title: Mouse large can modify complex N- and mucin O-glycans on alpha-dystroglycan to induce laminin binding.
abstract: The human LARGE gene encodes a protein with two putative glycosyltransferase domains and is required for the generation of functional alpha-dystroglycan (alpha-DG). Monoclonal antibodies IIH6 and VIA4-1 recognize the functional glycan epitopes of alpha-DG that are necessary for binding to laminin and other ligands. Overexpression of full-length mouse Large generated functionally glycosylated alpha-DG in Pro(-5) Chinese hamster ovary (CHO) cells, and the amount was increased by co-expression of protein:O-mannosyl N-acetylglucosaminyltransferase 1. However, functional alpha-DG represented only a small fraction of the alpha-DG synthesized by CHO cells or expressed from an alpha-DG construct. To identify features of the glycan epitopes induced by Large, the production of functionally glycosylated alpha-DG was investigated in several CHO glycosylation mutants. Mutants with defective transfer of sialic acid (Lec2), galactose (Lec8), or fucose (Lec13) to glycoconjugates, and the Lec15 mutant that cannot synthesize O-mannose glycans, all produced functionally glycosylated alpha-DG upon overexpression of Large. Laminin binding and the alpha-DG glycan epitopes were enhanced in Lec2 and Lec8 cells. In Lec15 cells, functional alpha-DG was increased by co-expression of core 2 N-acetylglucosaminyltransferase 1 with Large. Treatment with N-glycanase markedly reduced functionally glycosylated alpha-DG in Lec2 and Lec8 cells. The combined data provide evidence that Large does not transfer to Gal, Fuc, or sialic acid on alpha-DG nor induce the transfer of these sugars to alpha-DG. In addition, the data suggest that human LARGE may restore functional alpha-DG to muscle cells from patients with defective synthesis of O-mannose glycans via the modification of N-glycans and/or mucin O-glycans on alpha-DG.
authors.affiliation: Department of Cell Biology, Albert Einstein College of Medicine, New York, New York 10461, USA.
authors.forename: Santosh K
authors.initials: SK
authors.lastname: Patnaik
authors.forename: Pamela
authors.initials: P
authors.lastname: Stanley
mesh_terms: D000818:Animals; D019921:Biotinylation; D016466:CHO Cells; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000235:genetics; D049030:Dystroglycans / Q000737:chemistry / Q000378:metabolism*; D015870:Gene Expression; D006031:Glycosylation; D006801:Humans; D007163:Immunosorbent Techniques; D007797:Laminin / Q000378:metabolism*; D008099:Liver / Q000737:chemistry; D051379:Mice; D009077:Mucins / Q000378:metabolism*; D009154:Mutation; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011134:Polysaccharides / Q000378:metabolism*; D011994:Recombinant Proteins; D014162:Transfection
chemical_list: D018076:DNA, Complementary; D007797:Laminin; D009077:Mucins; D011134:Polysaccharides; D011994:Recombinant Proteins; D049030:Dystroglycans; C502747:Large1 protein, mouse; D017351:N-Acetylglucosaminyltransferases; C443106:protein O-mannose beta-1,2-N-acetylglucosaminyltransferase; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase