Dystroglycan is a highly glycosylated basement membrane receptor involved in a variety of physiological processes that maintain skeletal muscle membrane integrity as well as the structure and function of the central nervous system. Aberrant posttranslational modification of the α subunit of this protein and concomitant loss of dystroglycan function as an extracellular matrix (ECM) receptor have been observed in several forms of congenital/limb-girdle muscular dystrophies (also called dystroglycanopathies). Recent genetic data have shown that mutations in at least sixteen genes encoding known and putative glycosyltransferases disrupt O-glycosylation of dystroglycan and cause muscular dystrophy. Our current studies focus on the enzymatic function of these proteins, with the aim of understanding the structure and biosynthetic pathway of ECM ligand-binding glycan.
The Molecular Pathogenesis of Disorders of the Dystrophin-Glycoprotein Complex
Many neuromuscular conditions are characterized by an exaggerated exercise-induced fatigue response. This form of fatigue fails to correlate with either central or peripheral fatigue and is disproportionate to activity level—and is thus a major determinant of disability. We have investigated the molecular basis of the exercise-induced fatigue response using an interdisciplinary approach involving an integrated in vivo activity assay, genetically-defined mouse models, Laser Doppler imaging, magnetic resonance imaging, and patient biopsy analysis. We thereby demonstrated that loss of neuronal nitric oxide synthase (nNOS), a DGC component, from the skeletal muscle sarcolemma exacerbates the fatigue experienced after mild exercise. This loss leads to a deficiency in the normal contraction-induced cGMP-dependent attenuation of local vasoconstriction, resulting in post-exercise narrowing of the muscle vasculature. This decrease in blood flow manifests as an exaggerated fatigue response to mild activity, and in dystrophic muscle, causes exercise-induced muscle edema. Moreover, we showed that sarcolemmal nNOS levels are reduced in patient biopsies representing numerous distinct myopathies. This finding suggests a common mechanism of fatigue. In mouse models with mislocalized nNOS exercise-induced fatigue and muscle edema can be relieved pharmacologically by enhancing nitric oxide-cGMP signaling from active muscle. These findings reveal a novel therapeutic strategy for addressing fatigue symptoms in neuromuscular disease patients in whom nNOS is mislocalized or reduced. (Kobayashi, Y. et. al. Nature 2008).
Although the muscular dystrophies affect primarily skeletal muscle, the protein components of the DGC are expressed in many tissues and fat infiltration is seen in many muscular dystrophies. To understand the possible non-muscle function of the DGC, we studied white adipocytes, which share a common precursor with myocytes. Interestingly, white adiopcytes express a cell-specific sarcoglycan complex that contain β-, δ-, and ε-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and the laminin-binding form of dystroglycan. In studying multiple sarcoglycan null mouse models, we discovered that loss of α-sarcoglycan has no consequences for expression of the adipocyte sarcoglycan complex, but that in adipocytes the loss of either β- or δ-sarcoglycan leads to a concomitant loss of the sarcoglycan complex and sarcospan, as well as to a dramatic reduction in dystroglycan. Furthermore, in collaboration with Jeffrey Pessin at the Albert Einstein College of Medicine, we have demonstrated that mice lacking the sarcoglycan complex in both adipose tissue and skeletal muscle (β-sarcoglycan-null mice) are glucose-intolerant and exhibit whole-body insulin-resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. (Groh, S. et. al. J Biol Chem 2009).
The Mechanistic Basis of Maintaining Muscle Membrane Integrity
Compromised integrity of the muscle sarcolemma has been proposed to initiate muscle fiber pathology in muscular dystrophy, yet the molecular basis for this compromised integrity has never been clearly established. To study the possible role of dystroglycan in maintaining sarcolemmal integrity, we developed a novel in situ laser damage assay, and measured lengthening contraction-induced muscle damage. To directly test the function of the dystroglycan-mediated link between the basal lamina and the sarcolemma, we examined the sarcolemmal integrity of the muscle fibers from the Largemyd mouse, an animal model for dystroglycan hypoglycosylation that lacks only the laminin globular (LG) domain-binding O-glycan that is present in wild-type counterparts. We found that despite maintaining an intact dystrophin-glycoprotein complex, Largemyd muscle fibers have reduced sarcolemmal integrity and exhibit detachment from the basal lamina. This detachment makes Largemyd muscles highly susceptible to lengthening contraction-induced injury. Furthermore, we demonstrated that recombinant glycosylated α-dystroglycan can restore sarcolemma integrity of the Largemyd muscle fibers. Therefore, dystroglycan-dependent tight physical attachment of the basal lamina to the sarcolemma is important for transmission of the basal lamina’s structural strength to the sarcolemma, providing resistance to mechanical stress. Our findings establish—for the first time—a mechanism that accounts for the increased susceptibility of patients with hypoglycosylated dystroglycan to contraction-induced muscle injury, and highlights the importance of this protective basic cellular mechanism in the context of mechanical damage. (Han, R. et. al. Proc Natl Acad USA 2009).
