A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface

1985 ◽  
Vol 5 (11) ◽  
pp. 3074-3083
Author(s):  
C E Machamer ◽  
R Z Florkiewicz ◽  
J K Rose
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Glycosylation Sites ◽  
Vesicular Stomatitis ◽  
The Individual

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

scholarly journals A single N-linked oligosaccharide at either of the two normal sites is sufficient for transport of vesicular stomatitis virus G protein to the cell surface.

1985 ◽  
Vol 5 (11) ◽  
pp. 3074-3083 ◽  
Author(s):  
C E Machamer ◽  
R Z Florkiewicz ◽  
J K Rose
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Intracellular Transport ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Glycosylation Sites ◽  
Vesicular Stomatitis ◽  
The Individual

We investigated the role of glycosylation in intracellular transport and cell surface expression of the vesicular stomatitis virus glycoprotein (G) in cells expressing G protein from cloned cDNA. The individual contributions of the two asparagine-linked glycans of G protein to cell surface expression were assessed by site-directed mutagenesis of the coding sequence to eliminate one or the other or both of the glycosylation sites. One oligosaccharide at either position was sufficient for cell surface expression of G protein in transfected cells, and the rates of oligosaccharide processing were similar to the rate observed for wild-type protein. However, the nonglycosylated G protein synthesized when both glycosylation sites were eliminated did not reach the cell surface. This protein did appear to reach a Golgi-like region, as determined by indirect immunofluorescence microscopy, however, and was modified with palmitic acid. It was also apparently not subject to increased proteolytic breakdown.

Intracellular transport of the glycoprotein of VSV is inhibited by CCCP at a late stage of post-translational processing

1989 ◽  
Vol 92 (4) ◽  
pp. 633-642
Author(s):  
J.K. Burkhardt ◽  
Y. Argon
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Surface ◽  
G Protein ◽  
Vesicular Stomatitis Virus ◽  
Late Stage ◽  
Intracellular Transport ◽  
Post Translational Modifications ◽  
Glycoprotein G ◽  
Golgi Compartment ◽  
Vesicular Stomatitis

The appearance of newly synthesized glycoprotein (G) of vesicular stomatitis virus at the surface of infected BHK cells is inhibited reversibly by treatment with carbonylcyanide m-chlorophenylhydrazone (CCCP). Under the conditions used, CCCP treatment depleted the cellular ATP levels by 40–60%, consistent with inhibition of transport at energy-requiring stages. The G protein that accumulates in cells treated with CCCP is heterogeneous. Most of it is larger than the newly synthesized G protein, is acylated with palmitic acid, and is resistant to endoglycosidase H (Endo H). Most of the arrested G protein is also sensitive to digestion with neuraminidase, indicating that it has undergone at least partial sialylation. A minority of G protein accumulates under these conditions in a less-mature form, suggesting its inability to reach the mid-Golgi compartment. The oligosaccharides of this G protein are Endo-H-sensitive and seem to be partly trimmed. Whereas sialylated G protein was arrested intracellularly, fucose-labelled G protein was able to complete its transport to the cell surface, indicating that a late CCCP-sensitive step separates sialylation from fucosylation. These post-translational modifications indicate that G protein can be transported as far as the trans-Golgi in the presence of CCCP and is not merely arrested in the endoplasmic reticulum.

scholarly journals Two N-Linked Glycans Differentially Control Maturation, Trafficking and Proteolysis, but not Activity of the IL-11 Receptor

2018 ◽  
Vol 45 (5) ◽  
pp. 2071-2085 ◽  
Author(s):  
Maria Agthe ◽  
Yvonne Garbers ◽  
Joachim Grötzinger ◽  
Christoph Garbers
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Proteolytic Processing ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Target Cells ◽  
Post Translational Modification ◽  
Glycosylation Sites ◽  
D2 Domain

