scholarly journals A Linkage-specific Sialic Acid Labeling Strategy Reveals Different Site-specific Glycosylation Patterns in SARS-CoV-2 Spike Protein Produced in CHO and HEK Cell Substrates

2021 ◽  
Vol 9 ◽  
Author(s):  
Qiong Wang ◽  
Yan Wang ◽  
Shuang Yang ◽  
Changyi Lin ◽  
Lateef Aliyu ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Viral Envelope ◽  
Site Specific ◽  
S Protein ◽  
Hek Cells ◽  
High Mannose ◽  
The Impact ◽  
S Proteins ◽  
Spike Proteins

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus utilizes the extensively glycosylated spike (S) protein protruding from the viral envelope to bind to angiotensin-converting enzyme-related carboxypeptidase (ACE2) as its primary receptor to mediate host-cell entry. Currently, the main recombinant S protein production hosts are Chinese hamster ovary (CHO) and human embryonic kidney (HEK) cells. In this study, a recombinant S protein truncated at the transmembrane domain and engineered to express a C-terminal trimerization motif was transiently produced in CHO and HEK cell suspensions. To further evaluate the sialic acid linkages presenting on S protein, a two-step amidation process, employing dimethylamine and ammonium hydroxide reactions in a solid support system, was developed to differentially modify the sialic acid linkages on the glycans and glycopeptides from the S protein. The process also adds a charge to Asp and Glu which aids in ionization. We used MALDI-TOF and LC-MS/MS with electron-transfer/higher-energy collision dissociation (EThcD) fragmentation to determine global and site-specific N-linked glycosylation patterns. We identified 21 and 19 out of the 22 predicted N-glycosites of the SARS-CoV-2 S proteins produced in CHO and HEK, respectively. It was found that the N-glycosite at 1,158 position (N1158) and at 122, 282 and 1,158 positions (N122, N282 and N1158) were absent on S from CHO and HEK cells, respectively. The structural mapping of glycans of recombinant human S proteins reveals that CHO-Spike exhibits more complex and higher sialylation (α2,3-linked) content while HEK-Spike exhibits more high-mannose and a small amount of α2,3- and α2,6-linked sialic acids. The N74 site represents the most abundant glycosite on both spike proteins. The relatively higher amount of high-mannose abundant sites (N17, N234, N343, N616, N709, N717, N801, and N1134) on HEK-Spike suggests that glycan-shielding may differ among the two constructs. HEK-Spike can also provide different host immune system interaction profiles based on known immune system active lectins. Collectively, these data underscore the importance of characterizing the site-specific glycosylation of recombinant human spike proteins from HEK and CHO cells in order to better understand the impact of the production host on this complex and important protein used in research, diagnostics and vaccines.

scholarly journals An ultrasensitive method for analysis of viral spike N-glycoforms

2021 ◽  
Author(s):  
Sabyasachi Baboo ◽  
Jolene K. Diedrich ◽  
Salvador Martínez-Bartolomé ◽  
Xiaoning Wang ◽  
Torben Schiffner ◽  
...  
Keyword(s):  
Immune System ◽  
Glycosylation Site ◽  
Host Immune System ◽  
Site Specific ◽  
High Mannose ◽  
Complex Forms ◽  
Spike Proteins

Viruses can evade the host immune system by displaying numerous glycans on their surface "spike-proteins" that cover immune epitopes. We have developed an ultrasensitive "single pot" method to assess glycan occupancy and the extent of glycan processing from high-mannose to complex forms at each N-glycosylation site. Though aimed at characterizing glycosylation of viral spike proteins as potential vaccines, this method is applicable for analysis of site-specific glycosylation of any glycoprotein.

scholarly journals Site-specific N-glycosylation analysis of animal cell culture-derived Zika virus proteins

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexander Pralow ◽  
Alexander Nikolay ◽  
Arnaud Leon ◽  
Yvonne Genzel ◽  
Erdmann Rapp ◽  
...  
Keyword(s):  
Zika Virus ◽  
Animal Cell ◽  
Gradient Centrifugation ◽  
Viral Envelope ◽  
Protein Glycosylation ◽  
High Yield ◽  
Structural Protein ◽  
Site Specific ◽  
E Protein ◽  
The Impact