We are also interested in understanding the role of dystroglycan in maintaining sarcolemmal integrity in cardiac muscle since several forms of muscular dystrophy linked to dystroglycan hypoglycosylation are associated with the development of cardiomyopathy. We have shown that gene-targeted loss of dystroglycan function in ventricular cardiac myocytes is sufficient to induce a progressive cardiomyopathy (characterized by focal cardiac fibrosis, cardiac hypertrophy and dilation) that ultimately leads to heart failure. Our findings suggest that, in cardiac myocytes, the ability of dystroglycan to function as an extracellular matrix receptor is crucial for limiting the spread of myocardial membrane damage to neighboring cardiac myocytes, and that loss of dystroglycan matrix receptor function in cardiac muscle cells is central to the development of cardiomyopathy in glycosylation-deficient muscular dystrophies. (Michele, M.E. et. al. Circ Res 2009).
Muscle cells also utilize dystroglycan-independent mechanisms to maintain sarcolemmal integrity. One example is plasma membrane repair. It involves the patching of new membrane to the surface membrane at or near the site of membrane disruption. Our previous data demonstrated that the novel membrane protein dysferlin (a gene mutated in two forms of dystrophy: limb-girdle muscular dystrophy type 2B and Miyoshi myopathy) plays an important role in the Ca2+-dependent membrane repair of skeletal muscle. In order to study the role of membrane repair in muscular dystrophy, we generated mice deficient for both dystrophin and dysferlin. Notably, these mice exhibited not only more severe muscular dystrophy, but also an accelerated progression of cardiomyopathy. Since a number of inherited forms of dilated cardiomyopathy arise from mutations in genes encoding proteins that link the cytoskeleton to the extracellular matrix, we studied the possible role of dysferlin-dependent membrane repair in the heart. Using a modified membrane repair assay, we demonstrated that cardiac muscle possesses a Ca2+-regulated membrane resealing mechanism that repairs membrane damage and dysferlin plays an essential role in this process. In our study, the disruption of dysferlin-mediated membrane repair rendered the heart highly susceptible to stress-induced ventricular injury. In particular, stress exercise in dysferlin-null mice led to dysfunction of the left ventricle and to increased Evans blue dye uptake in dysferlin-deficient cardiomyocytes. Our results suggest that dysferlin-mediated membrane repair is necessary for maintaining the integrity of the cardiomyocyte membrane, particularly under conditions of mechanical stress. In addition, they provide a plausible explanation for the association of cardiomyopathy with most muscular dystrophy-associated mutations: contraction-induced injury causes myocyte necrosis because cumulative membrane damage cannot be effectively repaired. (Han, R. et. al. J Clin Invest 2007).
The Molecular Pathogenesis of Disorders in Dystroglycan Glycosylation
Previous work in my laboratory revealed that dystroglycan glycosylation plays a role in the pathogenesis of Fukuyama congenital muscular dystrophy, muscle-eye-brain disease and Walker-Warburg syndrome, each of which are congenital muscular dystrophies with a range of associated developmental brain and eye defects. Genetic data showed that mutations in proteins with homology to glycosyltransferases (POMT1, POMT2, POMGnT1, FKTN, FKRP and LARGE) are linked to these congenital muscular dystrophies (also called dystroglycanopathies). Biochemical analysis of muscle biopsies revealed a convergent role for these proteins in the glycosylation of a-dystroglycan, a process required for functional activity. We have confirmed these findings and extended our work on the dystroglycanopathies to milder forms of limb-girdle muscular dystrophy with or without brain involvement and confirmed that the more severe cases are characterized by a nearly complete lack of functional glycosylation while those that are milder show only reduced glycosylation. This work has been greatly facilitated by the Iowa Wellstone Muscular Dystrophy Cooperative Research Center which is under my direction.
