title: Differentiation-related glycan epitopes identify discrete domains of the muscle glycocalyx.
abstract: The neuromuscular junction (NMJ) is enriched with glycoproteins modified with N-acetylgalactosamine (GalNAc) residues, and four nominally GalNAc-specific plant lectins have historically been used to identify the NMJ and the utrophin-glycoprotein complex. However, little is known about the specific glycan epitopes on skeletal muscle that are bound by these lectins, the glycoproteins that bear these epitopes or how creation of these glycan epitopes is regulated. Here, we profile changes in cell surface glycosylation during muscle cell differentiation and identify distinct differences in the binding preferences of GalNAc-specific lectins, Wisteria floribunda agglutinin (WFA), Vicia villosa agglutinin (VVA), soybean agglutinin (SBA) and Dolichos biflorus agglutinin (DBA). While we find that all four GalNAc binding lectins specifically label the NMJ, each of the four lectins binds distinct sets of muscle glycoproteins; furthermore, none of the major adhesion complexes are required for binding of any of the four GalNAc-specific lectins. Analysis of glycosylation-related transcripts identified target glycosyltransferases and glycosidases that could potentially create GalNAc-containing epitopes; reducing expression of these transcripts by siRNA highlighted differences in lectin binding specificities. In addition, we found that complex N-glycans are required for binding of WFA and SBA to murine C2C12 myotubes and for WFA binding to wild-type skeletal muscle, but not for binding of VVA or DBA. These results demonstrate that muscle cell surface glycosylation is finely regulated during muscle differentiation in a domain- and acceptor-substrate-specific manner, suggesting that temporal- and site-specific glycosylation are important for skeletal muscle cell function.
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Brian J
authors.initials: BJ
authors.lastname: McMorran
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Francis E
authors.initials: FE
authors.lastname: McCarthy
authors.affiliation: Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Elizabeth M
authors.initials: EM
authors.lastname: Gibbs
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Mabel
authors.initials: M
authors.lastname: Pang
authors.affiliation: Program in Medical and Population Genetics, The Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA.
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Marshall
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Alison V
authors.initials: AV
authors.lastname: Nairn
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.
authors.forename: Kelley W
authors.initials: KW
authors.lastname: Moremen
authors.affiliation: Department of Integrative Biology and Physiology, University of California Los Angeles, Los Angeles, CA 90095, USA.
authors.forename: Rachelle H
authors.initials: RH
authors.lastname: Crosbie-Watson
authors.affiliation: Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California Los Angeles, Los Angeles, CA 90095, USA lbaum@mednet.ucla.edu.
authors.forename: Linda G
authors.initials: LG
authors.lastname: Baum
mesh_terms: D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D002645:Chickens; D000939:Epitopes / Q000276:immunology*; D019276:Glycocalyx / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000737:chemistry / Q000166:cytology / Q000276:immunology / Q000378:metabolism*; D011134:Polysaccharides / Q000276:immunology*
chemical_list: D000939:Epitopes; D011134:Polysaccharides
keywords: GalNAc; Wisteria floribunda agglutinin; muscle glycosylation; neuromuscular junction; utrophin–glycoprotein complex