N-linked glycosylation is essential for the stability but not the signaling function of the interleukin-6 signal transducer glycoprotein 130

Abstract

N-Linked glycosylation is an important determinant of protein structure and function. The interleukin-6 signal transducer glycoprotein 130 (gp130) is a common co-receptor for cytokines of the interleukin (IL)-6 family and is N-glycosylated at 9 of 11 potential sites. Whereas N-glycosylation of the extracellular domains D1-D3 of gp130 has been shown to be dispensable for binding of the gp130 ligand IL-6 and its cognate receptor in vitro, the role of the N-linked glycans on domains D4 and D6 is still unclear. We have mutated the asparagines of all nine functional N-glycosylation sites of gp130 to glutamine and systematically analyzed the consequences of deleted N-glycosylation (dNG) in both cellular gp130 and in a soluble gp130-IgG1-Fc fusion protein (sgp130Fc). Our results show that sgp130Fc-dNG is inherently unstable and degrades rapidly under conditions that do not harm wild-type sgp130Fc. Consistently, the bulk of cellular gp130-dNG is not transported to the plasma membrane but is degraded in the proteasome. However, the small quantities of gp130-dNG, which do reach the cell surface, are still able to activate the key gp130 signaling target signal transducer and activator of transcription-3 (STAT3) upon binding of the agonistic complex of IL-6 and soluble IL-6 receptor. In conclusion, N-linked glycosylation is required for the stability but not the signal-transducing function of gp130.

FIGURE 1.

Hexameric signaling complex of gp130, IL-6, and sIL-6R. In the tetrameric complex, only one molecule each of IL-6 and sIL-6R is present. 1–6, extracellular domains D1–D6 of gp130; transm., transmembrane.

FIGURE 2.

Schematic representation of the engineered sgp130Fc and gp130 variants. The dNG variant sgp130Fc-dNG was constructed to study the influence of N-glycans on the production, stability, and biological activity of sgp130Fc fusion proteins. Overexpressed cellular gp130 with or without N-glycans (gp130-dNG) was used to analyze the influence of N-glycans on the surface expression and signaling function of gp130. EYFP-tagged variants of gp130 and gp130-dNG were used for the same purposes and, in addition, for microscopic visualization. The molecular weight of the sgp130Fc(−dNG) proteins is 2 × 93 kDa (without signal peptides). The molecular weight, without signal peptide, of the cellular gp130(-dNG) and gp130-(dNG)-EYFP proteins is 101 and 129 kDa, respectively. The N-glycans on one complete extracellular part of gp130 (D1–D6) add about 25 kDa to the apparent molecular weight of the protein. N-Glycans are symbolized by rhombi. 1–6, extracellular domains D1–D6 of gp130; CBM, cytokine-binding module (D2 + D3); E, C-terminal EYFP tag; Fc, IgG1-Fc; IC, intracellular domain; TM, transmembrane domain.

FIGURE 3.

Enzymatic assessment of N- and O-glycosylation of sgp130Fc. Both sgp130Fc (upper panel) and the N- and O-glycosylated control protein fetuin (lower panel) were treated with either PNGase F alone (to completely remove N-glycans) or with PNGase in combination with a mixture of enzymes, which together remove most O-glycans: α-2(3,6,8,9)-neuraminidase, β-(1–4)-galactosidase, β-N-acetylglucosaminidase, and O-glycosidase. In contrast to fetuin, the apparent molecular weight of sgp130Fc was not changed by O-deglycosylating enzymes. The stronger high molecular weight bands in experimental 3rd lane represent glycosidase proteins. w/o, without.

FIGURE 4.