AbstractHere, we present for the first time, a site-specific N-glycosylation analysis of proteins from a Brazilian Zika virus (ZIKV) strain. The virus was propagated with high yield in an embryo-derived stem cell line (EB66, Valneva SE), and concentrated by g-force step-gradient centrifugation. Subsequently, the sample was proteolytically digested with different enzymes, measured via a LC–MS/MS-based workflow, and analyzed in a semi-automated way using the in-house developed glyXtoolMS software. The viral non-structural protein 1 (NS1) was glycosylated exclusively with high-mannose structures on both potential N-glycosylation sites. In case of the viral envelope (E) protein, no specific N-glycans could be identified with this method. Nevertheless, N-glycosylation could be proved by enzymatic de-N-glycosylation with PNGase F, resulting in a strong MS-signal of the former glycopeptide with deamidated asparagine at the potential N-glycosylation site N444. This confirmed that this site of the ZIKV E protein is highly N-glycosylated but with very high micro-heterogeneity. Our study clearly demonstrates the progress made towards site-specific N-glycosylation analysis of viral proteins, i.e. for Brazilian ZIKV. It allows to better characterize viral isolates, and to monitor glycosylation of major antigens. The method established can be applied for detailed studies regarding the impact of protein glycosylation on antigenicity and human pathogenicity of many viruses including influenza virus, HIV and corona virus.

scholarly journals Neutrophil azurophilic granule glycoproteins are distinctively decorated by atypical pauci- and phosphomannose glycans

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Karli R. Reiding ◽  
Yu-Hsien Lin ◽  
Floris P. J. van Alphen ◽  
Alexander B. Meijer ◽  
Albert J. R. Heck
Keyword(s):  
Immune System ◽  
Autoimmune Disease ◽  
Secreted Proteins ◽  
Receptor Interaction ◽  
Post Translational Modification ◽  
Site Specific ◽  
Healthy Donors ◽  
Asymmetric Hybrid ◽  
Membrane Bound ◽  
High Mannose

AbstractWhile neutrophils are critical first-responders of the immune system, they also cause tissue damage and act in a variety of autoimmune diseases. Many neutrophil proteins are N-glycosylated, a post-translational modification that may affect, among others, enzymatic activity, receptor interaction, and protein backbone accessibility. So far, a handful neutrophil proteins were reported to be decorated with atypical small glycans (paucimannose and smaller) and phosphomannosylated glycans. To elucidate the occurrence of these atypical glycoforms across the neutrophil proteome, we performed LC-MS/MS-based (glyco)proteomics of pooled neutrophils from healthy donors, obtaining site-specific N-glycan characterisation of >200 glycoproteins. We found that glycoproteins that are typically membrane-bound to be mostly decorated with high-mannose/complex N-glycans, while secreted proteins mainly harboured complex N-glycans. In contrast, proteins inferred to originate from azurophilic granules carried distinct and abundant paucimannosylation, asymmetric/hybrid glycans, and glycan phosphomannosylation. As these same proteins are often autoantigenic, uncovering their atypical glycosylation characteristics is an important step towards understanding autoimmune disease and improving treatment.

scholarly journals What Binds Cationic Photosensitizers Better: Brownian Dynamics Reveals Key Interaction Sites on Spike Proteins of SARS-CoV, MERS-CoV, and SARS-CoV-2

Viruses ◽  
2021 ◽  
Vol 13 (8) ◽  
pp. 1615
Author(s):  
Vladimir Fedorov ◽  
Ekaterina Kholina ◽  
Sergei Khruschev ◽  
Ilya Kovalenko ◽  
Andrew Rubin ◽  
...  
Keyword(s):  
Binding Site ◽  
Brownian Dynamics ◽  
Zinc Phthalocyanine ◽  
Heptad Repeat ◽  
Receptor Binding Domain ◽  
Large Area ◽  
S Protein ◽  
Interaction Sites ◽  
S Proteins ◽  
Spike Proteins

We compared the electrostatic properties of the spike proteins (S-proteins) of three coronaviruses, SARS-CoV, MERS-CoV, and SARS-CoV-2, and their interactions with photosensitizers (PSs), octacationic octakis(cholinyl)zinc phthalocyanine (Zn-PcChol8+) and monocationic methylene blue (MB). We found a major common PS binding site at the connection of the S-protein stalk and head. The molecules of Zn-PcChol8+ and MB also form electrostatic encounter complexes with large area of negative electrostatic potential at the head of the S-protein of SARS-CoV-2, between fusion protein and heptad repeat 1 domain. The top of the SARS-CoV spike head demonstrates a notable area of electrostatic contacts with Zn-PcChol8+ and MB that corresponds to the N-terminal domain. The S-protein protomers of SARS-CoV-2 in “open” and “closed” conformations demonstrate different ability to attract PS molecules. In contrast with Zn-PcChol8+, MB possesses the ability to penetrate inside the pocket formed as a result of SARS-CoV-2 receptor binding domain transition into the “open” state. The existence of binding site for cationic PSs common to the S-proteins of SARS-CoV, SARS-CoV-2, and MERS-CoV creates prospects for the wide use of this type of PSs to combat the spread of coronaviruses.

scholarly journals Modeling Coronavirus Spike Protein Dynamics: Implications for Immunogenicity and Immune Escape

