scholarly journals Role of N-Linked Glycosylation of the 5-HT2A Receptor in JC Virus Infection

2010 ◽  
Vol 84 (19) ◽  
pp. 9677-9684 ◽  
Author(s):  
Melissa S. Maginnis ◽  
Sheila A. Haley ◽  
Gretchen V. Gee ◽  
Walter J. Atwood
Keyword(s):  
Cell Surface ◽  
Demyelinating Disease ◽  
Jc Virus ◽  
Surface Expression ◽  
Host Cells ◽  
Human Polyomavirus ◽  
Cell Surface Expression ◽  
Tunicamycin Treatment ◽  
Infection Treatment ◽  
Glycosylation Sites

ABSTRACT JC virus (JCV) is a human polyomavirus and the causative agent of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV infection of host cells is dependent on interactions with cell surface asparagine (N)-linked sialic acids and the serotonin 5-hydroxytryptamine2A receptor (5-HT2AR). The 5-HT2AR contains five potential N-linked glycosylation sites on the extracellular N terminus. Glycosylation of other serotonin receptors is essential for expression, ligand binding, and receptor function. Also, glycosylation of cellular receptors has been reported to be important for JCV infection. Therefore, we hypothesized that the 5-HT2AR N-linked glycosylation sites are required for JCV infection. Treatment of 5-HT2AR-expressing cells with tunicamycin, an inhibitor of N-linked glycosylation, reduced JCV infection. Individual mutation of each of the five N-linked glycosylation sites did not affect the capacity of 5-HT2AR to support JCV infection and did not alter the cell surface expression of the receptor. However, mutation of all five N-linked glycosylation sites simultaneously reduced the capacity of 5-HT2AR to support infection and altered the cell surface expression. Similarly, tunicamycin treatment reduced the cell surface expression of 5-HT2AR. Mutation of all five N-linked glycosylation sites or tunicamycin treatment of cells expressing wild-type 5-HT2AR resulted in an altered electrophoretic mobility profile of the receptor. Treatment of cells with PNGase F, to remove N-linked oligosaccharides from the cell surface, did not affect JCV infection in 5-HT2AR-expressing cells. These data affirm the importance of 5-HT2AR as a JCV receptor and demonstrate that the sialic acid component of the receptor is not directly linked to 5-HT2AR.

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 The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

2006 ◽  
Vol 81 (2) ◽  
pp. 588-598 ◽  
Author(s):  
George Koutsoudakis ◽  
Eva Herrmann ◽  
Stephanie Kallis ◽  
Ralf Bartenschlager ◽  
Thomas Pietschmann
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Surface Expression ◽  
Hcv Infection ◽  
Host Cells ◽  
Productive Infection ◽  
Cell Surface Expression ◽  
Type I ◽  
Cell Clones

ABSTRACT Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 104 molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.

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.

scholarly journals N-glycosylation in the protease domain of trypsin-like serine proteases mediates calnexin-assisted protein folding

eLife ◽  
2018 ◽  
Vol 7 ◽  
Author(s):  
Hao Wang ◽  
Shuo Li ◽  
Juejin Wang ◽  
Shenghan Chen ◽  
Xue-Long Sun ◽  
...  
Keyword(s):  
Cell Surface ◽  
Serine Proteases ◽  
Surface Expression ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
General Mechanism ◽  
Common Mechanism ◽  
Physiological Processes ◽  
Glycosylation Sites ◽  
Underlying Mechanisms

Trypsin-like serine proteases are essential in physiological processes. Studies have shown that N-glycans are important for serine protease expression and secretion, but the underlying mechanisms are poorly understood. Here, we report a common mechanism of N-glycosylation in the protease domains of corin, enteropeptidase and prothrombin in calnexin-mediated glycoprotein folding and extracellular expression. This mechanism, which is independent of calreticulin and operates in a domain-autonomous manner, involves two steps: direct calnexin binding to target proteins and subsequent calnexin binding to monoglucosylated N-glycans. Elimination of N-glycosylation sites in the protease domains of corin, enteropeptidase and prothrombin inhibits corin and enteropeptidase cell surface expression and prothrombin secretion in transfected HEK293 cells. Similarly, knocking down calnexin expression in cultured cardiomyocytes and hepatocytes reduced corin cell surface expression and prothrombin secretion, respectively. Our results suggest that this may be a general mechanism in the trypsin-like serine proteases with N-glycosylation sites in their protease domains.

