N-terminal domain on dystroglycan enables LARGE1 to extend matriglycan on α-dystroglycan and prevents muscular dystrophy.

Dystroglycan (DG) requires extensive post-translational processing and O-glycosylation to function as a receptor for extracellular matrix (ECM) proteins containing laminin-G (LG) domains. Matriglycan is an elongated polysaccharide of alternating xylose (Xyl) and glucuronic acid (GlcA) that binds with high affinity to ECM proteins with LG domains and is uniquely synthesized on α-dystroglycan (α-DG) by like-acetylglucosaminyltransferase-1 (LARGE1). Defects in the post-translational processing or O-glycosylation of α-DG that result in a shorter form of matriglycan reduce the size of α-DG and decrease laminin binding, leading to various forms of muscular dystrophy. Previously, we demonstrated that protein O-mannose kinase (POMK) is required for LARGE1 to generate full-length matriglycan on α-DG (~150-250 kDa) (Walimbe et al., 2020). Here, we show that LARGE1 can only synthesize a short, non-elongated form of matriglycan in mouse skeletal muscle that lacks the DG N-terminus (α-DGN), resulting in an ~100-125 kDa α-DG. This smaller form of α-DG binds laminin and maintains specific force but does not prevent muscle pathophysiology, including reduced force production after eccentric contractions (ECs) or abnormalities in the neuromuscular junctions. Collectively, our study demonstrates that α-DGN, like POMK, is required for LARGE1 to extend matriglycan to its full mature length on α-DG and thus prevent muscle pathophysiology.

Identification of a short, single site matriglycan that maintains neuromuscular function in the mouse.

Matriglycan (-1,3-ß-glucuronic acid-1,3-α-xylose-) is a polysaccharide that is synthesized on α-dystroglycan, where it functions as a high-affinity glycan receptor for extracellular proteins, such as laminin, perlecan and agrin, thus anchoring the plasma membrane to the extracellular matrix. This biological activity is closely associated with the size of matriglycan. Using high-resolution mass spectrometry and site-specific mutant mice, we show for the first time that matriglycan on the T317/T319 and T379 sites of α-dystroglycan are not identical. T379-linked matriglycan is shorter than the previously characterized T317/T319-linked matriglycan, although it maintains its laminin binding capacity. Transgenic mice with only the shorter T379-linked matriglycan exhibited mild embryonic lethality, but those that survived were healthy. The shorter T379-linked matriglycan exists in multiple tissues and maintains neuromuscular function in adult mice. In addition, the genetic transfer of α-dystroglycan carrying just the short matriglycan restored grip strength and protected skeletal muscle from eccentric contraction-induced damage in muscle-specific dystroglycan knock-out mice. Due to the effects that matriglycan imparts on the extracellular proteome and its ability to modulate cell-matrix interactions, our work suggests that differential regulation of matriglycan length in various tissues optimizes the extracellular environment for unique cell types.

Large1 gene transfer in older myd mice with severe muscular dystrophy restores muscle function and greatly improves survival.

Muscular dystrophy is a progressive and ultimately lethal neuromuscular disease. Although gene editing and gene transfer hold great promise as therapies when administered before the onset of severe clinical symptoms, it is unclear whether these strategies can restore muscle function and improve survival in the late stages of muscular dystrophy. Largemyd/Largemyd (myd) mice lack expression of like-acetylglucosaminyltransferase-1 (Large1) and exhibit severe muscle pathophysiology, impaired mobility, and a markedly reduced life span. Here, we show that systemic delivery of AAV2/9 CMV Large1 (AAVLarge1) in >34-week-old myd mice with advanced disease restores matriglycan expression on dystroglycan, attenuates skeletal muscle pathophysiology, improves motor and respiratory function, and normalizes systemic metabolism, which collectively and markedly extends survival. Our results in a mouse model of muscular dystrophy demonstrate that skeletal muscle function can be restored, illustrating its remarkable plasticity, and that survival can be greatly improved even after the onset of severe muscle pathophysiology.

Muscular dystrophy-dystroglycanopathy in a family of Labrador retrievers with a LARGE1 mutation.

