N-glycans modulate the activation of gp130 in mouse embryonic neural precursor cells

Abstract

gp130 is a ubiquitously expressed glycoprotein and signal transducer of interleukin 6 family of cytokines. It has been reported that gp130 has 11 potential N-glycosylation sites in the extracellular domain, and nine of them are actually N-glycosylated. However, the structure and functional role of the carbohydrate chains carried by gp130 are totally unknown. In this study, we examined the functional role of N-glycans of gp130 in mouse neuroepithelial cells. In neuroepithelial cells treated with tunicamycin, an N-glycosylation inhibitor, unglycosylated form of gp130 was detected. The unglycosylated gp130 was not phosphorylated in response to leukemia inhibitory factor stimulation. Although the unglycosylated gp130 was found to be expressed on the cell surface, it could not form a heterodimer with leukemia inhibitory factor receptor. These results suggest that N-glycans are required for the activation, but not for the localization, of gp130 in neuroepithelial cells.

Fig. 1

Unglycosylated gp130 in NECs treated with tunicamycin. NECs were treated with tunicamycin for 8 h, stimulated with LIF (0 or 40 ng/ml) for 10 min and then lyzed. Lysates and immunoprecipitated gp130 were subjected to Western-blot analysis. NEC lysates were analyzed with anti-HNK-1 antibody, anti-β-actin antibody and anti-gp130 antibody. Anti-HNK-1 antibody was used to detect glycoproteins as a control. Anti-β-actin antibody was used to confirm that the same amount of proteins was applied to each lane. Immunoprecipitated gp130 was analyzed with anti-phospho-tyrosine antibody (4G10) to evaluate the activation. The molecular masses are indicated on the right of each panel.

Fig. 2

Cell surface localization of unglycosylated gp130 in NECs treated with tunicamycin. To confirm the cell surface localization of unglycosylated gp130, cell surface proteins expressed in NECs treated with or without tunicamycin (2 μg/ml) for 8 h were biotinylated by treating the cells with sulfo-NHS-LC-biotin. After lysis, biotinylated cell surface proteins were pulled down with streptavidin-conjugated agarose and then analyzed by Western-blot using anti-gp130 antibody (A) or anti-LIFR antibody (B).

Fig. 3

Heterodimerization of unglycosylated gp130 and LIFR in NECs. Lysates of NECs treated with tunicamycin (0 or 2μg/ml) for 8 h and then stimulated with LIF (0 or 40 ng/ml) for 10 min were immunoprecipitated with anti-LIFR antibody. gp130 co-immunoprecipitated with LIFR was analyzed by Western-blot analysis using anti-gp130 antibody.

Fig. 4

(A) Activation of the JAK-STAT pathway and the Ras-MAPK pathway in NECs treated with tunicamycin. Lysates prepared from NECs treated with tunicamycin (0 or 5 μg/ml) for 8 h and then stimulated with LIF (0 or 40 ng/ml) for 10 min were analyzed by Western-blot analysis. Stress-mediated cell death signaling was evaluated by measuring (B) LDH activities in culture media of tunicamycin-treated NECs and (C) caspase 3 activities in tunicamycin-treated NECs.