Xylosyl- and glucuronyltransferase functions of LARGE in α-dystroglycan modification are conserved in LARGE2

Abstract

LARGE-dependent modification enables α-dystroglycan (α-DG) to bind to its extracellular matrix ligands. Mutations in the LARGE gene and several others involved in O-mannosyl glycan synthesis have been identified in congenital and limb-girdle muscular dystrophies that are characterized by perturbed glycosylation and reduced ligand-binding affinity of α-DG. LARGE is a bifunctional glycosyltransferase that alternately transfers xylose and glucuronic acid, thereby generating the heteropolysaccharides on α-DG that confer its ligand binding. Although the LARGE paralog LARGE2 (also referred to as GYLTL1B) has likewise been shown to enhance the functional modification of α-DG in cultured cells, its enzymatic activities have not been identified. Here, we report that LARGE2 is also a bifunctional glycosyltransferase and compare its properties with those of LARGE. By means of a high-performance liquid chromatography-based enzymatic assay, we demonstrate that like LARGE, LARGE2 has xylosyltransferase (Xyl-T) and glucuronyltransferase (GlcA-T) activities, as well as polymerizing activity. Notably, however, the pH optima of the Xyl-T and GlcA-T of LARGE2 are distinct from one another and also from those of LARGE. Our results suggest that LARGE and LARGE2 catalyze the same glycosylation reactions for the functional modification of α-DG, but that they have different biochemical properties.

Fig. 1.

Overexpression of the LARGE paralog does not overcome the glycosylation defect in UXS1-deficient (pgsI-208) CHO cells. (A) Immunoblotting of WGA-enriched glycoproteins for functionally modified α-DG (IIH6) and β-DG, or of cell lysates for LARGE and LARGE2, from WT or pgsI-208 cells with or without overexpression of either LARGE or LARGE2. Mr, relative molecular mass. (B) Flow cytometry of the cells for surface staining with IIH6. Dotted line, secondary antibody alone.

Fig. 2.

Purification of LARGE2dTM. (A) Schematic representation of LARGE and the LARGEdTM construct as described previously (Inamori et al. 2012), and LARGE2 and the LARGE2dTM construct used in the enzymatic activity assay. The transmembrane (TM) sequence of LARGE2 was replaced with a FLAG (3x) tag sequence, and the C-terminus was modified with myc and Hisx6 tags. CC, coiled-coil domain. (B) Expression and purification of test proteins. (Left) Immunoblotting with anti-FLAG antibody. For each stable clone, 50 μL of the culture medium was analyzed. pCMV9, cell clone obtained by transfection of empty vector. (Right) Purification of recombinant LARGE2dTM protein expressed in the culture medium. Proteins were run over the Talon metal affinity resin and eluted, and the fractions were analyzed by Coomassie brilliant blue (CBB) staining.

Fig. 3.

LARGE2 is a bifunctional glycosyltransferase with Xyl-T and GlcA-T activities. (A) HPLC elution profile from the LC-18 column of the products obtained from the reaction of LARGE2dTM with GlcA-β-MU and UDP-Xyl. S, unreacted substrate. P, product. Dashed line, %Buffer B. (B) Q/TOF-MS analysis of the product peak detected in (A). Star and diamond indicate Xyl and GlcA, respectively. The MS/MS fragmentation pattern (Supplementary data, Figure S3A) confirmed that the ion with an m/z of 483.14 [M–H]⁻ is GlcA-β-MU with an added Xyl. (C) HPLC elution profile from the LC-18 column of the products obtained from the reaction of LARGE2dTM with Xyl-α-pNP and UDP-GlcA. (D) Q/TOF-MS analysis as in (B), for the product isolated from the reaction analyzed in (C). The MS/MS fragmentation pattern (Supplementary data, Figure S3B) confirmed that the ion with an m/z of 446.17 [M–H]⁻ is Xyl-α-pNP with an added GlcA. (E) Donor substrate specificity of LARGE2dTM. Representative data from two independent assays, showing relative activity (%) of Xyl-T toward GlcA-β-MU and of GlcA-T toward Xyl-α1,3-GlcA-β-MU. No other sugars were transferred to the acceptors.

Fig. 4.

LARGE2dTM has polymerizing activity. (A) HPLC profile of the reaction products generated when LARGE2dTM was added to GlcA-β-MU in the presence of both donors, UDP-Xyl and UDP-GlcA, on the amide column GlycoSep N. Peaks labeled 1–3 were subjected to further purification in (B). S, unreacted substrate. Dashed line, %Buffer B. (B) Purification of GlcA-β-MU derivatives on an LC-18 column. Each peak isolated in (A) was purified and the main peak was collected (solid bar). (C) Q/TOF-MS analysis of the peaks isolated in (B). Assigned mass spectra in each peak confirmed that LARGE2dTM added repeating units of Xyl and GlcA onto the acceptor, GlcA-β-MU. dp, degree of polymerization.

Fig. 5.

LARGEdTM and LARGE2dTM have distinct pH optima for the Xyl-T and GlcA-T activities. Data from two independent experiments are shown as relative activity (%). The highest activity in the dataset was arbitrarily set at 100%. The Xyl-T and the GlcA-T assays were carried out using GlcA-β-MU and Xyl-α1,3-GlcA-β-MU as the acceptors, respectively. The buffers used were: Acetate for pH 4.0–5.5 (empty circles), MES for pH 5.5–6.5 (closed circles), MOPS for pH 6.5–8.0 (empty triangles) and Tris–HCl for pH 8.0–9.0 (closed triangles). The details of the conditions are presented in the Materials and methods section.