We were also interested in determining if the loss of dystroglycan could result in the full spectrum of congenital brain and eye malformations observed in the dystroglycanopathies. Attempts to investigate the mechanism(s) underlying the brain and eye pathology have been technically challenging, since eliminating either dystroglycan or a glycosyltransferase involved in its post-translational modification causes early embryonic lethality in mice. We showed that mice with epiblast-specific loss of dystroglycan develop brain and eye defects that broadly resemble the clinical spectrum of the human disease. Specifically, we established that: 1) dystroglycan is the key substrate affected by loss of glycosyltransferase activity, and loss of its function is responsible for the full spectrum of brain and eye malformations observed in the dystroglycanopathies; and 2) dystroglycan stabilizes basement membrane structures in both the brain and eye, and disruption of the basement membrane structure is a common mechanism that underlies developmental defects in the dystroglycan-deficient brain and eye. These findings demonstrate that dystroglycan plays a central role in the dystroglycanopathies and suggest that novel defects in posttranslational processing or mutations of the dystroglycan gene itself may underlie disease cases in which no causative mutation has been found. (Satz, J.S. et. al. J Neurosci 2008).
To further our understanding of the biology of dystroglycan glycosylation, we have also investigated the hypoglycosylation of dystroglycan in epithelial cancer cells. The interaction between epithelial cells and the extracellular matrix is crucial for tissue architecture and function and is compromised during cancer progression. Dystroglycan is expressed in epithelial cells and mediates interactions between the cell membrane and basement membranes in various epithelia. In earlier studies in collaboration with Mina Bissel’s laboratory we had shown that many epithelium-derived cancers a-dystroglycan was not detected although the co-translated β-dystroglycan is. Our more recent study revealed that in a cohort of highly metastatic epithelial cell lines derived from breast, cervical, and lung cancers, α-dystroglycan is in fact correctly expressed and trafficked to the cell membrane but fails to bind laminin due to silencing of the LARGE gene. Exogenous expression of LARGE in these cancer cells restored the normal glycosylation and laminin binding of a-dystroglycan, leading to enhanced cell adhesion and reduced cell migration in vitro. Our findings suggest that LARGE repression is responsible for the defects in dystroglycan-mediated cell adhesion that are observed in epithelium-derived cancer cells. (Beltran, D. et. al. J Biol Chem 2009).
The Structural Basis of Dystroglycan Function as a Basement Membrane Receptor
We are also studying post-translational processing of dystroglycan by the novel glycosyltransferase LARGE. My laboratory found that the amino-terminal domain of α-dystroglycan is essential for recognition by LARGE. This enzyme-substrate recognition motif is critical for the post-translational modification of dystroglycan and is necessary for the functional maturation of the protein into a receptor for extracellular matrix components. We revealed the significance of this unique pathway in vivo, as gene transfer of LARGE into muscle-specific gene-knockout mice rescued domain-specific functions of dystroglycan in the whole animal. Thus, molecular recognition of dystroglycan by LARGE in the biosynthetic pathway is essential to the production of functional dystroglycan. (Kanagawa, M. et. al. Cell 2004).
We have explored the potential of LARGE overexpression as a treatment for glycosyltransferase-deficient muscular dystrophies. We showed that the enzyme LARGE modifies the sugar moieties of α-dystroglycan and prevents muscular dystrophy in myd mice. Importantly, we demonstrated that high levels of LARGE restore the function of α-dystroglycan and modulate its glycosylation in myoblasts and fibroblasts from patients afflicted with Fukuyama congenital muscular dystrophy, muscle-eye-brain disease, and Walker-Warburg syndrome. We also discovered that LARGE-dependent glycosylation of α-dystroglycan is required for dystroglycan function, and that induction of LARGE restores the function of α-dystroglycan, regardless of the type of glycosyltransferase that is mutated in patients. This work indicates that LARGE has a broad therapeutic potential. (Baressi, R. et. al. Nat Med 2004).
O-mannosyl Phosphorylation of α-Dystroglycan
Efforts in my laboratory to identify the ECM binding moiety on α-dystroglycan led to the isolation of a novel O-glycan on α-dystroglycan that results from a rare phosphodiester-linked modification. Nuclear magnetic resonance (NMR)-based analysis identified this O-glycan as a phosphorylated O-mannosyl trisaccharide (N-acetylgalactosamine-β3-N-acetylglucosamine-β4-mannose, designated as core M3). We further demonstrated that a hydroxyl residue of the phosphate at the C6 position of O-mannose is linked to the ECM ligand-binding motif. Recently, we identified the enzymes that synthesize this novel glycan.
First, we found that glycosyltransferase-like domain-containing 2 (GTDC2) is an endoplasmic reticulum (ER)-localized O-linked mannose β1,4-N-acetylglucosaminyltransferase (designated as POMGNT2). Second, we confirmed that GTDC2 and β1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) act coordinately on O-mannose to synthesize the core M3 glycan structure. Finally, we identified SGK196, which was previously thought to be an inactive protein kinase, as an active enzyme that phosphorylates the C6 position of O-mannose at the ER, specifically after the mannose is modified by both POMGNT2 and B3GALNT2. This strict specificity of SGK196 for α-dystroglycan-linked core M3 glycan explains why mutations in GTDC2 and B3GALNT2 cause muscular dystrophy although their products are not directly involved in recognition of the ECM ligand. Collectively, these findings demonstrate that the core M3 glycan is phosphorylated on mannose before LARGE glycosyltransferase-mediated extension to produce the ECM-binding motif. (Yoshida-Moriguchi, T. et.al. Science 2013).
