Role of Individual N-Linked Glycosylation Sites in the Function and Intracellular Transport of the Human α Folate Receptor

1998 ◽  
Vol 351 (2) ◽  
pp. 227-235 ◽  
Author(s):  
Susan J. Roberts ◽  
Maria Petropavlovskaja ◽  
Koong-Nah Chung ◽  
Clement B. Knight ◽  
Patrick C. Elwood
Keyword(s):  
Intracellular Transport ◽  
Folate Receptor ◽  
Glycosylation Sites

scholarly journals Role of glycosylation in transport and enzymic activity of neutral endopeptidase-24.11

1994 ◽  
Vol 302 (2) ◽  
pp. 451-454 ◽  
Author(s):  
M H Lafrance ◽  
C Vézina ◽  
Q Wang ◽  
G Boileau ◽  
P Crine ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Cell Surface ◽  
Intracellular Transport ◽  
Neutral Endopeptidase ◽  
Soluble Form ◽  
Cell Extracts ◽  
Glycosylation Sites ◽  
Sugar Addition ◽  
Membrane Bound

Neutral endopeptidase (NEP, EC 3.4.24.11) is a major ectoenzyme of the brush-border membrane. The ectodomain of NEP contains five putative N-glycosylation sites. In order to determine the role of the addition of sugar moieties on the activity and intracellular transport of NEP, we have used site-directed mutagenesis to remove all or some of the five potential sites of sugar addition in membrane-bound and secreted forms of the enzyme. Expression of NEP glycosylation mutants in COS-1 cells showed that all five sites are used for sugar addition. Immunoblotting of NEP in COS-1 cell extracts or culture media indicated that total expression of normal membrane-bound NEP was not affected by mutations at glycosylation sites, whereas this expression level appeared to be strictly dependent on the number of glycosylation sites retained on the soluble form. The transport to the cell surface was also reduced by decreased glycosylation, but again the phenomenon appeared more drastic in the case of the soluble form than for the membrane-bound enzyme. Enzyme activity was decreased by deglycosylation. However, the presence of either of two crucial sites (sites 1 and 5; numbered from the N-terminus of the protein) was sufficient to recover close-to-normal enzymic activities. Transport to the cell surface and enzyme activity of NEP are thus both dependent on sugar residues, probably through different conformational constraints. These constraints seem to be local for enzyme activity but more global for transport to the cell surface.

scholarly journals Analysis of N-Linked Glycosylation of Hantaan Virus Glycoproteins and the Role of Oligosaccharide Side Chains in Protein Folding and Intracellular Trafficking

2004 ◽  
Vol 78 (10) ◽  
pp. 5414-5422 ◽  
Author(s):  
Xiaohong Shi ◽  
Richard M. Elliott
Keyword(s):  
Protein Folding ◽  
Intracellular Trafficking ◽  
Intracellular Transport ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Hantaan Virus ◽  
Membrane Glycoproteins ◽  
Glycoprotein Gene ◽  
Glycosylation Sites

ABSTRACT The membrane glycoproteins Gn and Gc of Hantaan virus (HTNV) (family Bunyaviridae) are modified by N-linked glycosylation. The glycoproteins contain six potential sites for the attachment of N-linked oligosaccharides, five sites on Gn and one on Gc. The properties of the N-linked oligosaccharide chains were analyzed by treatment with endoglycosidase H, peptide:N-glycosidase F, tunicamycin, and deoxynojirimycin and were confirmed to be completely of the high-mannose type. Ten glycoprotein gene mutants were constructed by site-directed mutagenesis, including six single N glycosylation site mutants and four double-site mutants. We determined that four sites (N134, -235, -347, and -399) on Gn and the only site (N928) on Gc in their ectodomains are utilized, whereas the fifth site on Gn (N609), which faces the cytoplasm, is not glycosylated. The importance of individual N-oligosaccharide chains varied with respect to folding and intracellular transport. The oligosaccharide chain on residue N134 was found to be crucial for protein folding, whereas single mutations at the other glycosylation sites were better tolerated. Mutation at glycosylation sites N235 and N399 together resulted in Gn misfolding. The endoplasmic reticulum chaperones calnexin and calreticulin were found to be involved in HTNV glycoprotein folding. Our data demonstrate that N-linked glycosylation of HTNV glycoproteins plays important and differential roles in protein folding and intracellular trafficking.

scholarly journals CRISPR/Cas9 Mediated Knock Down of δ-ENaC Blunted the TNF-Induced Activation of ENaC in A549 Cells

