title: Endogenous glucuronyltransferase activity of LARGE or LARGE2 required for functional modification of α-dystroglycan in cells and tissues.
abstract: Mutations in the LARGE gene have been identified in congenital muscular dystrophy (CMD) patients with brain abnormalities. Both LARGE and its paralog, LARGE2 (also referred to as GYLTL1B) are bifunctional glycosyltransferases with xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, and are capable of forming polymers consisting of [-3Xyl-α1,3GlcAβ1-] repeats. LARGE-dependent modification of α-dystroglycan (α-DG) with these polysaccharides is essential for the ability of α-DG to act as a receptor for ligands in the extracellular matrix. Here we report on the endogenous enzymatic activities of LARGE and LARGE2 in mice and humans, using a newly developed assay for GlcA-T activity. We show that normal mouse and human cultured cells have endogenous LARGE GlcA-T, and that this activity is absent in cells from the Large(myd) (Large-deficient) mouse model of muscular dystrophy, as well as in cells from CMD patients with mutations in the LARGE gene. We also demonstrate that GlcA-T activity is significant in the brain, heart, and skeletal muscle of wild-type and Large2(-/-) mice, but negligible in the corresponding tissues of the Large(myd) mice. Notably, GlcA-T activity is substantial, though reduced, in the kidneys of both the Large(myd) and Large2(-/-) mice, consistent with the observation of α-DG/laminin binding in these contexts. This study is the first to test LARGE activity in samples as small as cryosections and, moreover, provides the first direct evidence that not only LARGE, but also LARGE2, is vital to effective functional modification of α-DG in vivo.
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101, Division of Glycopathology, Institute of Molecular Biomembrane and Glycobiology, Tohoku Pharmaceutical University, Sendai, Miyagi 981-8558, Japan.
authors.forename: Kei-ichiro
authors.initials: K
authors.lastname: Inamori
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101.
authors.forename: Tobias
authors.initials: T
authors.lastname: Willer
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Hara
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101.
authors.forename: David
authors.initials: D
authors.lastname: Venzke
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Anderson
authors.affiliation: Institute for Neuroscience and Muscle Research, The Children's Hospital at Westmead, University of Sydney, Sydney, Australia.
authors.forename: Nigel F
authors.initials: NF
authors.lastname: Clarke
authors.affiliation: Inserm, U1166, Faculté de Médecine Pierre et Marie Curie, Institute of Cardiometabolism and Nutrition, ICAN, Paris, France, Sorbonne Universités, UPMC Univ Paris 06, UMR_S1166, Paris, France.
authors.forename: Pascale
authors.initials: P
authors.lastname: Guicheney
authors.affiliation: Neurogenetics Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20892.
authors.forename: Carsten G
authors.initials: CG
authors.lastname: Bönnemann
authors.affiliation: Department of Pathology, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Moore
authors.affiliation: From the Howard Hughes Medical Institute, Department of Molecular Physiology and Biophysics, Department of Neurology, Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, Iowa City, Iowa 52242-1101, kevin-campbell@uiowa.edu.
authors.forename: Kevin P
authors.initials: KP
authors.lastname: Campbell
mesh_terms: D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology / Q000473:pathology; D002478:Cells, Cultured; D002648:Child; D004195:Disease Models, Animal; D049030:Dystroglycans / Q000235:genetics / Q000378:metabolism*; D057075:Enzyme Assays; D005260:Female; D005347:Fibroblasts / Q000201:enzymology / Q000473:pathology; D005786:Gene Expression Regulation; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007668:Kidney / Q000201:enzymology / Q000473:pathology; D007797:Laminin / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018482:Muscle, Skeletal / Q000201:enzymology / Q000473:pathology; D009136:Muscular Dystrophies / Q000201:enzymology* / Q000235:genetics / Q000473:pathology; D009206:Myocardium / Q000201:enzymology / Q000473:pathology; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009928:Organ Specificity; D011485:Protein Binding
chemical_list: D007797:Laminin; D049030:Dystroglycans; D016695:Glycosyltransferases; C502747:Large1 protein, mouse; C507407:Large2 protein, mouse; D017351:N-Acetylglucosaminyltransferases
keywords: Dystroglycan; Extracellular Matrix; Glucuronyltransferase; Glycosyltransferase; LARGE; Laminin; Muscular Dystrophy; Post-translational Modification (PTM)