Protein Glycosylation in Mammalian Cells

Glycosylation is a conserved posttranslational modification that is found in all eukaryotes, which helps generate proteins with multiple functions. Our knowledge of glycosylation mainly comes from the investigation of the yeastSaccharomyces cerevisiaeand mammalian cells. However, during the last decade, glycosylation in the human pathogenic moldAspergillus fumigatushas drawn significant attention. It has been revealed that glycosylation inA. fumigatusis crucial for its growth, cell wall synthesis, and development and that the process is more complicated than that found in the budding yeastS. cerevisiae. The present paper implies that the investigation of glycosylation inA. fumigatusis not only vital for elucidating the mechanism of fungal cell wall synthesis, which will benefit the design of new antifungal therapies, but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes. This paper describes the advances in functional analysis of protein glycosylation inA. fumigatus.

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A glycosylated peptide in the West Nile virus envelope protein is immunogenic during equine infection

Journal of General Virology ◽

10.1099/vir.0.2008/003731-0 ◽

2008 ◽

Vol 89 (12) ◽

pp. 3063-3072 ◽

Cited By ~ 20

Author(s):

Jody Hobson-Peters ◽

Philip Toye ◽

Melissa D. Sánchez ◽

Katharine N. Bossart ◽

Lin-Fa Wang ◽

...

Keyword(s):

West Nile Virus ◽

Fusion Protein ◽

Synthetic Peptides ◽

Mammalian Cells ◽

Protein Glycosylation ◽

The West ◽

West Nile ◽

High Sequence Identity ◽

Recombinant Peptide ◽

E Protein

Using a monoclonal antibody directed to domain I of the West Nile virus (WNV) envelope (E) protein, we identified a continuous (linear) epitope that was immunogenic during WNV infection of horses. Using synthetic peptides, this epitope was mapped to a 19 aa sequence (WN19: E147–165) encompassing the WNV NY99 E protein glycosylation site at position 154. The inability of WNV-positive horse and mouse sera to bind the synthetic peptides indicated that glycosylation was required for recognition of peptide WN19 by WNV-specific antibodies in sera. N-linked glycosylation of WN19 was achieved through expression of the peptide as a C-terminal fusion protein in mammalian cells and specific reactivity of WNV-positive horse sera to the glycosylated WN19 fusion protein was shown by Western blot. Additional sera collected from horses infected with Murray Valley encephalitis virus (MVEV), which is similarly glycosylated at position E154 and exhibits high sequence identity to WNV NY99 in this region, also recognized the recombinant peptide. Failure of most WNV- and MVEV-positive horse sera to recognize the epitope as a deglycosylated fusion protein confirmed that the N-linked glycan was important for antibody recognition of the peptide. Together, these results suggest that the induction of antibodies to the WN19 epitope during WNV infection of horses is generally associated with E protein glycosylation of the infecting viral strain.

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The Role of HCV E2 Protein Glycosylation in Functioning of Virus Envelope Proteins in Insect and Mammalian Cells

Acta Naturae ◽

10.32607/20758251-2015-7-1-87-97 ◽

2015 ◽

Vol 7 (1) ◽

pp. 87-97 ◽

Cited By ~ 5

Author(s):

O. V. Orlova ◽

V. L. Drutsa ◽

P. V. Spirin ◽

A. V. Ivanov ◽

V. S. Prasolov ◽

...

Keyword(s):