2021 ◽  
Vol 22 (4) ◽  
pp. 1858
Author(s):  
Waheed Shabbir ◽  
Nermina Topcagic ◽  
Mohammed Aufy ◽  
Murat Oz
Keyword(s):  
A549 Cells ◽  
Na Channel ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Whole Cell ◽  
Knock Down ◽  
Whole Cell Patch Clamp ◽  
Glycosylation Sites ◽  
Epithelial Na Channel

Tumor necrosis factor (TNF) is known to activate the epithelial Na+ channel (ENaC) in A549 cells. A549 cells are widely used model for ENaC research. The role of δ-ENaC subunit in TNF-induced activation has not been studied. In this study we hypothesized that δ-ENaC plays a major role in TNF-induced activation of ENaC channel in A549 cells which are widely used model for ENaC research. We used CRISPR/Cas 9 approach to knock down (KD) the δ-ENaC in A549 cells. Western blot and immunofluorescence assays were performed to analyze efficacy of δ-ENaC protein KD. Whole-cell patch clamp technique was used to analyze the TNF-induced activation of ENaC. Overexpression of wild type δ-ENaC in the δ-ENaC KD of A549 cells restored the TNF-induced activation of whole-cell Na+ current. Neither N-linked glycosylation sites nor carboxyl terminus domain of δ-ENaC was necessary for the TNF-induced activation of whole-cell Na+ current in δ-ENaC KD of A549 cells. Our data demonstrated that in A549 cells the δ-ENaC plays a major role in TNF-induced activation of ENaC.

scholarly journals Role of N-linked oligosaccharides in processing and intracellular transport of E2 glycoprotein of rubella virus.

1992 ◽  
Vol 66 (6) ◽  
pp. 3514-3521 ◽  
Author(s):  
Z Qiu ◽  
T C Hobman ◽  
H L McDonald ◽  
N O Seto ◽  
S Gillam
Keyword(s):  
Rubella Virus ◽  
Intracellular Transport ◽  
E2 Glycoprotein

scholarly journals Functional Role of N-Linked Glycosylation in Pseudorabies Virus Glycoprotein gH

2018 ◽  
Vol 92 (9) ◽  
pp. e00084-18 ◽  
Author(s):  
Melina Vallbracht ◽  
Sascha Rehwaldt ◽  
Barbara G. Klupp ◽  
Thomas C. Mettenleiter ◽  
Walter Fuchs
Keyword(s):  
Pseudorabies Virus ◽  
Viral Envelope ◽  
Fusion Activity ◽  
Virus Particles ◽  
Glycosylation Sites ◽  
Functional Relevance ◽  
And Function ◽  
Cell Spread

ABSTRACTMany viral envelope proteins are modified by asparagine (N)-linked glycosylation, which can influence their structure, physicochemical properties, intracellular transport, and function. Here, we systematically analyzed the functional relevance of N-linked glycans in the alphaherpesvirus pseudorabies virus (PrV) glycoprotein H (gH), which is an essential component of the conserved core herpesvirus fusion machinery. Upon gD-mediated receptor binding, the heterodimeric complex of gH and gL activates gB to mediate fusion of the viral envelope with the host cell membrane for viral entry. gH contains five potential N-linked glycosylation sites at positions 77, 162, 542, 604, and 627, which were inactivated by conservative mutations (asparagine to glutamine) singly or in combination. The mutated proteins were tested for correct expression and fusion activity. Additionally, the mutated gH genes were inserted into the PrV genome for analysis of function during virus infection. Our results demonstrate that all five sites are glycosylated. Inactivation of the PrV-specific N77 or the conserved N627 resulted in significantly reducedin vitrofusion activity, delayed penetration kinetics, and smaller virus plaques. Moreover, substitution of N627 greatly affected transport of gH in transfected cells, resulting in endoplasmic reticulum (ER) retention and reduced surface expression. In contrast, mutation of N604, which is conserved in theVaricellovirusgenus, resulted in enhancedin vitrofusion activity and viral cell-to-cell spread. These results demonstrate a role of the N-glycans in proper localization and function of PrV gH. However, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles.IMPORTANCEHerpesvirus infection requires fusion of the viral envelope with cellular membranes, which involves the conserved fusion machinery consisting of gB and the heterodimeric gH/gL complex. The bona fide fusion protein gB depends on the presence of the gH/gL complex for activation. Viral envelope glycoproteins, such as gH, usually contain N-glycans, which can have a strong impact on their folding, transport, and functions. Here, we systematically analyzed the functional relevance of all five predicted N-linked glycosylation sites in the alphaherpesvirus pseudorabies virus (PrV) gH. Despite the fact that mutation of specific sites affected gH transport,in vitrofusion activity, and cell-to-cell spread and resulted in delayed penetration kinetics, even simultaneous inactivation of all five N-glycosylation sites of gH did not severely inhibit formation of infectious virus particles. Thus, our results demonstrate a modulatory but nonessential role of N-glycans for gH function.

