A molecular overview of the primary dystroglycanopathies

Abstract Dystroglycan is a major non‐integrin adhesion complex that connects the cytoskeleton to the surrounding basement membranes, thus providing stability to skeletal muscle. In Vertebrates, hypoglycosylation of α‐dystroglycan has been strongly linked to muscular dystrophy phenotypes, some of which also show variable degrees of cognitive impairments, collectively termed dystroglycanopathies. Only a small number of mutations in the dystroglycan gene, leading to the so called primary dystroglycanopathies, has been described so far, as opposed to the ever‐growing number of identified secondary or tertiary dystroglycanopathies (caused by genetic abnormalities in glycosyltransferases or in enzymes involved in the synthesis of the carbohydrate building blocks). The few mutations found within the autonomous N‐terminal domain of α‐dystroglycan seem to destabilise it to different degrees, without influencing the overall folding and targeting of the dystroglycan complex. On the contrary other mutations, some located at the α/β interface of the dystroglycan complex, seem to be able to interfere with its maturation, thus compromising its stability and eventually leading to the intracellular engulfment and/or partial or even total degradation of the dystroglycan uncleaved precursor.

| 3059 BRANCACCIO calcium to establish additional coordination contacts with the sugar moieties protruding from it. 13 The DG affinity towards these ligands is generally high (K d s within the nanomolar range) and can be also influenced by the heterogeneous glycosylation of α-DG. 12 The α-DG/laminin interaction is considered crucial for the stability of basement membranes. Intracellularly, the transmembrane β-DG subunit does establish contacts with dystrophin and the cytoskeleton (see Figure 1). Due to these pivotal structural functions, DG and its associated proteins, as well as the enzymes responsible for its post-translational maturation, are heavily involved in several forms of muscular dystrophy. 14,15 As a matter of fact sugar moieties, including a crucial phosphorylated O-linked mannose, 17,18 that protrude from the central mucin-like domain of α-DG have been recently found to be important for efficient binding to matrix partners, and hypoglycosylation of α-DG is thought to represent a distinctive molecular trait leading to several human pathologies, in particular to an increasing number of neuromuscular disorders.

| THE E XPAND ING G AL A X Y OF DYS TROG LYC ANOPATHIE S
Dystroglycanopathies are genetic diseases often arising from the hypoglycosylation of α-DG and, depending on the affected genes they originate from, they are classified in the following main groups: (a) primary dystroglycanopathies, which occur when mutations of the DAG1 gene alter the state of the DG core protein with potential repercussions on the glycosylation state of α-DG; (b) secondary dystroglycanopathies, which depend on genetic abnormalities of POMGnT1, POMT1 or LARGE1 among others. These result in malfunctioning of the corresponding enzymes involved in the decoration with sugars of the DG core protein in the endoplasmic reticulum (ER) and Golgi, often affecting severely the glycosylation of α-DG; (c) tertiary dystroglycanopathies, possibly involving genes (such as ISPD or GMPPB) and their corresponding enzymes responsible for the fabrication of the carbohydrate building blocks in the cytosol, thus indirectly modifying α-DG glycosylation. 19 The spectrum of secondary/tertiary dystroglycanopathies is likely to be even wider, since a link has been recently found between a dystrophic phenotype F I G U R E 1 Schematic representation of the dystrophinglycoprotein complex (DGC) in skeletal muscle. The two dystroglycan subunits interact non-covalently to form a bridge between the extracellular matrix and the actin cytoskeleton. α-DG and β-DG are non-covalently connected and they also interact with numerous other proteins. The cytosolic domain of β-DG is anchored to actin through the interaction with dystrophin and β-DG also constitutes a scaffold for proteins involved in signal transduction such as Gbr2 and ERK. α-DG is a so-called peripheral membrane protein that interacts with the ectodomain of β-DG on the extracellular side of the plasma membrane. α-DG acts as a receptor for extracellular matrix proteins such as laminins (reported in the scheme), perlecan, neurexins and agrin among others  Moreover, no data have been collected to clarify whether α-DG is abnormally glycosylated in this family.
depending on α-DG hypoglycosylation and mutations in protein complexes responsible for localizing proteins to the Golgi compartment. 20 As opposed to the constantly growing number of secondary and tertiary dystroglycanopathies so far identified, only a few cases of primary dystroglycanopathies have been found in human patients as well as in zebrafish. In Table 1 is reported a re-collection of the relevant pathologic and genetic details behind the mutations identified to date. Different phenotypes have been observed, ranging from mild muscular dystrophy with asymptomatic hyperCKemia to more severe limb-girdle muscular dystrophy or Muscle-Eye-Brain disease.

