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.
Highlights
Dystrophy and neuronal cell migration defects [3, 4]
The loss of ␣-DG glycosylation and function in the dystroglycanopathies suggests that the combined activity of Protein:O-mannosyltransferase 1 (POMT1), POMT2, protein:O-mannosyl N-acetylglucosaminyltransferase 1 (POMGnT1), fukutin, fukutin-related protein (FKRP), and LARGE is required for the functional glycosylation of ␣-DG and that the glycans imparting function are formed on GlcNAc1,2Man-O-Ser/Thr in the mucin domain of ␣-DG
Overexpression of LARGE in cultured cells causes the appearance of functional ␣-DG that binds to laminin, neurexin, and agrin and carries glycan epitopes recognized by the VIA4-1 and IIH6 monoclonal antibodies [17, 35]
Summary
MEB, and WWS [27,28,29,30,31,32] and belong to a family of enzymes that transfer phosphoryl ligands to lipopolysaccharides and glycoproteins [33]. The loss of ␣-DG glycosylation and function in the dystroglycanopathies suggests that the combined activity of POMT1, POMT2, POMGnT1, fukutin, FKRP, and LARGE is required for the functional glycosylation of ␣-DG and that the glycans imparting function are formed on GlcNAc1,2Man-O-Ser/Thr in the mucin domain of ␣-DG. Overexpression of LARGE in cultured cells causes the appearance of functional ␣-DG that binds to laminin, neurexin, and agrin and carries glycan epitopes recognized by the VIA4-1 and IIH6 monoclonal antibodies [17, 35]. Overexpression of LARGE, but not POMGnT1, “rescues” functional glycosylation of ␣-DG in dystrophic muscle cells derived from patients with WWS that cannot synthesize O-Man glycans [17]. We show that ␣-DG glycan epitopes are sensitive to N-glycanase in Lec and Lec cells and are enhanced by core 2 N-acetylglucosaminyltransferase 1 (C2GnT1), indicating mechanisms by which LARGE may rescue muscle cells with dystroglycanopathies
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