Quantitative proteomics identifies FOLR1 to drive sorafenib resistance via activating autophagy in hepatocellular carcinoma cells

2021 ◽  
Author(s):  
Hongwei Chu ◽  
Changqing Wu ◽  
Qun Zhao ◽  
Rui Sun ◽  
Kuo Yang ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Folate Receptor ◽  
Mass Spectrometry Analysis ◽  
Label Free ◽  
Spectrometry Analysis ◽  
Underlying Mechanisms ◽  
Related Proteins ◽  
Hcc Cells

Abstract Sorafenib is commonly used to treat advanced human hepatocellular carcinoma (HCC). However, clinical efficacy has been limited by drug resistance. In this study, we used label-free quantitative proteomic analysis to systematically investigate the underlying mechanisms of sorafenib resistance in HCC cells. A total of 1709 proteins were confidently quantified. Among them, 89 were differentially expressed, and highly enriched in the processes of cell-cell adhesion, negative regulation of apoptosis, response to drug and metabolic processes involving in sorafenib resistance. Notably, folate receptor α (FOLR1) was found to be significantly upregulated in resistant HCC cells. In addition, in-vitro studies showed that overexpression of FOLR1 decreased the sensitivity of HCC cells to sorafenib, whereas siRNA-directed knockdown of FOLR1 increased the sensitivity of HCC cells to sorafenib. Immunoprecipitation-mass spectrometry analysis suggested a strong link between FOLR1 and autophagy related proteins. Further biological experiments found that FOLR1-related sorafenib resistance was accompanied by the activation of autophagy, whereas inhibition of autophagy significantly reduced FOLR1-induced cell resistance. These results suggest the driving role of FOLR1 in HCC resistance to sorafenib, which may be exerted through FOLR1-induced autophagy. Therefore, this study may provide new insights into understanding the mechanism of sorafenib resistance.

ADP-ribosylation and intracellular traffic: an emerging role for PARP enzymes

2019 ◽  
Vol 47 (1) ◽  
pp. 357-370 ◽  
Author(s):  
Giovanna Grimaldi ◽  
Daniela Corda
Keyword(s):  
Intracellular Transport ◽  
Post Translational Modification ◽  
Cellular Functions ◽  
Intracellular Traffic ◽  
Adp Ribosylation ◽  
Cell Responses ◽  
Physiological Processes ◽  
Dependent Cell ◽  
Ribose Moiety

AbstractADP-ribosylation is an ancient and reversible post-translational modification (PTM) of proteins, in which the ADP-ribose moiety is transferred from NAD+ to target proteins by members of poly-ADP-ribosyl polymerase (PARP) family. The 17 members of this family have been involved in a variety of cellular functions, where their regulatory roles are exerted through the modification of specific substrates, whose identification is crucial to fully define the contribution of this PTM. Evidence of the role of the PARPs is now available both in the context of physiological processes and of cell responses to stress or starvation. An emerging role of the PARPs is their control of intracellular transport, as it is the case for tankyrases/PARP5 and PARP12. Here, we discuss the evidence pointing at this novel aspect of PARPs-dependent cell regulation.

scholarly journals A Series of Tubes: The C. elegans Excretory Canal Cell as a Model for Tubule Development

2020 ◽  
Vol 8 (3) ◽  
pp. 17 ◽  
Author(s):  
Matthew Buechner ◽  
Zhe Yang ◽  
Hikmat Al-Hashimi
Keyword(s):  
Intracellular Transport ◽  
Renal Tubules ◽  
Genetic Screens ◽  
C Elegans ◽  
Brain Vasculature ◽  
Canal Cell ◽  
Mammalian Tissues ◽  
Cytoskeletal Elements ◽  
Excretory Canal

Formation and regulation of properly sized epithelial tubes is essential for multicellular life. The excretory canal cell of C. elegans provides a powerful model for investigating the integration of the cytoskeleton, intracellular transport, and organismal physiology to regulate the developmental processes of tube extension, lumen formation, and lumen diameter regulation in a narrow single cell. Multiple studies have provided new understanding of actin and intermediate filament cytoskeletal elements, vesicle transport, and the role of vacuolar ATPase in determining tube size. Most of the genes discovered have clear homologues in humans, with implications for understanding these processes in mammalian tissues such as Schwann cells, renal tubules, and brain vasculature. The results of several new genetic screens are described that provide a host of new targets for future studies in this informative structure.

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