Background/Aims: The cytokine interleukin-11 (IL-11) has important pro- and anti-inflammatory functions. It activates its target cells through binding to the IL-11 receptor (IL-11R), and the IL-11/IL-11R complex recruits a homodimer of glycoprotein 130 (gp130). N-linked glycosylation, a post-translational modification where complex oligosaccharides are attached to the side chain of asparagine residues, is often important for stability, folding and biological function of cytokine receptors. Methods: We generated different IL-11R mutants via site-directed mutagenesis and analyzed them in different cell lines via Western blot, flow cytometry, confocal microscopy and proliferation assays. Results: In this study, we identified two functional N-glycosylation sites in the D2 domain of the IL-11R at N127 and N194. While mutation of N127Q only slightly affects cell surface expression of the IL-11R, mutation of N194Q broadly prevents IL-11R appearance at the plasma membrane. Accordingly, IL-11R mutants lacking N194 are retained within the ER, whereas the N127 mutant is transported through the Golgi complex to the cell surface, uncovering a differential role of the two N-glycan sequons for IL-11R maturation. Interestingly, IL-11R mutants devoid of one or both N-glycans are still biologically active. Furthermore, the IL-11RN127Q/N194Q mutant shows no inducible shedding by ADAM10, but is rather constitutively released into the supernatant. Conclusion: Our results show that the two N-glycosylation sites differentially influence stability and proteolytic processing of the IL-11R, but that N-linked glycosylation is not a prerequisite for IL-11 signaling.

scholarly journals Mutational analysis of the N-linked glycosylation sites of the human insulin receptor

2000 ◽  
Vol 347 (3) ◽  
pp. 771-779 ◽  
Author(s):  
Thomas C. ELLEMAN ◽  
Maurice J. FRENKEL ◽  
Peter A. HOYNE ◽  
Neil M. MCKERN ◽  
Leah COSGROVE ◽  
...  
Keyword(s):  
Cell Surface ◽  
Mutational Analysis ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Insulin Binding ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Expression ◽  
Triple Mutant ◽  
Glycosylation Sites

Site-directed mutagenesis has been used to remove 15 of the 18 potential N-linked glycosylation sites, in 16 combinations, from the human exon 11-minus receptor isoform. The three glycosylation sites not mutated were asparagine residues 25, 397 and 894, which are known to be important in receptor biosynthesis or function. The effects of these mutations on proreceptor processing into α and β subunits, cell-surface expression, insulin binding and receptor autophosphorylation were assessed in Chinese hamster ovary cells. The double mutants 16+78, 16+111, 16+215, 16+255, 337+418, the triple mutants 295+337+418, 295+418+514, 337+418+514 and 730+743+881 and the quadruple mutants 606+730+743+881 and 671+730+743+881 seemed normal by all criteria examined. The triple mutant 16+215+255 showed only low levels of correctly processed receptor on the cell surface, this processed receptor being autophosphorylated in response to insulin. The quadruple mutant 624+730+743+881 showed normal processing and ligand binding but exhibited a constitutively active tyrosine kinase as judged by autophosphorylation. Three higher-order mutants were constructed, two of which, 16+337+418+730+743+881 (∆6) and 16+295+337+418+730+743+881 (∆7a), seemed normal. The third construct, 16+337+418+514+730+743+881 (∆7b), was expressed at high levels on the cell surface, essentially as uncleaved proreceptor with only the small proportion of ∆7b that was correctly processed showing insulin-stimulated autophosphorylation. The mutations of ∆6 and ∆7a were incorporated into soluble ectodomains, which had affinities for insulin that were 4-fold that of wild-type ectodomain. The ∆6 ectodomain expressed in Lec8 cells was produced in quantity in a bioreactor for subsequent structural analysis.

scholarly journals Role of Asparagine-Linked Glycosylation in Cell Surface Expression and Function of the Human Adrenocorticotropin Receptor (Melanocortin 2 Receptor) in 293/FRT Cells

Endocrinology ◽  
2010 ◽  
Vol 151 (2) ◽  
pp. 660-670 ◽  
Author(s):  
Simon Roy ◽  
Benoît Perron ◽  
Nicole Gallo-Payet
Keyword(s):  
Cell Surface ◽  
Fold Increase ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Cell Surface Receptor ◽  
Cell Surface Expression ◽  
Camp Production ◽  
Glycosylation Sites ◽  
Surface Receptor

