- What is the structural basis for the interaction of α-DG with laminin?
DG is a widely expressed high-affinity extracellular matrix receptor that requires extensive glycosylation and additional post-translational processing1-3. In skeletal muscle, DG is part of the dystrophin-glycoprotein complex, which establishes a continuous link between laminin-G-like (LG) domains of extracellular matrix-resident proteins (laminin, agrin, and perlecan) and the cytoskeleton4-6. Extracellular matrix proteins that contain LG domains bind to α-DG via a novel heteropolysaccharide [-GlcA-β1,3-Xyl-α1,3-]n called matriglycan, which is synthesized by the bifunctional enzyme known as Like-acetylglucosaminyltransferase (LARGE)7,8. How this unusual carbohydrate structure is recognized with high affinity by LG domain-containing proteins is one of the major questions we addressed9.
In this study we used a multidisciplinary approach to provide the first atomic-level insights into the structure of a unique protein-carbohydrate interaction that is essential for the binding of α-DG to the extracellular matrix9. In my laboratory, we purified LARGE and synthesized matriglycan chains of defined lengths in sufficient quantities to perform both biochemical and structural studies. Using NMR spectroscopy, we found that matriglycan bound to laminin-α2 LG4,5 with high affinity (Kd=0.23 µM) and that this binding was calcium dependent.
Next, we initiated a collaboration with Erhard Hohenester (Imperial College, London), who had previously crystallized laminin-α2 LG4,510. We provided him with enough matriglycan to soak into crystals of LG4,5 and to produce a high-resolution crystal structure of LG4,5 bound to matriglycan. This analysis revealed that the LG4 domain is a Ca2+-dependent lectin with specificity for GlcA-β1,3-Xyl disaccharides, and that a single glucuronic acid-β1,3-xylose disaccharide repeat straddles a Ca2+ ion in the LG4 domain, with oxygen atoms from both sugars replacing Ca2+-bound water molecules. This chelating binding mode is unprecedented among animal lectins and accounts for the high affinity of this protein-carbohydrate interaction as measured by NMR.
In my laboratory we also identified two novel exoglycosidases that, collectively, can degrade matriglycan on α-DG in skeletal muscle. As a result, we provided direct evidence that native α-DG contains unmodified matriglycan and numerous GlcA-Xyl repeats. The multiple tandem repeats in matriglycan are predicted to increase the apparent affinity of the protein for LG domains by favoring rapid rebinding after dissociation. Abnormalities in the post-translational processing of DG lead to an absence or reduction of the matriglycan modification on α-DG, and thereby lead to various forms of muscular dystrophy (secondary dystroglycanopathies)11. Our results represent a major conceptual advance regarding protein-carbohydrate interactions and shed light on the mechanism leading to the molecular pathogenesis underlying the dystroglycanopathies.
- How does LARGE modify the phosphorylated mannose glycan?
Previously, we had assigned functions to the POMGNT2, B3GALNT2, and POMK gene products, all of which contribute to synthesis of a phosphorylated, O-mannosyl-linked trisaccharide on α-DG that we have described as core M3 (GalNAc-β3-GlcNAc-β4-Man-α Ser/Thr)12. Synthesized in the ER, this structure is thought to serve as a platform for further functional modification of α-DG as it passes through the secretory pathway; however, our previous findings did not reveal how the matriglycan synthesized by LARGE is attached to phosphorylated core M3.
Using a multidisciplinary approach, we showed that initiation of production of the LARGE glycan on α-DG requires B4GAT1-dependent synthesis of a novel glucuronyl-xylosyl acceptor and that B4GAT1 is a novel xylose β1,4 glucuronyltransferase14. This activity contributes to production of the post-phosphoryl glycan linker by transferring a GlcA residue to a Xyl acceptor, thereby forming the glucuronyl-β1,4-xylosyl disaccharide primer. This primer is required by the glycosyltransferase LARGE to initiate polymerization of the terminal heteropolysaccharide that enables ligand binding. Our findings demonstrate how the laminin-binding glycan synthesized by LARGE is attached to core M3 and how LARGE modifies a novel glucuronyl-xylosyl acceptor to initiate synthesis of the laminin binding polysaccharide.
- What is the pathophysiology that leads to muscle degeneration and death in muscular dystrophy?
By the start of the past appointment period, the association between increased susceptibility to contraction-induced injury and muscular dystrophy had been well characterized in mouse models in which the dystrophic mutation was present throughout development. However, a major limitation of this early work and subsequent related studies is that they did not resolve whether the observed susceptibility to injury was a primary defect or a secondary consequence of either abnormal development and growth or cycles of degeneration and regeneration of muscle fibers. To address this limitation, we directly assessed muscle performance and susceptibility to contraction-induced injury in an inducible mouse model of muscular dystrophy in which DG is disrupted at maturity16.
Notably, an increase in susceptibility to such injury was the first pathological feature observed following the decrease in the levels of DG, and it was not accompanied by increases in necrosis, excitation-contraction uncoupling, or fragility of the sarcolemma. Rather, a severe reduction in passive stiffness and titin immunofluorescence was observed, suggesting that the sarcomeric cytoskeleton was disrupted. These results revealed a novel role for DG in maintaining stability of the sarcomeric cytoskeleton during contraction, and they provided mechanistic insight into the cause of the reduction in strength that occurs in muscular dystrophy following lengthening contractions.
Although progress is also being made toward the development of therapies for muscular dystrophy, this disease remains lethal and most patients die in their late teens or early twenties. The latter outcome is consistent with a high susceptibility of muscular dystrophy patients to influenza. However, it remains unclear whether increase incidence of influenza is caused by muscle pathophysiology and/or a heightened susceptibility to influenza infection. We noticed that in lung tissue from mice infected with Influenza A virus (IAV), which causes airway inflammation, α-DG glycosylation was aberrant. This modification is dependent on the N-terminal domain of α-DG (DGN) and its interaction with LARGE1, and DGN can be cleaved from α-DG by the proprotein convertase furin during inflammation and secreted18.
We have now shown that IAV infection promotes the expression of furin in the lungs, as well as secretion of DGN into murine bronchoalveolar lavage fluid17. In mice lacking DGN, the lung IAV titer after infection was significantly higher than in wild-type counterparts, suggesting that the ability to control viral load was impaired. In mice overexpressing DGN in the lungs both prior to and during IAV infection, viral load was significantly reduced. Moreover, recombinant DGN disrupted hemagglutination-mediated by the influenza virus, demonstrating that DGN can neutralize IAV in vitro. Our findings reveal that DGN is protective in the context of IAV infection. If DGN competes for receptors on lung epithelial cells, it could potentially also play a protective role during infection of the respiratory tract by other viruses.
Although we have made great progress determining the structure, function and biosynthesis of the laminin binding glycan on α-DG over the past five years, significant questions remain to be answered. The glycosylation and post-translational processing of DG requires over eighteen genes and likely more will be found in the next few years. Our multifaceted experimental approach is also expected to provide new insights into the unique post-translational processing of DG, as well as to identify and delineate the pathogenetic mechanisms that lead to muscular dystrophy, including CNS abnormalities. Given that perturbation of the functional glycosylation of α-DG affects a variety of physiological processes – including maintenance of the integrity of the skeletal-muscle membrane, development and function of the CNS, the progression of epithelial tumors, and infection by arenaviruses – our studies will broaden our knowledge of principles that are fundamental to a wide range of research fields. In summary, our field is rich with opportunity for discovery.
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