Mammalian Cells ◽

Expression System ◽

Point Mutations ◽

Envelope Proteins ◽

Protein Glycosylation ◽

Virus Envelope ◽

Mutant Proteins ◽

Virus Like Particles ◽

E2 Glycoprotein ◽

Glycosylation Sites

The hepatitis C virus (HCV) envelope proteins E1 and E2, being virion components, are involved in the formation of infectious particles in infected cells. The detailed structure of the infectious particle of HCV remains poorly understood. Moreover, the virion assembly and release of virions by the cell are the least understood processes. It is believed that virion properties depend on glycosylation of the virus envelope proteins in a cell, while glycansat several glycosylation sites of these proteins play a pivotal role in protein functioning and the HCV life cycle. N-glycans of glycoproteins can influence viral particle formation, virus binding to cell surface, and HCV pathogenesis. We studied the effect of glycans on the folding ofthe E2 glycoprotein, formation of functional glycoprotein complexes and virus particles in insect and mammalian cells. In order to investigate these processes, point mutations of the N-glycosylation sites of HCV protein E2 (genotype 1b strain 274933RU) were generated and the mutant proteins were further analyzed in the baculovirus expression system. Elimination of the single glycosylation sites of the E2 glycoprotein, except for the N6 site, did not affect its synthesis efficiency in Sf9 insect cells, while the electrophoretic mobility of mutant proteins increased in proportion to the decrease in the number of glycosylation sites. The level of synthesis of HCV glycoprotein E2 in human HEK293T cells depended on the presence of glycans at the N1 and N8 glycosylation sites in contrast to Sf9 cells. At the same time, elimination of glycans at the N1, N2, and N10 sites led to the accumulation of unproductive E1E2 dimers as aggregates and productive assembly suppression of virus-like particles both in insect and mammalian cells. In addition, elimination of single glycosylation sites of HCV E2 had no impact on the RNA synthesis of structural proteins and formation of virus-like particles in insect and mammalian cells.

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Role of Protein Glycosylation in Interactions of Medically Relevant Fungi with the Host

Journal of Fungi ◽

10.3390/jof7100875 ◽

2021 ◽

Vol 7 (10) ◽

pp. 875

Author(s):

Manuela Gómez-Gaviria ◽

Ana P. Vargas-Macías ◽

Laura C. García-Carnero ◽

Iván Martínez-Duncker ◽

Héctor M. Mora-Montes

Keyword(s):

Protein Trafficking ◽

Mammalian Cells ◽

Fungal Infections ◽

Fungal Species ◽

Protein Glycosylation ◽

Amino Acid Side Chain ◽

Post Translational Modification ◽

Fungal Cell ◽

The Relationship

Protein glycosylation is a highly conserved post-translational modification among organisms. It plays fundamental roles in many biological processes, ranging from protein trafficking and cell adhesion to host–pathogen interactions. According to the amino acid side chain atoms to which glycans are linked, protein glycosylation can be divided into two major categories: N-glycosylation and O-glycosylation. However, there are other types of modifications such as the addition of GPI to the C-terminal end of the protein. Besides the importance of glycoproteins in biological functions, they are a major component of the fungal cell wall and plasma membrane and contribute to pathogenicity, virulence, and recognition by the host immunity. Given that this structure is absent in host mammalian cells, it stands as an attractive target for developing selective compounds for the treatment of fungal infections. This review focuses on describing the relationship between protein glycosylation and the host–immune interaction in medically relevant fungal species.

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Cex1 is a component of the COPI intracellular trafficking machinery

Biology Open ◽

10.1242/bio.058528 ◽

2021 ◽

Vol 10 (3) ◽

Author(s):

Ludovic Enkler ◽

Bruno Rinaldi ◽

Johan Owen de Craene ◽

Philippe Hammann ◽

Osamu Nureki ◽

...

Keyword(s):

Neurodegenerative Disorders ◽

Intracellular Trafficking ◽

Complex I ◽

Mammalian Cells ◽

Deletion Mutant ◽

Protein Glycosylation ◽

Coated Vesicles ◽

Human Homologue ◽

Membrane Compartments ◽

Mutant Cells

ABSTRACT COPI (coatomer complex I) coated vesicles are involved in Golgi-to-ER and intra-Golgi trafficking pathways, and mediate retrieval of ER resident proteins. Functions and components of the COPI-mediated trafficking pathways, beyond the canonical set of Sec/Arf proteins, are constantly increasing in number and complexity. In mammalian cells, GORAB, SCYL1 and SCYL3 proteins regulate Golgi morphology and protein glycosylation in concert with the COPI machinery. Here, we show that Cex1, homologous to the mammalian SCYL proteins, is a component of the yeast COPI machinery, by interacting with Sec27, Sec28 and Sec33 (Ret1/Cop1) proteins of the COPI coat. Cex1 was initially reported to mediate channeling of aminoacylated tRNA outside of the nucleus. Our data show that Cex1 localizes at membrane compartments, on structures positive for the Sec33 α-COP subunit. Moreover, the Wbp1 protein required for N-glycosylation and interacting via its di-lysine motif with the Sec27 β′-COP subunit is mis-targeted in cex1Δ deletion mutant cells. Our data point to the possibility of developing Cex1 yeast-based models to study neurodegenerative disorders linked to pathogenic mutations of its human homologue SCYL1.