Asparagine-linked glycosylation (N-glycosylation) of G protein-coupled receptors may be necessary for functions ranging from agonist binding, folding, maturation, stability, and internalization. Human melanocortin 2 receptor (MC2R) possesses putative N-glycosylation sites in its N-terminal extracellular domain; however, to date, the role of MC2R N-glycosylation has yet to be investigated. The objective of the present study is to examine whether N-glycosylation is essential or not for cell surface expression and cAMP production in native and MC2R accessory protein (MRAPα, -β, or -dCT)-expressing cells using 293/FRT transfected with Myc-MC2R. Western blot analyses performed with or without endoglycosidase H, peptide:N-glycosidase F or tunicamycin treatments and site-directed mutagenesis revealed that MC2R was glycosylated in the N-terminal domain at its two putative N-glycosylation sites (Asn12-Asn13-Thr14 and Asn17-Asn18-Ser19). In the absence of human MRAP coexpression, N-glycosylation of at least one of the two sites was necessary for MC2R cell surface expression. However, when MRAP was present, cell surface expression of MC2R mutants was either rescued entirely with the N17-18Q (QQNN) and N12-13Q (NNQQ) mutants or partially with the unglycosylated N12-13, 17-18Q (QQQQ) mutant. Functional and expression analyses revealed a discrepancy between wild-type (WT) and QQQQ cell surface receptor levels and maximal cAMP production with a 4-fold increase in EC50 values. Taken together, these results indicate that the absence of MC2R N-glycosylation abrogates to a large extent MC2R cell surface expression in the absence of MRAPs, whereas when MC2R is N-glycosylated, it can be expressed at the plasma membrane without MRAP assistance.

Role ofN-glycosylation in cell surface expression and protection against proteolysis of the intestinal anion exchanger SLC26A3

2012 ◽  
Vol 302 (5) ◽  
pp. C781-C795 ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Yukari Yamashita
Keyword(s):  
Cell Surface ◽  
Surface Expression ◽  
Site Directed Mutagenesis ◽  
Tryptic Digestion ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Biochemical Modification ◽  
Glycosylation Sites ◽  
Increased Susceptibility

SLC26A3 is a Cl−/HCO3−exchanger that plays a major role in Cl−absorption from the intestine. Its mutation causes congenital chloride-losing diarrhea. It has been shown that SLC26A3 are glycosylated, with the attached carbohydrate being extracellular and perhaps modulating function. However, the role of glycosylation has yet to be clearly determined. We used the approaches of biochemical modification and site-directed mutagenesis to prevent glycosylation. Deglycosylation experiments with glycosidases indicated that the mature glycosylated form of SLC26A3 exists at the plasma membrane, and a putative large second extracellular loop contains all of the N-linked carbohydrates. Deglycosylation of SLC26A3 causes depression of transport activity compared with wild-type, although robust intracellular pH changes were still observed, suggesting that N-glycosylation is not absolutely necessary for transport activity. To localize glycosylation sites, we mutated the five consensus sites by replacing asparagine (N) with glutamine. Immnoblotting suggests that SLC26A3 is glycosylated at N153, N161, and N165. Deglycosylation of SLC26A3 causes a defect in cell surface processing with decreased cell surface expression. We also assessed whether SLC26A3 is protected from tryptic digestion. While the mature glycosylated SLC26A3 showed little breakdown after treatment with trypsin, deglycosylated SLC26A3 exhibited increased susceptibility to trypsin, suggesting that the oligosaccharides protect SLC26A3 from tryptic digestion. In conclusion, our data indicate that N-glycosylation of SLC26A3 is important for cell surface expression and for protection from proteolytic degradation that may contribute to the understanding of pathogenesis of congenital disorders of glycosylation.

scholarly journals Newly synthesized G protein of vesicular stomatitis virus is not transported to the cell surface during mitosis.

1983 ◽  
Vol 97 (5) ◽  
pp. 1623-1628 ◽  
Author(s):  
G Warren ◽  
C Featherstone ◽  
G Griffiths ◽  
B Burke
Keyword(s):  
Cell Surface ◽  
G Protein ◽  
Immunoelectron Microscopy ◽  
Vesicular Stomatitis Virus ◽  
Viral Protein ◽  
Intracellular Transport ◽  
Mitotic Cell ◽  
Interphase Cells ◽  
Vesicular Stomatitis ◽  
Simultaneous Inhibition

Indirect immunofluorescence, immunoelectron microscopy, and digestion by protease were used to study intracellular transport of the G protein of vesicular stomatitis virus in mitotic and interphase cells. Quantitation showed that the appearance of G protein on the surface of mitotic cells was inhibited at least 10-fold when compared with that on interphase cells, even though similar amounts of viral protein were being synthesized. This dramatic inhibition, taken together with the simultaneous inhibition of endocytosis (Berlin, R. D., and J. M. Oliver, 1980, J. Cell Biol. 85: 660-671), points to a general cessation of membrane traffic in the mitotic cell.

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