Alpha-dystroglycan (αDG) is a highly glycosylated cell surface protein with a significant role in cell-to-extracellular matrix interactions in muscle. αDG interaction with extracellular ligands relies on the activity of the LARGE1 glycosyltransferase that synthesizes and extends the heteropolysaccharide matriglycan. Abnormalities in αDG glycosylation and formation of matriglycan are the pathogenic mechanisms for the dystroglycanopathies, a group of congenital muscular dystrophies. Muscle biopsies were evaluated from related 6-week-old Labrador retriever puppies with poor suckling, small stature compared to normal litter mates, bow-legged stance and markedly elevated creatine kinase activities. A dystrophic phenotype with marked degeneration and regeneration, multifocal mononuclear cell infiltration and endomysial fibrosis was identified on muscle cryosections. Single nucleotide polymorphism (SNP) array genotyping data on the family members identified three regions of homozygosity in 4 cases relative to 8 controls. Analysis of whole genome sequence data from one of the cases identified a stop codon mutation in the LARGE1 gene that truncates 40% of the protein. Immunofluorescent staining and western blotting demonstrated the absence of matriglycan in skeletal muscle and heart from affected dogs. Compared to control, LARGE enzyme activity was not detected. This is the first report of a dystroglycanopathy in dogs.

POMK regulates dystroglycan function via LARGE1-mediated elongation of matriglycan.

Matriglycan [-GlcA-ß1,3-Xyl-α1,3-]n serves as a scaffold in many tissues for extracellular matrix proteins containing laminin-G domains including laminin, agrin, and perlecan. Like-acetyl-glucosaminyltransferase 1 (LARGE1) synthesizes and extends matriglycan on α-dystroglycan (α-DG) during skeletal muscle differentiation and regeneration; however, the mechanisms which regulate matriglycan elongation are unknown. Here, we show that Protein O-Mannose Kinase (POMK), which phosphorylates mannose of core M3 (GalNAc-ß1,3-GlcNAc-ß1,4-Man) preceding matriglycan synthesis, is required for LARGE1-mediated generation of full-length matriglycan on α-DG (~150 kDa). In the absence of Pomk gene expression in mouse skeletal muscle, LARGE1 synthesizes a very short matriglycan resulting in a ~ 90 kDa α-DG which binds laminin but cannot prevent eccentric contraction-induced force loss or muscle pathology. Solution NMR spectroscopy studies demonstrate that LARGE1 directly interacts with core M3 and binds preferentially to the phosphorylated form. Collectively, our study demonstrates that phosphorylation of core M3 by POMK enables LARGE1 to elongate matriglycan on α-DG, thereby preventing muscular dystrophy.

HNK-1 sulfotransferase modulates α-dystroglycan glycosylation by 3-O-sulfation of glucuronic acid on matriglycan.

Mutations in multiple genes required for proper O-mannosylation of α-dystroglycan are causal for congenital/limb-girdle muscular dystrophies and abnormal brain development in mammals. Previously, we and others further elucidated the functional O-mannose glycan structure that is terminated by matriglycan, [(-GlcA-ß3-Xyl-α3-)n]. This repeating disaccharide serves as a receptor for proteins in the extracellular matrix. Here, we demonstrate in vitro that HNK-1 sulfotransferase (HNK-1ST/carbohydrate sulfotransferase) sulfates terminal glucuronyl residues of matriglycan at the 3-hydroxyl and prevents further matriglycan polymerization by the LARGE1 glycosyltransferase. While α-dystroglycan isolated from mouse heart and kidney is susceptible to exoglycosidase digestion of matriglycan, the functional, lower molecular weight α-dystroglycan detected in brain, where HNK-1ST expression is elevated, is resistant. Removal of the sulfate cap by a sulfatase facilitated dual-glycosidase digestion. Our data strongly support a tissue specific mechanism in which HNK-1ST regulates polymer length by competing with LARGE for the 3-position on the nonreducing GlcA of matriglycan.

Protective role for the N-terminal domain of α-dystroglycan in Influenza A virus proliferation.

α-Dystroglycan (α-DG) is a highly glycosylated basement membrane receptor that is cleaved by the proprotein convertase furin, which releases its N-terminal domain (α-DGN). Before cleavage, α-DGN interacts with the glycosyltransferase LARGE1 and initiates functional O-glycosylation of the mucin-like domain of α-DG. Notably, α-DGN has been detected in a wide variety of human bodily fluids, but the physiological significance of secreted α-DGN remains unknown. Here, we show that mice lacking α-DGN exhibit significantly higher viral titers in the lungs after Influenza A virus (IAV) infection (strain A/Puerto Rico/8/1934 H1N1), suggesting an inability to control virus load. Consistent with this, overexpression of α-DGN before infection or intranasal treatment with recombinant α-DGN prior and during infection, significantly reduced IAV titers in the lungs of wild-type mice. Hemagglutination inhibition assays using recombinant α-DGN showed in vitro neutralization of IAV. Collectively, our results support a protective role for α-DGN in IAV proliferation.

Structure of protein O-mannose kinase reveals a unique active site architecture.

