Inhibition of dystroglycan cleavage causes muscular dystrophy in transgenic mice
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.
References (47)
- et al.
Membrane organization of the dystrophin–glycoprotein complex
Cell
(1991) - et al.
Dystroglycan: inside and out
Curr Opin Cell Biol
(1999) Mutations in the fukutin-related protein gene (FKRP) cause a form of congenital muscular dystrophy with secondary laminin α2 deficiency and abnormal glycosylation of α-dystroglycan
Am J Hum Genet
(2001)Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1
Dev Cell
(2001)- et al.
Detection of O-mannosyl glycans in rabbit skeletal muscle alpha-dystroglycan
Biochim Biophys Acta
(1998) - et al.
Structures of sialylated O-linked oligosaccharides of bovine peripheral nerve alpha-dystroglycan. The role of a novel O-mannosyl-type oligosaccharide in the binding of alpha-dystroglycan with laminin
J Biol Chem
(1997) O-mannosyl glycans in mammals
Biochim Biophys Acta
(1999)- et al.
The fukutin protein family – predicting enzymes modifying cell-surface molecules
Curr Biol
(1999) - et al.
Overexpression of the CT GalNAc transferase in skeletal muscle alters myofiber growth, neuromuscular structure, and laminin expression
Dev Biol
(2002) - et al.
The α-dystroglycan-β-dystroglycan complex: membrane organization and relationship to an agrin receptor
J Biol Chem
(1995)
Purification of cranin, a laminin binding membrane protein
J Biol Chem
Identification and purification of an agrin receptor from Torpedo postsynaptic membranes: a heteromeric complex related to the dystroglycans
Neuron
Dystroglycan-alpha, a dystrophin-associated glycoprotein, is a functional agrin receptor
Cell
Dystroglycan binds nerve and muscle agrin
Neuron
Localization of the dystrophin binding site at the carboxyl terminus of beta-dystroglycan
Biochem Biophys Res Commun
Identification and characterization of the dystrophin anchoring site on beta-dystroglycan
J Biol Chem
WW and EF hand domains of dystrophin-family proteins mediate dystroglycan binding
Mol Cell Biol Res Commun
Evidence for in situ and in vitro association between beta-dystroglycan and the subsynaptic 43K rapsyn protein. Consequence for acetylcholine receptor clustering at the synapse
J Biol Chem
Rapsyn may function as a link between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex
Neuron
A role for dystroglycan in basement membrane assembly
Cell
Biosynthesis of dystroglycan: processing of a precursor polypeptide
FEBS Lett
Characteristics of skeletal muscle in mdx mutant mice
Int Rev Cytol
A role for the dystrophin–glycoprotein complex as a transmembrane linker between laminin and actin
J Cell Biol
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