pathway and product required for functional binding of laminin are unknown.

To solve this mystery, the authors extracted glycoproteins from muscle and used several treatments to determine which modification of α-DG is required for laminin binding. The receptor is both N- and O-glycosylated, including Core1 O-glycans and an O-mannosyl tetrasaccharide. Glycosidase enzymes were used to degrade these known glycans, and an overlay assay confirmed that none are essential to bind laminin. Chemical treatment to cleave phosphoester bonds, however, resulted in the loss of laminin binding, and significantly reduced the relative molecular mass of α-DG, but not β-DG. The reduction was not observed when α-DG from CMD-model mice, which cannot bind to laminin due to the mutation in large, was similarly treated, indicating that the phosphoester cleavage specifically degrades the laminin binding moiety.

Imaging of labeled phosphate on secreted recombinant α-DG showed that the mucin-like region was phosphorylated, and careful hydrolysis indicated that this was likely to be linked to a carbohydrate, not a peptide. Further evidence that an O-mannosyl glycan is phosphorylated came from cells derived from human cases of CMD, as well as fibroblasts from mice with mutated large. Cells with mutated POMT1 were the only ones tested that were unable to secrete phosphorylated recombinant α-DG. Using affinity chromatography, the authors found that in cells with mutations in either Large or another putative glycosyltransferase gene, fukutin, the phosphorylated O-linked mannose did not undergo any further modification. This suggests that both LARGE and fukutin function in a common pathway to assemble the laminin-binding moiety onto the phosphorylated monosaccharide.

The authors isolated the phosphorylated O-mannosyl glycan to determine its structure. With a combination of linear trap quadrupole mass spectrometry, high performance anion exchange chromatography, and NMR, a trisaccharide structure of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and mannitol (Man-ol) was revealed (GalNAc-β-1,3-GlcNAc-β-1,4-Man-ol). The phosphate group is attached to the C6 position of the mannitol, and this occurs within the Golgi complex independently of GlcNAc-1-phosphotransferase.

This study identifies the first vertebrate protein, other than glycosylphosphatidylinositol-anchored proteins, known to be modified with a phosphodiester-linked glycan, and increases understanding of the mechanisms that underlie CMD.

Author: Emma Leah