The 'pseudokinase' SgK196 is a protein O-mannose kinase (POMK) that catalyzes an essential phosphorylation step during biosynthesis of the laminin-binding glycan on α-dystroglycan. However, the catalytic mechanism underlying this activity remains elusive. Here we present the crystal structure of Danio rerio POMK in complex with Mg2+ ions, ADP, aluminum fluoride, and the GalNAc-ß3-GlcNAc-ß4-Man trisaccharide substrate, thereby providing a snapshot of the catalytic transition state of this unusual kinase. The active site of POMK is established by residues located in non-canonical positions and is stabilized by a disulfide bridge. GalNAc-ß3-GlcNAc-ß4-Man is recognized by a surface groove, and the GalNAc-ß3-GlcNAc moiety mediates the majority of interactions with POMK. Expression of various POMK mutants in POMK knockout cells further validated the functional requirements of critical residues. Our results provide important insights into the ability of POMK to function specifically as a glycan kinase, and highlight the structural diversity of the human kinome.

Structural basis of laminin binding to the LARGE glycans on dystroglycan.

Dystroglycan is a highly glycosylated extracellular matrix receptor with essential functions in skeletal muscle and the nervous system. Reduced matrix binding by α-dystroglycan (α-DG) due to perturbed glycosylation is a pathological feature of several forms of muscular dystrophy. Like-acetylglucosaminyltransferase (LARGE) synthesizes the matrix-binding heteropolysaccharide [-glucuronic acid-ß1,3-xylose-α1,3-]n. Using a dual exoglycosidase digestion, we confirm that this polysaccharide is present on native α-DG from skeletal muscle. The atomic details of matrix binding were revealed by a high-resolution crystal structure of laminin-G-like (LG) domains 4 and 5 (LG4 and LG5) of laminin-α2 bound to a LARGE-synthesized oligosaccharide. A single glucuronic acid-ß1,3-xylose disaccharide repeat straddles a Ca(2+) ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca(2+)-bound water molecules. The chelating binding mode accounts for the high affinity of this protein-carbohydrate interaction. These results reveal a previously uncharacterized mechanism of carbohydrate recognition and provide a structural framework for elucidating the mechanisms underlying muscular dystrophy.

The glucuronyltransferase B4GAT1 is required for initiation of LARGE-mediated α-dystroglycan functional glycosylation.

Dystroglycan is a cell membrane receptor that organizes the basement membrane by binding ligands in the extracellular matrix. Proper glycosylation of the α-dystroglycan (α-DG) subunit is essential for these activities, and lack thereof results in neuromuscular disease. Currently, neither the glycan synthesis pathway nor the roles of many known or putative glycosyltransferases that are essential for this process are well understood. Here we show that FKRP, FKTN, TMEM5 and B4GAT1 (formerly known as B3GNT1) localize to the Golgi and contribute to the O-mannosyl post-phosphorylation modification of α-DG. Moreover, we assigned B4GAT1 a function as a xylose ß1,4-glucuronyltransferase. Nuclear magnetic resonance studies confirmed that a glucuronic acid ß1,4-xylose disaccharide synthesized by B4GAT1 acts as an acceptor primer that can be elongated by LARGE with the ligand-binding heteropolysaccharide. Our findings greatly broaden the understanding of α-DG glycosylation and provide mechanistic insight into why mutations in B4GAT1 disrupt dystroglycan function and cause disease.

Endogenous glucuronyltransferase activity of LARGE or LARGE2 required for functional modification of α-dystroglycan in cells and tissues.

Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAß1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.

LARGE glycans on dystroglycan function as a tunable matrix scaffold to prevent dystrophy.

The dense glycan coat that surrounds every cell is essential for cellular development and physiological function, and it is becoming appreciated that its composition is highly dynamic. Post-translational addition of the polysaccharide repeating unit [-3-xylose-α1,3-glucuronic acid-ß1-]n by like-acetylglucosaminyltransferase (LARGE) is required for the glycoprotein dystroglycan to function as a receptor for proteins in the extracellular matrix. Reductions in the amount of [-3-xylose-α1,3-glucuronic acid-ß1-]n (hereafter referred to as LARGE-glycan) on dystroglycan result in heterogeneous forms of muscular dystrophy. However, neither patient nor mouse studies has revealed a clear correlation between glycosylation status and phenotype. This disparity can be attributed to our lack of knowledge of the cellular function of the LARGE-glycan repeat. Here we show that coordinated upregulation of Large and dystroglycan in differentiating mouse muscle facilitates rapid extension of LARGE-glycan repeat chains. Using synthesized LARGE-glycan repeats we show a direct correlation between LARGE-glycan extension and its binding capacity for extracellular matrix ligands. Blocking Large upregulation during muscle regeneration results in the synthesis of dystroglycan with minimal LARGE-glycan repeats in association with a less compact basement membrane, immature neuromuscular junctions and dysfunctional muscle predisposed to dystrophy. This was consistent with the finding that patients with increased clinical severity of disease have fewer LARGE-glycan repeats. Our results reveal that the LARGE-glycan of dystroglycan serves as a tunable extracellular matrix protein scaffold, the extension of which is required for normal skeletal muscle function.