| HE TEROG ENEIT Y OF PRIMARY DYS TROG LYC ANOPATHIE S
Contrary to the increasing number of described secondary and ter- On the other hand, both in human patients and zebrafish, phenotypes can also arise when mutations are found at the interface formed by α-DG and β-DG that is ultimately responsible for the non-covalent interaction between the two subunits. 33 In one case a missense mutation within the second Ig-like domain of α-DG, V567D, was shown to induce the patchy-tail phenotype in zebrasfish typically caused by the total absence of DG. 25 A complete lack of DG has also been observed in another zebrafish mutant in which a nonsense mutation was found within the mucin-like region of α-DG. 24 It is worth to note that the case identified by Riemersma and colleagues (with a nonsense stop codon at the level of the S6 domain of α-DG resulting in the full depletion of the whole DG complex) might represent the nearest human counterpart to these mutations. 10 Our group has a long-standing tradition of molecular studies on DG, for example by modelling and molecular dynamics, we have shown that the V567D zebrafish mutation, as well as its murine topological counterpart I591D, is likely to introduce a degree of instability/ collapse within the α-DG IG-like β-sandwich structure, leading to the exposure of some hydrophobic internal residues. 34 In another case, the C669F mutation affecting the ectodomain of β-DG was shown to cause a severe Muscle-Eye-Brain disease with a relevant phenotype involving the white matter in the brain displaying as multicystic leukodystrophy. 26 Recently, we have shown that such conditions could depend on the intracellular engulfment within the ER of the DG unprocessed precursor, eventually leading to its likely ubiquitination and consequent degradation by the proteasome. 35 It has yet to be assessed whether the pathologic consequences of this mutation depend on (a) the absence of DG properly targeted at the sarcolemma/ plasma membrane, or on (b) the accumulation of intracellular DG due to its engulfment into the ER The evidence that no dominant negative effects have been observed in heterozygous carriers of the mutation seems to make the latter hypothesis less likely. 26 It is perhaps not surprising that genetic abnormalities within the area responsible for the maturation of the DG complex, in which po- Very recently a first mutation within the cytodomain of β-DG, namely R776C, has been identified, causing a late-onset form of limb-girdle muscular dystrophy (see Table 1). 27 This arginine is the first residue of the cytosolic domain of β-DG, that is, it is part of its nuclear localization peptide and might represent a putative docking site for MAPK. 27 Interestingly, the effect of R776C could also depend on it being a mutation in the basic sequence that governs membrane orientation of transmembrane proteins. 38

| CON CLUS IONS
New mutations in DAG1 are likely to be identified in the future, and it will be interesting to assess their effect in view of the ongoing domain structural assessment and the possible collection of further additional structural information. A system and rationale for the classification of a larger amount of information (ie, mutations) based on the molecular structure of DG is likely to become a priority in the future, once a statistically significant amount of mutations has been characterised.

ACK N OWLED G EM ENTS
The author acknowledges his AFM-Téléthon 20009 grant "Establishing new models for primary dystroglycanopathies" for supporting research carried on dystroglycan in his laboratory.

CO N FLI C T S O F I NTE R E S T
The Author declares that he has no competing interests.