Structural Basis of Dystroglycan Function and the Pathogenesis of Muscular Dystrophy

Kevin P Campbell

doi:10.1096/fasebj.2020.34.s1.00139

(DG) is a widely expressed high‐affinity extracellular matrix receptor that requires extensive glycosylation and additional post‐translational processing. 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 cytoskeleton. 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). We have 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 matrix. We first 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, who had previously crystallized laminin‐α2 LG4,5. We provided Dr. Hohenester 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. Finally, we used two novel exoglycosidases to provide 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). Our results represent a major conceptual advancement regarding a functional carbohydrate and shed light on the mechanism of protein‐carbohydrate interactions.

Structural Basis of Dystroglycan Function and the Pathogenesis of Muscular Dystrophy

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