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 Importance of N-glycosylation positioning for cell-surface expression, targeting, affinity and quality control of the human AT1 receptor

2005 ◽  
Vol 390 (1) ◽  
pp. 367-376 ◽  
Author(s):  
Pascal M. Lanctot ◽  
Patrice C. Leclerc ◽  
Martin Clément ◽  
Mannix Auger-Messier ◽  
Emanuel Escher ◽  
...  
Keyword(s):  
Quality Control ◽  
Cell Surface ◽  
Functional Expression ◽  
Surface Expression ◽  
Glycosylation Site ◽  
At1 Receptor ◽  
Cell Surface Expression ◽  
Receptor Subtype ◽  
Angiotensin Ii Receptor ◽  
Glycosylation Sites

GPCRs (G-protein-coupled receptors) are preferentially N-glycosylated on ECL2 (extracellular loop 2). We previously showed that N-glycosylation of ECL2 was crucial for cell-surface expression of the hAT1 receptor (human angiotensin II receptor subtype 1). Here, we ask whether positioning of the N-glycosylation sites within the various ECLs of the receptor is a vital determinant in the functional expression of hAT1 receptor at the cell surface. Artificial N-glycosylation sequons (Asn-Xaa-Ser/Thr) were engineered into ECL1, ECL2 and ECL3. N-glycosylation of ECL1 caused a very significant decrease in affinity and cell surface expression of the resulting receptor. Shifting the position of the ECL2 glycosylation site by two residues led to the synthesis of a misfolded receptor which, nevertheless, was trafficked to the cell surface. The misfolded nature of this receptor is supported by an increased interaction with the chaperone HSP70 (heat-shock protein 70). Introduction of N-glycosylation motifs into ECL3 yielded mutant receptors with normal affinity, but low levels of cell surface expression caused by proteasomal degradation. This behaviour differed from that observed for the aglycosylated receptor, which accumulated in the endoplasmic reticulum. These results show how positioning of the N-glycosylation sites altered many properties of the AT1 receptor, such as targeting, folding, affinity, cell surface expression and quality control.

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.

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.

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 Functional Analysis of Glycoprotein L (gL) from Rhesus Lymphocryptovirus in Epstein-Barr Virus-Mediated Cell Fusion Indicates a Direct Role of gL in gB-Induced Membrane Fusion

2009 ◽  
Vol 83 (15) ◽  
pp. 7678-7689 ◽  
Author(s):  
Aileen E. Plate ◽  
Jasmina Smajlović ◽  
Theodore S. Jardetzky ◽  
Richard Longnecker
Keyword(s):  
Cell Surface ◽  
Cell Fusion ◽  
Epstein Barr Virus ◽  
Surface Expression ◽  
Host Cells ◽  
Cell Surface Expression ◽  
Direct Role ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Glycoprotein L (gL), which complexes with gH, is a conserved herpesvirus protein that is essential for Epstein-Barr virus (EBV) entry into host cells. The gH/gL complex has a conserved role in entry among herpesviruses, yet the mechanism is not clear. To gain a better understanding of the role of gL in EBV-mediated fusion, chimeric proteins were made using rhesus lymphocryptovirus (Rh-LCV) gL (Rh gL), which shares a high sequence homology with EBV gL but does not complement EBV gL in mediating fusion with B cells. A reduction in fusion activity was observed with chimeric gL proteins that contained the amino terminus of Rh gL, although they retained their ability to process and transport gH/gL to the cell surface. Amino acids not conserved within this region in EBV gL when compared to Rh gL were further analyzed, with the results mapping residues 54 and 94 as being functionally important for EBV-mediated fusion. All chimeras and mutants displayed levels of cell surface expression similar to that of wild-type gL and interacted with gH and gp42. Our data also suggest that the role of gL involves the activation or recruitment of gB with the gH/gL complex, as we found that reduced fusion of Rh gL, EBV/Rh-LCV chimeras, and gL point mutants could be restored by replacing EBV gB with Rh gB. These observations demonstrate a distinction between the role of gL in the processing and trafficking of gH to the cell surface and a posttrafficking role in cell-cell fusion.

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