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Dystroglycan Function Requires Xylosyl- And Glucuronyltransferase Activities of LARGE

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Dystroglycan Function Requires Xylosyl- And Glucuronyltransferase Activities of LARGE

Kei-ichiro Inamori et al. Science.

Abstract

Posttranslational modification of alpha-dystroglycan (α-DG) by the like-acetylglucosaminyltransferase (LARGE) is required for it to function as an extracellular matrix (ECM) receptor. Mutations in the LARGE gene have been identified in congenital muscular dystrophy patients with brain abnormalities. However, the precise function of LARGE remains unclear. Here we found that LARGE could act as a bifunctional glycosyltransferase, with both xylosyltransferase and glucuronyltransferase activities, which produced repeating units of [-3-xylose-α1,3-glucuronic acid-β1-]. This modification allowed α-DG to bind laminin-G domain-containing ECM ligands.

Figures

Fig. 1
Fig. 1
Xylosylation is required for functional modification of α-DG. (A) Compositional sugar analysis by gas chromatography-MS with trimethylsilyl derivatization using α-DG produced by LARGE-overexpressing HEK293 cells digested with peptide: N-glycosidase F, O-glycosidase, and neuraminidase. Peaks shown in red represent Xyl, those in blue represent GlcA, and numbers indicate 1, Man; 2, Gal; 3, Glc; 4, GlcNAc; 5, GalNAc; and 6, inositol (internal control). Some of the detected Gal was derived from the Jacalin elution buffer we used for the purification. (B) Flow cytometry of WT or UXS1-deficient (pgsI-208) CHO cells surface stained with antibodies against the laminin-binding epitope of α-DG (IIH6), the α-DG core protein (CORE), or heparan sulfate (HS). Dashed line, secondary antibody alone. (C) Functional modification of α-DG in cells overexpressing UXS1. (Left) Immunoblotting or laminin overlay (OL) assays of glycoproteins. Mr, relative molecular mass. Asterisk indicates nonfunctional α-DG that appeared as a sharp band because of hypomodification by CORE staining. (Right) Flow cytometry for surface staining with HS or IIH6. (D) Immunoblotting for IIH6 and β-DG–specific antibody reactivity in WT and pgsI-208 cells with or without overexpression of LARGE. (E) Immunoblotting for IIH6, CORE, or β-DG–specific antibody reactivity in LARGE-expressing WT cells treated with or without Xyl-α-pNP.
Fig. 2
Fig. 2
LARGE is a bifunctional glycosyltransferase that has Xyl-T and GlcA-T activities. (A) HPLC elution profile from the amide column GlycoSep N of the products obtained from the reaction of LARGEdTM with Xyl-α-pNP and UDP-GlcA. S, unreacted substrate. P, product. (B) Linear trap quadrupole (LTQ)–MS analysis of the product peak detected in (A). Stars and diamonds indicate Xyl and GlcA, respectively. MS/MS fragmentation pattern (fig. S7) confirmed that the ion with m/z of 446.3 [M-H] is Xyl-α-pNP with an added GlcA. (C) HPLC elution profile from GlycoSep N of the products obtained from the reaction of LARGEdTM with GlcA-β-MU and UDP-Xyl. (D) As in the legend to (B), for the product isolated from the reaction analyzed in (C). The MS/MS fragmentation pattern (fig. S9) confirmed that the ion with m/z of 483.3 [M-H] is GlcA-β-MU with an added Xyl. Asterisks indicate background ions. (E) Schematic representation of the WT and DXD mutants of LARGEdTM constructs used in the assay. The locations of the mutations in the DXD motifs are symbolized by stars. (F) GlcA-T and Xyl-T activities of WT and mutant LARGEdTMs. Relative activity (%) with respect to WT, and the standard deviation in triplicate experiments, are shown. TM, transmembrane domain. CC, coiled-coil domain. N.D., not detected.
Fig. 3
Fig. 3
Polymerizing activity of LARGE confers ligand-binding ability on α-DG. HPLC profile of the reaction products of LARGEdTM generated from GlcA-β-MU (A) and Xyl-α-pNP (B) in the presence of both donors, UDP-Xyl and UDP-GlcA, on the amide column. S, unreacted substrate. (C) Glycoproteins extracted from Largemyd mouse skeletal muscle were incubated with LARGEdTM, with or without UDP-GlcA and UDP-Xyl, and analyzed by immunoblotting with IIH6 or the CORE antibody, or by overlay assays (OLs) using laminin-G domain–containing ECM ligands (laminin, agrin, or neurexin).
Fig. 4
Fig. 4
MS and NMR analyses of products obtained from in vitro LARGE enzymatic reaction products, using GlcA-β-MU as the acceptor and UDP-GlcA and UDP-Xyl as the donors. (A) Separation profile of polymeric oligosaccharide by Superdex Peptide 10/300. S, unreacted substrate. (B) Matrix-assisted laser desorption–ionization tandem MS analysis of products dp3 to dp6. (C) HMQC spectrum of dp5 at 15°C. Assigned cross peaks are labeled with a letter representing the subunit [as designated in (F)], and a number representing the position on that subunit. The cross peak derived from sample impurities is marked by an asterisk. (D) TOCSY spectrum of dp5, collected with a mixing time of 120 ms at 15°C. (E) ROESY spectrum of dp5, collected with a mixing time of 300 ms at 15°C for the assignment of interglycosidic linkages (indicated by blue circles). The first letter in each label refers to the sugar subunit and the second to the hydrogen position of that subunit. (F) Structures of the polymeric oligosaccharides produced by the LARGE enzymatic reaction in vitro, with the sugar subunits labeled a to f.

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