Identification of Matriglycan by Dual Exoglycosidase Digestion of -Dystroglycan

Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-alpha 1,3-GlcA-beta 1,3](n) that is uniquely synthesized on alpha-dystroglycan (alpha-DG) and is essential for neuromuscular function and brain development. It binds several extracellular matrix proteins that contain laminin-globular domains and is a receptor for Old World arenaviruses such as Lassa Fever virus. Monoclonal antibodies such as IIH6 are commonly used to detect matriglycan on alpha-DG. However, endogenous expression levels are not sufficient to detect and analyze matriglycan by mass spectrometry approaches. Thus, there is a growing need to independently confirm the presence of matriglycan on alpha-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: beta-Glucuronidase from Thermotoga maritima and alpha-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on alpha-DG by studying post-digestion changes in the molecular weight of alpha-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core alpha-DG antibodies, and/or laminin binding assay. In some tissues, matriglycan is capped by a sulfate group, which renders it resistant to digestion by these dual exoglycosidases. Thus, this method can be used to determine the capping status of matriglycan. To date, matriglycan has only been identified on vertebrate alpha-DG. We anticipate that this method will facilitate the discovery of matriglycan on alpha-DG in other species and possibly on other proteins.