SGK196 is a glycosylation-specific O-mannose kinase required for dystroglycan function.

Phosphorylated O-mannosyl trisaccharide [N-acetylgalactosamine-ß3-N-acetylglucosamine-ß4-(phosphate-6-)mannose] is required for dystroglycan to bind laminin-G domain-containing extracellular proteins with high affinity in muscle and brain. However, the enzymes that produce this structure have not been fully elucidated. We found that glycosyltransferase-like domain-containing 2 (GTDC2) is a protein O-linked mannose ß 1,4-N-acetylglucosaminyltransferase whose product could be extended by ß 1,3-N-acetylgalactosaminyltransferase2 (B3GALNT2) to form the O-mannosyl trisaccharide. Furthermore, we identified SGK196 as an atypical kinase that phosphorylated the 6-position of O-mannose, specifically after the mannose had been modified by both GTDC2 and B3GALNT2. These findings suggest how mutations in GTDC2, B3GALNT2, and SGK196 disrupt dystroglycan receptor function and lead to congenital muscular dystrophy.

ISPD loss-of-function mutations disrupt dystroglycan O-mannosylation and cause Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is clinically defined as congenital muscular dystrophy that is accompanied by a variety of brain and eye malformations. It represents the most severe clinical phenotype in a spectrum of diseases associated with abnormal post-translational processing of a-dystroglycan that share a defect in laminin-binding glycan synthesis1. Although mutations in six genes have been identified as causes of WWS, only half of all individuals with the disease can currently be diagnosed on this basis2. A cell fusion complementation assay in fibroblasts from undiagnosed individuals with WWS was used to identify five new complementation groups. Further evaluation of one group by linkage analysis and targeted sequencing identified recessive mutations in the ISPD gene (encoding isoprenoid synthase domain containing). The pathogenicity of the identified ISPD mutations was shown by complementation of fibroblasts with wild-type ISPD. Finally, we show that recessive mutations in ISPD abolish the initial step in laminin-binding glycan synthesis by disrupting dystroglycan O-mannosylation. This establishes a new mechanism for WWS pathophysiology.

Sarcoglycan complex: implications for metabolic defects in muscular dystrophies.

The sarcoglycans are known as an integral subcomplex of the dystrophin glycoprotein complex, the function of which is best characterized in skeletal muscle in relation to muscular dystrophies. Here we demonstrate that the white adipocytes, which share a common precursor with the myocytes, express a cell-specific sarcoglycan complex containing beta-, delta-, and epsilon-sarcoglycan. In addition, the adipose sarcoglycan complex associates with sarcospan and laminin binding dystroglycan. Using multiple sarcoglycan null mouse models, we show that loss of alpha-sarcoglycan has no consequence on the expression of the adipocyte sarcoglycan complex. However, loss of beta- or delta-sarcoglycan leads to a concomitant loss of the sarcoglycan complex as well as sarcospan and a dramatic reduction in dystroglycan in adipocytes. We further demonstrate that beta-sarcoglycan null mice, which lack the sarcoglycan complex in adipose tissue and skeletal muscle, are glucose-intolerant and exhibit whole body insulin resistance specifically due to impaired insulin-stimulated glucose uptake in skeletal muscles. Thus, our data demonstrate a novel function of the sarcoglycan complex in whole body glucose homeostasis and skeletal muscle metabolism, suggesting that the impairment of the skeletal muscle metabolism influences the pathogenesis of muscular dystrophy.

A different conformation for EGC stator subcomplex in solution and in the assembled yeast V-ATPase: possible implications for regulatory disassembly.

Vacuolar ATPases (V-ATPases) are ATP-dependent proton pumps that maintain the acidity of cellular compartments. They are composed of a membrane-integrated proton-translocating V(0) and an extrinsic cytoplasmic catalytic domain V(1), joined by several connecting subunits. To clarify the arrangement of these peripheral connections and their interrelation with other subunits of the holocomplex, we have determined the solution structures of isolated EG and EGC connecting subcomplexes by small angle X-ray scattering and the 3D map of the yeast V-ATPase by electron microscopy. In solution, EG forms a slightly kinked rod, which assembles with subunit C into an L-shaped structure. This model is supported by the microscopy data, which show three copies of EG with two of these linked by subunit C. However, the relative arrangement of the EG and C subunits in solution is more open than that in the holoenzyme, suggesting a conformational change of EGC during regulatory assembly and disassembly.