Production and characterization of sgp130Fc-dNG in CHO-K1 cells. A, reverse transcription-PCR of sgp130Fc(-dNG) mRNA normalized by simultaneous amplification of cDNA from the neomycin resistance gene (NeoR) of the expression vector in the same PCR. sgp130Fc wild-type (wt) and sgp130Fc-dNG were amplified by the same primer pair. To avoid amplification of endogenous gp130 transcripts, the forward primer binds to gp130, and the reverse primer binds to Fc. The results show that sgp130Fc and sgp130Fc-dNG have the same mRNA expression levels. c, control; mock, only expression vector pcDNA-DEST40 (expressing NeoR). B, Western blots with anti-human IgG-Fc to detect wild-type (wt) sgp130Fc or sgp130Fc-dNG in denatured whole cell extracts (left panels) or in direct immunoprecipitations (IP) of the Fc part of the secreted sgp130Fc(-dNG) proteins from cell supernatants by protein A/G-agarose beads (right panels). Results from two (of five) experiments (exp. 1 and 2) with duplicate samples (#1 and #2) are shown to demonstrate the range of sgp130Fc-dNG protein levels. Whereas sgp130Fc-dNG protein can be readily detected in whole cell lysates, albeit at lower levels than wild-type sgp130Fc, almost no sgp130Fc-dNG is present in the cell supernatant (only visible with massive overexposure), indicating retention and degradation in the cell. C, silver-stained SDS-PAGE of wild-type (wt) sgp130Fc, sgp130Fc-dNG (dNG), control-Fc (i.e. IgG1-Fc alone), or IgG1 heavy chain (hc) after incubation without (−) or with (+) PNGase F to remove N-glycans. Shifts in apparent molecular weight are indicated below the panels. Gel strengths were 7.5% for sgp130Fc(−dNG) and 12% for control-Fc and IgG1 heavy chain. D, native and SDS-PAGE analysis of wild-type (wt) sgp130Fc and sgp130Fc-dNG preparations after elution from protein A. Molecular weight (MW) marker refers only to SDS-PAGE. The stronger band labeled monomers of dNG has the same apparent molecular weight as the single band detected in Western blots of whole cell extracts separated by SDS-PAGE under the same running conditions (B).

FIGURE 5.

Localization and degradation of gp130-dNG. A and B, fluorescence micrographs of gp130(-dNG)-EYFP in HeLa cells. Whereas wild-type (wt) gp130-EYFP is localized on the cell surface (A), gp130-dNG-EYFP largely remains within the ER·Golgi complex (B). C, Western blots of plasma membrane proteins (PM) and of the complete transmembrane protein fraction (TM) from HeLa and CHO-K1 cells transiently transfected with gp130-EYFP or gp130-dNG-EYFP (detection by anti-EYFP). D, Western blots of denatured whole cell protein extracts from CHO-K1 cells transiently transfected with gp130-EYFP or gp130-dNG-EYFP (detection by anti-EYFP). Proteasome inhibition by MG132 significantly increased protein levels of gp130-dNG-EYFP (normalized by probing the stripped blot membrane with anti-β-actin). Lane 4 (dNG without MG132) does not show a signal due to short exposure time. c, control; mock, only expression vector pcDNA-DEST40. Controls for the experiments shown in C and D included Western blots with anti-gp130 (sc-655, directed against the intracellular domain of gp130) and confirmation of equal protein loading and transfer by Ponceau S staining (data not shown).

FIGURE 6.

Characterization of gp130-dNG expression and function in stably transduced BAF3 cell pools. A, flow cytometry analysis of total gp130-EYFP and gp130-dNG-EYFP expression (fluorescein isothiocyanate/GFP channel; black) versus the nonspecific signal of untransduced BAF3 cells (gray). B and C, Western blot detection of wild-type (wt) gp130(-EYFP) and gp130-dNG(-EYFP) in complete transmembrane protein (prot.) fractions produced by detergent extraction (B) or plasma membrane protein fractions produced by surface biotinylation (C). In the experiments represented by B and C, gp130 was detected by an anti-gp130 (sc-655) directed against the intracellular domain of gp130. D, colorimetric cell proliferation assay of the BAF3 cell pools after stimulation with 10 ng/ml Hyper-IL-6 in the absence of the essential growth factor IL-3. E, EYFP tag; GFP, pool expressing green fluorescent protein but no gp130 (negative control); **, p < 0.01 versus unstimulated control. E, Western blot analysis of STAT3 activation (P, phosphorylation at Tyr⁷⁰⁵) in denatured whole cell protein extracts produced 15 min after stimulation with 10 ng/ml Hyper-IL-6 (H-IL-6) in the absence of IL-3 and after 6 h of serum starvation. BAF3 cells expressing no gp130 served as a negative control. After stripping, the blot membrane was probed with anti-STAT3 for normalization. Arrows mark weak phospho-STAT3 band in BAF3/gp130-dNG-EYFP extracts. F, STAT3 phosphorylation of BAF3/gp130-dNG-EYFP cells was determined as in E, but after a 5-min stimulation.