Journal article
Identification of Matriglycan by Dual Exoglycosidase Digestion of α-Dystroglycan
Bio-protocol, Vol.13(18), e4827
09/20/2023
DOI: 10.21769/BioProtoc.4827
PMCID: PMC10518772
PMID: 37753476
Abstract
Matriglycan is a linear polysaccharide of alternating xylose and glucuronic acid units [-Xyl-α1,3-GlcA-β1,3]
n that is uniquely synthesized on α-dystroglycan (α-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 α-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 α-DG and possibly other proteins. We used an enzymatic approach to detect matriglycan, which involved digesting it with two thermophilic exoglycosidases: β-Glucuronidase from Thermotoga maritima and α-xylosidase from Sulfolobus solfataricus. This allowed us to identify and categorize matriglycan on α-DG by studying post-digestion changes in the molecular weight of α-DG using SDS-PAGE followed by western blotting with anti-matriglycan antibodies, anti-core α-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 α-DG. We anticipate that this method will facilitate the discovery of matriglycan on α-DG in other species and possibly on other proteins.
Key features
• Analysis of endogenous matriglycan on dystroglycan from any animal tissue.
• Matriglycan is digested using thermophilic enzymes, which require optimum thermophilic conditions.
• Western blotting is used to assay the success and extent of digestion.
• Freshly purified enzymes work best to digest matriglycan.
Details
- Title: Subtitle
- Identification of Matriglycan by Dual Exoglycosidase Digestion of α-Dystroglycan
- Creators
- Ishita Chandel - University of IowaKevin P. Campbell - Howard Hughes Medical Institute, Senator Paul D. Wellstone Muscular Dystrophy Specialized Research Center, Department of Molecular Physiology and Biophysics and Department of Neurology, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA, USA
- Resource Type
- Journal article
- Publication Details
- Bio-protocol, Vol.13(18), e4827
- Publisher
- Bio-Protocol
- DOI
- 10.21769/BioProtoc.4827
- PMID
- 37753476
- PMCID
- PMC10518772
- ISSN
- 2331-8325
- eISSN
- 2331-8325
- Language
- English
- Date published
- 09/20/2023
- Academic Unit
- Neurology; Molecular Physiology and Biophysics; Iowa Neuroscience Institute
- Record Identifier
- 9984471941602771
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