Inhibition of dystroglycan cleavage causes muscular dystrophy in transgenic mice

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Abstract

Dystroglycan (DG) is an essential component of the dystrophin–glycoprotein complex, a molecular scaffold that links the extracellular matrix to the actin cytoskeleton. Dystroglycan protein is post-translationally cleaved into α dystroglycan, a highly glycosylated peripheral membrane protein, and β dystroglycan, a transmembrane protein. Despite clear evidence of the importance of dystroglycan and its associated proteins in muscular dystrophy, the purpose of dystroglycan proteolysis is unclear. By introducing a point mutation at the normal site of proteolysis (serine 654 to alanine, DGS654A), we have created a dystroglycan protein that is severely inhibited in its cleavage. Transgenic expression of DGS654A in mouse skeletal muscles inhibited the expression of endogenously cleaved dystroglycan, while overexpression of wild type dystroglycan by similar amounts did not. DGS654A animals had increased serum creatine kinase activity and most muscles had increased numbers of central nuclei. Overexpression of wild type dystroglycan, by contrast, caused no dystrophy by these measures. Dystrophy in DGS654A muscles correlated with reduced binding of antibodies that recognize glycosylated forms of α dystroglycan. Lastly, neuromuscular junctions in DGS654A muscles were aberrant in structure. These data show that aberrant processing of the dystroglycan polypeptide causes muscular dystrophy and suggest that dystroglycan processing is important for the proper glycosylation of α dystroglycan.

Introduction

Dystroglycan is an essential component of the dystrophin–glycoprotein complex that links the extracellular matrix surrounding myofibers to the actin cytoskeleton [1], [2], [3]. Deficiencies in many of the proteins that bind to dystroglycan cause forms of muscular dystrophy, including dystrophin (Duchenne muscular dystrophy, DMD), sarcoglycans (forms of limb–girdle muscular dystrophy, LGMD), and laminin α2 (merosin-dependent congenital muscular dystrophy, CMD) [3]. In mice, loss of dystroglycan is lethal at an early embryonic stage [4]. Thus, persons with mutations resulting in complete loss of dystroglycan expression would probably not survive to the point of developing muscular dystrophy [4]. Chimeric mice in which only some muscles lack dystroglycan, however, do have muscular dystrophy [5]. Moreover, a number of recent studies have implicated changes in the post-translational modification of α dystroglycan in forms of muscular dystrophy [6], [7], [8], [9], [10], [11], [12], [13]. Therefore, an understanding of structure–function relationships in the dystroglycan protein is essential for understanding the mechanism responsible for the pathology in many neuromuscular disorders.

Defects in at least five genes that cause muscular dystrophy alter post-translational modifications on α dystroglycan [6], [7], [8], [9], [10], [11], [12], [13]. Insertions or mutations in the fukutin gene cause Fukuyama-type CMD [6], mutations in the fukutin-related protein (FKRP) gene cause CMD MDC1C [7], and limb–girdle muscular dystrophy 2I [8], POMGnT1 mutations cause muscle–eye–brain (MEB) disease [9], a deletion in the Large gene is responsible for muscular dystrophy in the myodystrophy (myd) mouse [10], and mutations in POMT1 cause Walker–Warburg syndrome [11]. Mutations in FKRP correlate with aberrant migration of the muscle form of α dystroglycan on sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels [7]. Since α dystroglycan is roughly one half carbohydrate by molecular weight [2], this change in α dystroglycan migration suggests an alteration in its glycosylation. Defects in fukutin [12], [13], Large [10], [13], and POMGnT1 [13] abolish binding of monoclonal antibodies that require carbohydrate structures on α dystroglycan [2] and create forms of α dystroglycan that bind very poorly to extracellular matrix ligands such as laminin [13]. As the carbohydrate-dependent antibodies used to define changes in α dystroglycan expression appear to be very specific for α dystroglycan, it is likely that these changes occur in the unusual NeuAcα2, 3Galβ1, 4GlcNAcβ1, 2Manα-O-Ser structure in the mucin-like domain of the protein [14], [15], [16], [17]. POMGnT1 encodes an UDP-GlcNAc: O-mannose β1,2 N-acetylglucosaminyl transferase that can synthesize the second glycan in this chain [9], [18], while POMT1 encodes an O-mannosyltransferase that could synthesize the first glycan [11]. Large and fukutin also have structures that suggest that they are glycosyltransferases [10], [19]. The association of mutations in these glycosyltransferases with muscular dystrophy supports the contention that glycan structures that are O-linked via mannose are lost on α dystroglycan in these disorders. We have recently identified a potential β1,4-linked synaptic GalNAc modification on α dystroglycan called the CT carbohydrate antigen [20]. Overexpression of the glycosyltransferase that creates this antigen in skeletal muscle inhibits muscular dystrophy in the mdx mouse model for DMD [21]. Because modifying skeletal muscle glycosylation can inhibit muscular dystrophy [21] and deficits in glycosylation can cause muscular dystrophy [6], [7], [8], [9], [10], [11], [12], [13], we were particularly interested in the relationship between dystroglycan processing, glycosylation, and dystrophy.