2021 ◽  
Author(s):  
Genevieve Kunkel ◽  
Mohammad Madani ◽  
S. J. White ◽  
Paulo Verardi ◽  
Anna Tarakanova
Keyword(s):  
Protein Dynamics ◽  
Antibody Binding ◽  
Phase Iii ◽  
Coarse Grained ◽  
Mode Analysis ◽  
S Protein ◽  
Viral Attachment ◽  
Binding Behavior ◽  
The Impact ◽  
S Proteins

The ongoing COVID-19 pandemic is a global public health emergency requiring urgent development of efficacious vaccines. While concentrated research efforts are underway to develop antibody-based vaccines that would neutralize SARS-CoV-2, and several first-generation vaccine candidates are currently in Phase III clinical trials or have received emergency use authorization, it is forecasted that COVID-19 will become an endemic disease requiring second-generation vaccines. The SARS-CoV-2 surface Spike (S) glycoprotein represents a prime target for vaccine development because antibodies that block viral attachment and entry, i.e. neutralizing antibodies, bind almost exclusively to the receptor binding domain (RBD). Here, we develop computational models for a large subset of S proteins associated with SARS-CoV-2, implemented through coarse-grained elastic network models and normal mode analysis. We then analyze local protein domain dynamics of the S protein systems and their thermal stability to characterize structural and dynamical variability among them. These results are compared against existing experimental data, and used to elucidate the impact and mechanisms of SARS-CoV-2 S protein mutations and their associated antibody binding behavior. We construct a SARS-CoV-2 antigenic map and offer predictions about the neutralization capabilities of antibody and S mutant combinations based on protein dynamic signatures. We then compare SARS-CoV-2 S protein dynamics to SARS-CoV and MERS-CoV S proteins to investigate differing antibody binding and cellular fusion mechanisms that may explain the high transmissibility of SARS-CoV-2. The outbreaks associated with SARS-CoV, MERS-CoV, and SARS-CoV-2 over the last two decades suggest that the threat presented by coronaviruses is ever-changing and long-term. Our results provide insights into the dynamics-driven mechanisms of immunogenicity associated with coronavirus S proteins, and present a new approach to characterize and screen potential mutant candidates for immunogen design, as well as to characterize emerging natural variants that may escape vaccine-induced antibody responses.

scholarly journals Mucin-type O-glycosylation Landscapes of SARS-CoV-2 Spike Proteins

2020 ◽  
Author(s):  
Yong Zhang ◽  
Wanjun Zhao ◽  
Yonghong Mao ◽  
Yaohui Chen ◽  
Jingqiang Zhu ◽  
...  
Keyword(s):  
High Resolution Mass Spectrometry ◽  
Targeted Drugs ◽  
Cell Type ◽  
Glycosylation Pattern ◽  
Site Specific ◽  
S Protein ◽  
Viral Attachment ◽  
Viral Binding ◽  
Pngase F ◽  
S Proteins

ABSTRACTThe densely glycosylated spike (S) proteins that are highly exposed on the surface of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) facilitate viral attachment, entry, and membrane fusion. We have previously reported all the 22 N-glycosites and site-specific N-glycans in the S protein protomer. Herein, we report the comprehensive and precise site-specific O-glycosylation landscapes of SARS-CoV-2 S proteins, which were characterized using high-resolution mass spectrometry. Following digestion using trypsin and trypsin/Glu-C, and de-N-glycosylation using PNGase F, we determined the mucin-type (GalNAc-type) O-glycosylation pattern of S proteins, including unambiguous O-glycosites and the 6 most common O-glycans occupying them, via Byonic identification and manual validation. Finally, 43 O-glycosites were identified in the insect cell-expressed S protein. Most glycosites were modified by non-sialylated O-glycans such as HexNAc(1) and HexNAc(1)Hex(1). In contrast, 30 O-glycosites were identified in the human cell-expressed S protein S1 subunit. Most glycosites were modified by sialylated O-glycans such as HexNAc(1)Hex(1)NeuAc(1) and HexNAc(1)Hex(1)NeuAc(2). Our results are the first to reveal that the SARS-CoV-2 S protein is a mucin-type glycoprotein; clustered O-glycans often occur in the N- and the C-termini of the S protein, and the O-glycosite and O-glycan compositions vary with the host cell type. These site-specific O-glycosylation landscapes of the SARS-CoV-2 S protein are expected to provide novel insights into the viral binding mechanism and present a strategy for the development of vaccines and targeted drugs.

scholarly journals Combinatorial approach with mass spectrometry and lectin microarray dissected glycoproteomic features of virion-derived spike protein of SARS-CoV-2

