Muscular dystrophies involving the Dystrophin-Glycoprotein Complex

Muscular dystrophy is a general term that describes a group of inherited and gradually progressing myogenic disorders. Genetically, the pattern of inheritance can be X-linked recessive as in Duchenne or Becker muscular dystrophies (DMD/BMD), autosomal dominant as in limb-girdle muscular dystrophy type 1 (LGMD type 1), or autosomal recessive as in limb-girdle muscular dystrophy type 2 (LGMD type 2) (Campbell, 1995; Groh et al., 2009; Moore et al., 2008; Straub and Campbell, 1997). DMD is a severe progressive muscle-wasting disease affecting approximately 1 out of 3500 males (Blake et al., 2002). Patients with DMD have a childhood onset phenotype and die before their early twenties as a result of either respiratory or cardiac failure, whereas patients with BMD have moderate weakness in adulthood and may have normal lifespan (Campbell, 1995;

Wagner et al., 2007). The limb-girdle muscular dystrophies initially involve the shoulder and pelvic girdle muscles. Moreover, muscular dystrophies may or may not be associated with cardiomyopathy (Bushby, 1999). One of the most important advances in understanding the molecular genetics of neuromuscular diseases has been the cloning of the gene encoding dystrophin, the protein absent in muscle of DMD patients (reviewed in (Davies et al., 1983)). In the last few years the role of the dystrophin in skeletal muscle has been studied, and several dystrophin-associated proteins have been identified, which are the members of Dystrophin-Glycoprotein Complex (DGC). The DGC is a large complex of membrane-associated proteins that is critical for integrity of skeletal muscle fibers. This complex consists of dystrophin, the dystroglycans (α and β), the sarcoglycans (α, β, γ and δ), sarcospan, the syntrophins (α1, β1, β2, γ1- and γ2) and α-dystrobrevin (Figure 1, (Durbeej and Campbell, 2002)). Components of the DGC are now being characterized and evidence is beginning to indicate that proteins of this complex could be responsible for other forms of muscular dystrophy (Figure 1). Members of the dystrophin protein family perform a critical but incompletely characterized role in the maintenance of membrane-associated complexes at points of intercellular contact in many vertebrate cell types. Dystrophin is a large (427 kDa) protein, which is highly conserved in vertebrates. Its largest isoform comprises an N-terminal domain, which binds to F-actin, 24 spectrin-like repeats and a characteristic C-terminal set of domains. Dystrophin is functionally similar to cytolinkers, as it links the multiple components of the cellular cytoskeleton to the transmembrane dystroglycan complex. The direct link between dystrophin and microtubules

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(MTs) was reported recently (Prins et al., 2009). A number of dystrophin-related vertebrate proteins, whose function is even less clear, have been described (reviewed in (Ilsley et al., 2002)).

Figure 1. Structure of Dystrophin-Glycoprotein Complex. Arrows indicate disorders caused by the disruption in corresponding component.

Utrophin is very similar in overall structure to dystrophin itself, and its disruption in mice causes subtle abnormalities of the neuromuscular junction. Dystrophin-related protein 2 (DRP 2) resembles certain small (approximately 110 kDa) isoforms of dystrophin and utrophin in that it comprises the last two spectrin-like repeats and the C-terminal region. Dystrophin, utrophin and DRP 2 are expressed in distinct, but partially overlapping regions of the vertebrate body. A number of motifs are recognizable in the C-terminal region of this family of proteins. The WW domain has been implicated in mediating the interaction between dystrophin and the transmembrane protein β-dystroglycan. Motifs in the remaining C-terminal sequences comprise of two EF hands, a ZZ domain with the ability to bind Zn²⁺, and two leucine heptads, which form a region involved in binding members of the syntrophin family of PDZ-domain containing proteins, which in turn bind to neuronal nitric oxide synthase (reviewed in (Ilsley et al., 2002)).

Another major component of the DGC is dystroglycan. Interestingly, the expression of dystroglycan is ubiquitous and not simply restricted to muscle cells. Besides muscle, dystroglycan is expressed at high levels in both, developing and adult tissues. Typically dystroglycan is present in the cell types that adjoin basal membranes such as epithelial and neural tissues (Durbeej and

Campbell, 2002). Dystroglycan provides a continuous link between laminin-2 in the extracellular matrix and dystrophin that is attached to the intracellular cytoskeleton. Dystroglycan is synthesized as a precursor protein and later it is proteolytically cleaved into two interacting subunits, α- and β-dystroglycan. α-dystroglycan is a heavily glycosylated membrane protein that interacts directly with laminin-2; in contrast, β-dystroglycan is an integral membrane glycoprotein that can bind both dystrophin and utrophin. The dystrophin-anchoring site on β-dystroglycan is localized to the extreme C-terminus at amino acids 880-895 (Huang et al., 2000). The knock-down of dystroglycan in mouse embryos leads to early embryonic lethality prior to gastrulation, long before any muscle has formed (reviewed in (Durbeej and Campbell, 2002)). Therefore it is not possible to analyze the consequences of dystroglycan deficiency in muscle. To overcome this Carbonetto and co-workers generated chimeric mice, lacking dystroglycan in skeletal muscles (Cote et al., 1999). Interestingly, these mice develop progressive muscle pathology and have disrupted neuromuscular junctions.

Thus, dystroglycan is necessary for myofiber stability and differentiation.

Recently a group of human muscular dystrophies have been demonstrated to have defective dystroglycan glycosylation and are hence termed dystroglycanopathies. Thus far, six dystroglycanopathy genes have been identified: POMT1, POMT2, POMGnT1, fukutin, FKRP and LARGE. Although a paralogue of LARGE, (LARGE2) has been identified and is likely to be involved in dystroglycan glycosylation, no mutations have yet been identified in this gene (Muntoni, 2004). The dystroglycanopathies can be divided into several clinical disorders that range in severity from Walker-Warburg syndrome (WWS), a severe form of congenital muscular dystrophy (CMD), which is also associated with ocular abnormalities and CNS defects, to forms of LGMD that have an onset later in life and have no CNS involvement. Muscle-eye-brain disease (MEB) is a type of CMD associated with loss-of-function mutations in the gene encoding a glycosyltransferase, POMGnT1 and resulting in eye defects, epilepsy and lissencephaly (smooth brain appearance as a result of abnormal neuronal migration) (reviewed in (Moore et al., 2008)).

The mechanistic bases for these disorders are yet to be determined.

The sarcoglycan complex is a group of single-pass transmembrane proteins (α-, β-, γ- and δ-sarcoglycans) that are tightly associated with the sarcospan to form the subcomplex within the DGC (Crosbie et al., 1999). Although the exact function of the sarcoglycan-sarcospan complex is not known, it is well established that mutations in any of α-, β-, γ- and δ-sarcoglycan genes result in the distinct forms of muscular dystrophy now collectively called sarcoglycanopaties (LGMD2C-F) (Groh et al., 2009). The sarcoglycans are primary expressed in muscles and correct expression of sarcoglycans in skeletal muscle is a necessity for their normal function. Furthermore, the proper

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expression of sarcoglycans in smooth muscles is important for normal skeletal and cardiac muscle function (Straub et al., 1999).