LARGE is a Bifunctional Glycosyltransferase
We are also studying post-translational processing of dystroglycan by the novel glycosyltransferase LARGE (the like-acetylglucosaminyltransferase). Previously, my laboratory found that the amino-terminal domain of α-dystroglycan is required for recognition by LARGE. Also, LARGE-mediated modification of α-dystroglycan is essential for its binding to various ECM-localized ligands such as laminin, agrin, and neurexin. In 2012 we found that xylose (Xyl) and glucuronic acid (GlcA) are component sugars of α-dystroglycan produced in LARGE-overexpressing cells and that mutant cells deficient for UDP-xylose synthase (and thus lacking cellular xylosylation) are defective for functional modification of α-dystroglycan.
Next we discovered that LARGE is a bifunctional glycosyltransferase with both xylosyltransferase and glucuronyltransferase activities that produce repeating units of [-3-Xyl-α1,3-GlcA-β1-]. Using skeletal muscle glycoproteins from the Largemyd mouse (a mutation in the Large gene causes defects in α-dystroglycan glycosylation) as the acceptor substrate, we further demonstrated that LARGE can assemble a polysaccharide with ligand-binding activity onto the immature glycan of the Largemyd α-dystroglycan. These results and previous studies demonstrate that LARGE synthesizes a (Xyl-GlcA)n polymer on a phosphorylated O-mannosyl glycan of α-dystroglycan, thereby conferring the ability to bind ECM ligands. (Inamori, K. et.al. Science 2012).
Another area of research focuses on the cellular significance of the LARGE-mediated glycosylation of dystroglycan, and on gaining novel insights into how defects in this post-translational modification cause diseases of varying severity. Binding between dystroglycan and its matrix-localized ligands is mediated through the disaccharide repeat added to dystroglycan by LARGE. The amount of this LARGE-glycan that decorates the nearly-ubiquitous dystroglycan is remarkably tissue-specific. In this study we demonstrated that the levels of LARGE-glycan in muscle are established during myogenesis.
Using a novel Large knockdown mouse (LargeKD) we interrupted extension of the LARGE-glycan during muscle regeneration in vivo, and as a result were able to assess the primary cellular impacts of this treatment on the muscle and its disposition to the disease state. Although dystroglycan maintained the ability to bind ligands in the matrix, the dystroglycan that formed in regenerated LargeKD muscles had a significantly reduced ligand-binding capacity. This was a direct consequence of reducing the quantity of LARGE-glycan repeats in each chain, a finding that was confirmed using synthesized LARGE-glycan repeats. Ligand saturation due to insufficiency of the LARGE-glycan in LargeKD-regenerated muscle resulted in reduced basement membrane compaction, defective maturation of the neuromuscular junction, and functionally deficient muscle predisposed to dystrophy. Consistent with these findings, disease severity in patients correlates directly with the degree to which extension of the LARGE glycan is reduced. We propose that ultrastructural organization of the basement membrane can be modified during tissue establishment by extension of the LARGE-glycan. These findings both redefine the cellular significance of dystroglycan and support a new model for the underpinnings of dystroglycan-related disease. (Goddeeris, M.M. et.al. Nature 2013).
A Dystroglycan Mutation Associated with Muscular Dystrophy
Despite recent advances in our understanding of the glycosylation defects underlying these dystroglycanopathies, it remains unclear whether mutated glycosyltransferases are the only causes of these diseases. Recently, we identified—for the first time—a missense mutation (c. 575C->T, T192M) in the dystroglycan gene of a patient with limb-girdle muscular dystrophy and cognitive impairment. Our in vitro analysis revealed that this mutation does not affect the expression of dystroglycan; instead it impairs the glycosylation of, and thus its ability to bind, laminin. A mouse model harboring this mutation recapitulates immunohistochemical and neuromuscular abnormalities observed in the patient, and the affected residue selectively impairs the modification of dystroglycan’s post-phosphoryl chains, owing to disruption of the interaction between dystroglycan and LARGE. These findings led us to propose a novel pathogenic mechanism to account for muscular dystrophy: disruption of the enzyme-substrate complex that is required to initiate maturation of phosphorylated O-mannosyl glycans on dystroglycan. (Hara, Y. et. al. N. Eng. J. Med. 2011).