title: High throughput screening for compounds that alter muscle cell glycosylation identifies new role for N-glycans in regulating sarcolemmal protein abundance and laminin binding.
abstract: Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function.
authors.affiliation: Department of Pathology and Laboratory Medicine, UCLA School of Medicine, Los Angeles, California 90095, USA.
authors.forename: Paula V
authors.initials: PV
authors.lastname: Cabrera
authors.forename: Mabel
authors.initials: M
authors.lastname: Pang
authors.forename: Jamie L
authors.initials: JL
authors.lastname: Marshall
authors.forename: Raymond
authors.initials: R
authors.lastname: Kung
authors.forename: Stanley F
authors.initials: SF
authors.lastname: Nelson
authors.forename: Stephanie H
authors.initials: SH
authors.lastname: Stalnaker
authors.forename: Lance
authors.initials: L
authors.lastname: Wells
authors.forename: Rachelle H
authors.initials: RH
authors.lastname: Crosbie-Watson
authors.forename: Linda G
authors.initials: LG
authors.lastname: Baum
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D054794:Glycomics / Q000295:instrumentation / Q000379:methods*; D006031:Glycosylation; D007797:Laminin / Q000378:metabolism*; D008120:Lobeline / Q000494:pharmacology; D008297:Male; D008364:Mannosyltransferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D018101:Mice, Inbred mdx; D018485:Muscle Fibers, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D009124:Muscle Proteins / Q000378:metabolism; D020388:Muscular Dystrophy, Duchenne / Q000378:metabolism / Q000473:pathology; D032448:Myoblasts, Skeletal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D018722:Nicotinic Agonists / Q000494:pharmacology; D037121:Plant Lectins / Q000494:pharmacology; D011134:Polysaccharides / Q000378:metabolism*; D052097:Quadriceps Muscle / Q000166:cytology / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics; D034781:Receptors, N-Acetylglucosamine; D012508:Sarcolemma / Q000187:drug effects / Q000378:metabolism*
chemical_list: D007797:Laminin; D009124:Muscle Proteins; D018722:Nicotinic Agonists; D037121:Plant Lectins; D011134:Polysaccharides; D034741:RNA, Small Interfering; D034781:Receptors, N-Acetylglucosamine; C040129:wisteria lectin; D008120:Lobeline; D008364:Mannosyltransferases