Large induces functional glycans in an O-mannosylation dependent manner and targets GlcNAc terminals on alpha-dystroglycan

Abstract

Alpha-dystroglycan (α-DG) is a ubiquitously expressed receptor for extracellular matrix proteins and some viruses, and plays a pivotal role in a number of pathological events, including cancer progression, muscular dystrophies, and viral infection. The O-glycans on α-DG are essential for its ligand binding, but the biosynthesis of the functional O-glycans remains obscure. The fact that transient overexpression of LARGE, a putative glycosyltransferase, up-regulates the functional glycans on α-DG to mediate its ligand binding implied that overexpression of LARGE may be a novel strategy to treat disorders with hypoglycosylation of α-DG. In this study, we focus on the effects of stable overexpression of Large on α-DG glycosylation in Chinese hamster ovary (CHO) cell and its glycosylation deficient mutants. Surprisingly, stable overexpression of Large in an O-mannosylation null deficient Lec15.2 CHO cells failed to induce the functional glycans on α-DG. Introducing the wild-type DPM2 cDNA, the deficient gene in the Lec15.2 cells, fully restored the Large-induced functional glycosylation, suggesting that Large induces the functional glycans in a DPM2/O-mannosylation dependent manner. Furthermore, stable overexpression of Large can effectively induce functional glycans on N-linked glycans in the Lec8 cells and ldlD cells growing in Gal deficient media, in both of which circumstances galactosylation are deficient. In addition, supplement of Gal to the ldlD cell culture media significantly reduces the amount of functional glycans induced by Large, suggested that galactosylation suppresses Large to induce the functional glycans. Thus our results revealed a mechanism by which Large competes with galactosyltransferase to target GlcNAc terminals to induce the functional glycans on α-DG.

Figure 1. Stable overexpressing Large induces functional glycans in CHO cells.

(A) Equal amount of lysates (60 µg proteins each lane) extracted from the cells stably expressing Large as indicated. The immuno-blotting procedure was described in methods section. The glycan antibody IIH6 and anti-MYC antibody (9E10) were used to detect the functional glycans and Large–MYC, respectively. In addition, an anti-β-DG (MANDAG2) antibody was used for detecting dystroglycan as the loading control. (B) The cells as indicated were seeded in 96-well plates and immuno-fluorescent staining was performed as described in the methods section. The images in top panel present the negative controls, while the images of bottom panel present the Large positive cells. A rabbit polyclonal anti-MYC antibody and the IIH6 antibody were used to stain the Large-MYC and glycosylated α-DG, respectively. The cells were also stained with DAPI to visualize the nucleus. The bar is 50 µm. (C) The cells as indicated were seeded in 96-well plate for 24 hour growth. The protocol of laminin clustering assay is described in the methods section. The laminins-DyLight488 was added to the cell culture for 6 hours incubation. A rabbit polyclonal anti-MYC antibody was used to stain Large-MYC protein followed by staining with secondary anti-rabbit antibodies conjugated with Alex594. In addition, DAPI was added to each well to stain the DNA for cells counting. The images were captured with a fluorescent microscope as described in methods section. The bar is 50 µm.

Figure 2. Stable overexpression levels of Large on glycosylation of α-DG in the Lec15 cells.

Pro-5, B421 clones overexpressing (+) or without (−) Large-MYC or Lec15.2 cells overexpressing Large-MYC at high +(H), modest +(M) low +(L) or without Large-MYC (−) were tested by immuno-blot assay. The blot of the Lec15.2 samples had 10 mins exposure, while all other blot had about 10 seconds' exposure time.

Figure 3. Overexpressing DPM2 restores the Large function in the Lec15.2 cells.

(A) Transient transfection was conducted to introduce mouse DPM2 cDNA into the Lec 15.2-LG cells. 48 hours after transfection, the cell lysates were harvested and immuno-blotting assay and laminins overlay assay were conducted as described above. (B) A transient transfection was conducted to introduce DPM-GFP cDNA into the cells in 96-well plate. 48 hours the after transfection, cells were fixed and an indirect immuno-fluorescent staining with IIH6 antibody was conducted with the protocol as described in the methods section. The images were captured with a fluorescent microscope as described in methods section. The bar is 50 µm.

Figure 4. Effects of Large-induced pathway on N-linked glycans.

(A) All cells were grown in the completed media as described in the methods section and the lysates harvested from the indicated cells with stably expressing LARGE were treated with pNGase F for 4 hours, while it is absent in the negative controls reactions as described in the methods section. IIH6 antibody was used to determine the LARGE induced glycans, and the anti-β-DG antibody was used to determine the β-DG proteins as described above. The blot of the Lec15.2 samples with the IIH6 antibody had 30 mins exposure, while all other blots had about 10 seconds' exposure time.

Figure 5. LARGE modifies both mucin type O-glycans and N-linked glycans on α-DG in ldlD cells while without galactosylation.

(A) Predicted structures of the glycans in N-linked, Mucin and O-mannosyl pathway in the ldlD cells. (B)The ldlD-LG cells were maintained in F12 nutrition mix media with 3% lipoprotein deficient bovine serum for more than 48 hours prior to the experiment. The cells were seeded into 6-well plates one day before addition of indicated sugars (Gal at 10 µM and GalNAc at 200 µM). After being treated with the sugar(s) for 24 hour, the cell lysates were harvested and equal amount of the lysates were loaded. An immuno-blotting assay was performed to detect the functional glycans and β-DG with the IIH6 and β-DG antibody, respectively. (C) The quantitative data of the expression levels of the functional glycans were obtained with AlphaImage AIC software based on densitometers followed the manufacture instructions. The IIH6 expression levels were normalized with the expression levels of β-DG (N = 3). (D) The lysates harvested from the ldlD-LG cells growing in the conditions as indicated and the experimental procedure is the same as described in (Fig. 4A). (E) Quantitative analysis of the data of (D) (N = 3).

Figure 6. Illustration of the role of Large in the glycosylation of α-DG.

LGIFG: Large-induced functional glycans.