While much is known about the proteins that associate with dystroglycan, very little is understood about the mechanism involved in the proteolytic processing of the dystroglycan polypeptide. The dystroglycan protein is synthesized as a single polypeptide, which is made from a singly spliced mRNA [22], [23]. This polypeptide is cleaved into α dystroglycan, a membrane-associated extracellular protein, and β dystroglycan, a transmembrane protein [1], [24], [25]. The cleavage site between the α and the β chain has been identified, and the N-terminus of the β chain begins at serine 654 [24], [25]. The protease or proteases involved in this processing, however, are unknown, as is the role of the cleavage event in the first place. The α and β dystroglycans can bind tightly to one another via non-covalent interactions, making an α/β complex [1], [24], [25]. The α dystroglycan is heavily glycosylated, in large part due to a serine/threonine-rich mucin domain, and binds to extracellular matrix proteins such as laminins [13], [22], [26], agrins [13], [27], [28], [29], [30], perlecan [26], and biglycan [31], as well as to viruses [32], bacteria [33], and neurexins [13], [34]. The β dystroglycan, in turn, binds to α dystroglycan [1], [3] via its extracellular domain and to dystrophin [35], [36], [37], utrophin [37], and rapsyn [38], [39] via its intracellular domain. The α/β dystroglycan also associates with a complex of other transmembrane proteins called sarcoglycans [40]. Through this complex series of intermolecular interactions, α/β dystroglycan serves as a link between the extracellular matrix and the cytoskeleton, and can mediate both matrix signaling [41], [42] and matrix deposition [43], [44].

To test the role of dystroglycan cleavage into two polypeptides, we have transgenically expressed a mutated form of dystroglycan in the skeletal muscles of mice. This mutation (Ser654Ala) not only inhibits the cleavage of dystroglycan protein made by the transgene, but also inhibits the expression of endogenously cleaved dystroglycan as well, thereby allowing us to study muscles where all, or very close to all, dystroglycan exist as a single polypeptide. We show that lack of proper processing of dystroglycan correlates with changes in glycosylation of α dystroglycan that are analogous to those seen in several congenital forms of muscular dystrophy. These experiments suggest that dystroglycan cleavage is required for normal muscle function and may be an as yet unidentified cause of muscular dystrophy.

Section snippets

Materials

IIH6, a monoclonal antibody that requires glycans on the α dystroglycan protein [2], OR12, a polyclonal antiserum against α/β dystroglycan polypeptide, and a cDNA for dystroglycan [23] were gifts from Kevin Campbell (HHMI, University of Iowa) provided in part by Elizabeth Apel (Washington University). VIA4-1, a second monoclonal antibody that requires glycans on α dystroglycan [2], was purchased from Upstate Biotechnology (Lake Placid, NY). A polyclonal rabbit antiserum to the C-terminal 15

Creation and characterization of uncleaved dystroglycan

To study dystroglycan proteolysis, we created cDNA constructs for dystroglycan (α/β) and its α and β chain (Fig. 1A). An eight amino FLAG epitope tag was placed at the N-terminus of the coding sequence for each of these constructs (Fig. 1A). In addition, we created two constructs where DG cleavage was inhibited (Fig. 1A). DG Esp was made by inserting FLAG at the EspI restriction site in the β chain, which is near the normal site proteolysis [24], [25]. DGSer654Ala (DGS654A) was made by mutating

Discussion

Transgenic expression of a cleavage-resistant form of dystroglycan (DGS654A) in mice has allowed us to investigate what happens in skeletal muscle when dystroglycan is not properly cleaved into an α and a β polypeptide. The most significant finding of these studies is that most muscles lacking dystroglycan cleavage are dystrophic. Most muscles have increased levels of central nuclei when compared to age-matched wild type mice, and the level of increase is roughly equivalent to that seen in

Acknowledgements

We would like to thank Kevin Campbell (HHMI, University of Iowa), Elizabeth Apel (Washington University), Joshua Sanes (Washington University), Jeffery Chamberlain (HHMI, University of Washington), and Palmer Taylor (University of California, San Diego, CA) for gifts of reagents. This work was supported by grants from the Muscular Dystrophy Association, March of Dimes, and NIH (NS37214) to P.T.M.

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