Peripheral stator of the yeast V-ATPase: stoichiometry and specificity of interaction between the EG complex and subunits C and H.

V-ATPases are multisubunit membrane protein complexes that use the energy provided by ATP hydrolysis to generate a proton gradient across various intracellular and plasma membranes. In doing so, they maintain an acidic pH in the lumen of intracellular organelles and acidify extracellular milieu to support specific cellular functions. V-ATPases are structurally similar to the F1F0-ATP synthase, with an intrinsic membrane domain (V0) and an extrinsic peripheral domain (V1) joined by several connecting elements. To gain a clear functional understanding of the catalytic mechanism, and of the stability requirements for regulatory processes in the enzyme, a clear topology of the enzyme has to be established. In particular, the composition and arrangement of the peripheral stator subunits must be firmly settled, as these play specific roles in catalysis and regulation. We have designed a strategy allowing us to coexpress different combinations of these subunits to delineate specific interactions. In this study, we report the interaction between the peripheral stator EG complex and subunits C and H of the V-ATPase from the yeast Saccharomyces cerevisae. A combination of analytical gel filtration, native gel electrophoresis, and ultracentrifugation analysis allowed us to ascertain the homogeneity and molar mass of the purified EGC complex as well as of the EG complex, supporting the formation of 1:1(:1) stoichiometric complexes. The EGC complex can be formed in vitro by combining equimolar amounts of subunit C and the EG subcomplex and results most likely from the initial interaction between subunits E and C.

Elucidation of the stator organization in the V-ATPase of Neurospora crassa.

V-ATPases are membrane protein complexes that pump protons in the lumen of various subcellular compartments at the expense of ATP. Proton pumping is done by a rotary mechanism that requires a static connection between the membrane pumping domain (V(0)) and the extrinsic catalytic head (V(1)). This static connection is composed of several known subunits of the V-ATPase, but their location and topological relationships are still a matter of controversy. Here, we propose a model for the V-ATPase of Neurospora crassa on the basis of single-particle analysis by electron microscopy. Comparison of the resulting map to that of the A-ATPase from Thermus thermophilus allows the positioning of two subunits in the static connecting region that are unique to eukaryotic V-ATPases (C and H). These two subunits seem to be located on opposite sides of a semicircular arrangement of the peripheral connecting elements, suggesting a role in stabilizing the stator in V-ATPases.

Building the stator of the yeast vacuolar-ATPase: specific interaction between subunits E and G.

The vacuolar (H+)-ATPase (or V-ATPase) is a membrane protein complex that is structurally related to F1 and F0 ATP synthases. The V-ATPase is composed of an integral domain (V0) and a peripheral domain (V1) connected by a central stalk and up to three peripheral stalks. The number of peripheral stalks and the proteins that comprise them remain controversial. We have expressed subunits E and G in Escherichia coli as maltose binding protein fusion proteins and detected a specific interaction between these two subunits. This interaction was specific for subunits E and G and was confirmed by co-expression of the subunits from a bicistronic vector. The EG complex was characterized using size exclusion chromatography, cross-linking with short length chemical cross-linkers, circular dichroism spectroscopy, and electron microscopy. The results indicate a tight interaction between subunits E and G and revealed interacting helices in the EG complex with a length of about 220 angstroms. We propose that the V-ATPase EG complex forms one of the peripheral stators similar to the one formed by the two copies of subunit b in F-ATPase.

Three-dimensional map of a plant V-ATPase based on electron microscopy.

V-ATPases pump protons into the interior of various subcellular compartments at the expense of ATP. Previous studies have shown that these pumps comprise a membrane-integrated, proton-translocating (V(0)), and a soluble catalytic (V(1)) subcomplex connected to one another by a thin stalk region. We present two three-dimensional maps derived from electron microscopic images of the complete V-ATPase complex from the plant Kalanchoë daigremontiana at a resolution of 2.2 nm. In the presence of a non-hydrolyzable ATP analogue, the details of the stalk region between V(0) and V(1) were revealed for the first time in their three-dimensional organization. A central stalk was surrounded by three peripheral stalks of different sizes and shapes. In the absence of the ATP analogue, the tilt of V(0) changed with respect to V(1), and the stalk region was less clearly defined, perhaps due to increased flexibility and partial detachment of some of the peripheral stalks. These structural changes corresponded to decreased stability of the complex and might be the initial step in a controlled disassembly.