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Glycoproteomics-Based Identification of Cancer Biomarkers

International Journal of Proteomics ◽

10.1155/2011/601937 ◽

2011 ◽

Vol 2011 ◽

pp. 1-10 ◽

Cited By ~ 30

Author(s):

Evelyn H. Kim ◽

David E. Misek

Keyword(s):

Posttranslational Modifications ◽

Mammalian Cells ◽

Cancer Biomarkers ◽

Biological Pathways ◽

Protein Glycosylation ◽

Lectin Affinity Chromatography ◽

Early Detection Of Disease ◽

Structural Alterations ◽

Lectin Affinity ◽

Technological Developments

Protein glycosylation is one of the most common posttranslational modifications in mammalian cells. It is involved in many biological pathways and molecular functions and is well suited for proteomics-based disease investigations. Aberrant protein glycosylation may be associated with disease processes. Specific glycoforms of glycoproteins may serve as potential biomarkers for the early detection of disease or as biomarkers for the evaluation of therapeutic efficacy for treatment of cancer, diabetes, and other diseases. Recent technological developments, including lectin affinity chromatography and mass spectrometry, have provided researchers the ability to obtain detailed information concerning protein glycosylation. These in-depth investigations, including profiling and quantifying glycoprotein expression, as well as comprehensive glycan structural analyses may provide important information leading to the development of disease-related biomarkers. This paper describes methodologies for the detection of cancer-related glycoprotein and glycan structural alterations and briefly summarizes several current cancer-related findings.

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Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2974-2976.1987 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2974-2976

Author(s):

Y K Kim ◽

A S Lee

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Protein Glycosylation ◽

Temperature Sensitive ◽

Ionophore A23187 ◽

Grp78 Promoter ◽

Steady State Levels ◽

Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

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The Effect of Bioreactor pH and Temperature on Protein Glycosylation in Perfusion Cultures of Mammalian Cells

Cells and Culture ◽

10.1007/978-90-481-3419-9_136 ◽

2010 ◽

pp. 785-788 ◽

Cited By ~ 1

Author(s):

Eva Sajan ◽

Ricaredo Matanguihan ◽

Rüdiger Heidemann ◽

Susan Abu-Absi ◽

Wilfred Asuncion ◽

...

Keyword(s):

Mammalian Cells ◽

Protein Glycosylation ◽

Perfusion Cultures

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Ogt-Dependent X-Chromosome-Linked Protein Glycosylation Is a Requisite Modification in Somatic Cell Function and Embryo Viability

Molecular and Cellular Biology ◽

10.1128/mcb.24.4.1680-1690.2004 ◽

2004 ◽

Vol 24 (4) ◽

pp. 1680-1690 ◽

Cited By ~ 277

Author(s):

Niall O'Donnell ◽

Natasha E. Zachara ◽

Gerald W. Hart ◽

Jamey D. Marth

Keyword(s):

Somatic Cell ◽

Mammalian Cells ◽

Cell Function ◽

Embryonic Stem ◽

Cell Types ◽

Protein Glycosylation ◽

Embryo Viability ◽

Altered Expression ◽

T Cell Apoptosis

ABSTRACT The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling pathways.

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Transcriptional activation of the glucose-regulated protein genes and their heterologous fusion genes by beta-mercaptoethanol.

Molecular and Cellular Biology ◽

10.1128/mcb.7.8.2974 ◽

1987 ◽

Vol 7 (8) ◽

pp. 2974-2976 ◽

Cited By ~ 20

Author(s):

Y K Kim ◽

A S Lee

Keyword(s):

Transcriptional Activation ◽

Mammalian Cells ◽

Calcium Ionophore ◽

Calcium Ionophore A23187 ◽

Protein Glycosylation ◽

Temperature Sensitive ◽

Ionophore A23187 ◽

Grp78 Promoter ◽

Steady State Levels ◽

Tata Sequence

The sulfhydryl-reducing agent beta-mercaptoethanol preferentially stimulates the synthesis of glucose-regulated proteins (GRPs) in mammalian cells. The rapid and large increase in GRPs is due to transcriptional activation of GRP94 and GRP78 genes, resulting in a rapid increase in the steady-state levels of GRP transcripts. From analysis of 5'-deletion mutants, the region of beta-mercaptoethanol responsiveness in the GRP78 promoter was mapped within 450 nucleotides upstream of the TATA sequence. This same general region was demonstrated to be important for induction of the GRP78 gene by the calcium ionophore A23187, glucose starvation, and a temperature-sensitive mutation in a K12 cell line defective in protein glycosylation.

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