2021 ◽  
Author(s):  
Takahiro Hiono ◽  
Azusa Tomioka ◽  
Hiroyuki Kaji ◽  
Michihito Sasaki ◽  
Yasuko Orba ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Viral Envelope ◽  
The Novel ◽  
Combinatorial Approach ◽  
Viral Protein Synthesis ◽  
Site Specific ◽  
S Protein ◽  
Lectin Microarray ◽  
Global Impact ◽  
Novel Coronavirus

The COVID-19 pandemic caused by the novel coronavirus, SARS-CoV-2, has a global impact on public health. Since glycosylation of the viral envelope glycoproteins is known to be deeply associated with their immunogenicity, intensive studies on the glycans of its major glycoprotein, S protein, have been conducted. Nevertheless, the detailed site-specific glycan compositions of virion-associated S protein have not yet been clarified. Here, we conducted intensive glycoproteomic analyses of SARS-CoV-2 S protein using a combinatorial approach with two different technologies: mass spectrometry (MS) and lectin microarray. Using our unique MS1-based glycoproteomic technique, Glyco-RIDGE, in addition to MS2-based Byonic search, we identified 1,759 site-specific glycan compositions. The most frequent was HexNAc:Hex:Fuc:NeuAc:NeuGc = 6:6:1:0:0, suggesting a tri-antennary N-glycan terminating with LacNAc and having bisecting GlcNAc and a core fucose, which was found in 20 of 22 glycosylated sites. The subsequent lectin microarray analysis emphasized intensive outer arm fucosylation of glycans, which efficiently complemented the glycoproteomic features. The present results illustrate the high-resolution glycoproteomic features of SARS-CoV-2 S protein and significantly contribute to vaccine design, as well as the understanding of viral protein synthesis.

scholarly journals Hyposialylation Must Be Considered to Develop Future Therapies in Autoimmune Diseases

2021 ◽  
Vol 22 (7) ◽  
pp. 3402
Author(s):  
Anne Bordron ◽  
Marie Morel ◽  
Cristina Bagacean ◽  
Maryvonne Dueymes ◽  
Pierre Pochard ◽  
...  
Keyword(s):  
Immune System ◽  
Sialic Acid ◽  
Autoimmune Disease ◽  
Disease Development ◽  
Detailed Characterization ◽  
Multiple Factors ◽  
Innovative Therapies ◽  
Future Treatments ◽  
The Impact

Autoimmune disease development depends on multiple factors, including genetic and environmental. Abnormalities such as sialylation levels and/or quality have been recently highlighted. The adjunction of sialic acid at the terminal end of glycoproteins and glycolipids is essential for distinguishing between self and non-self-antigens and the control of pro- or anti-inflammatory immune reactions. In autoimmunity, hyposialylation is responsible for chronic inflammation, the anarchic activation of the immune system and organ lesions. A detailed characterization of this mechanism is a key element for improving the understanding of these diseases and the development of innovative therapies. This review focuses on the impact of sialylation in autoimmunity in order to determine future treatments based on the regulation of hyposialylation.

scholarly journals Intracellular Targeting Signals Contribute to Localization of Coronavirus Spike Proteins near the Virus Assembly Site

2004 ◽  
Vol 78 (11) ◽  
pp. 5913-5922 ◽  
Author(s):  
Erik Lontok ◽  
Emily Corse ◽  
Carolyn E. Machamer
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Virus Assembly ◽  
Intracellular Localization ◽  
Cytoplasmic Tail ◽  
Viral Envelope ◽  
Reporter Protein ◽  
S Protein ◽  
Cytoplasmic Tails ◽  
S Proteins

ABSTRACT Coronavirus budding at the endoplasmic reticulum-Golgi intermediate compartment (ERGIC) requires accumulation of the viral envelope proteins at this point in the secretory pathway. Here we demonstrate that the spike (S) protein from the group 3 coronavirus infectious bronchitis virus (IBV) contains a canonical dilysine endoplasmic reticulum retrieval signal (-KKXX-COOH) in its cytoplasmic tail. This signal can retain a chimeric reporter protein in the ERGIC and when mutated allows transport of the full-length S protein as well as the chimera to the plasma membrane. Interestingly, the IBV S protein also contains a tyrosine-based endocytosis signal in its cytoplasmic tail, suggesting that any S protein that escapes the ERGIC will be rapidly endocytosed when it reaches the plasma membrane. We also identified a novel dibasic motif (-KXHXX-COOH) in the cytoplasmic tails of S proteins from group 1 coronaviruses and from the newly identified coronavirus implicated in severe acute respiratory syndrome. This dibasic motif also retained a reporter protein in the ERGIC, similar to the dilysine motif in IBV S. The cytoplasmic tails of S proteins from group 2 coronaviruses lack an intracellular localization signal. The inherent differences in S-protein trafficking could point to interesting variations in pathogenesis of coronaviruses, since increased levels of surface S protein could promote syncytium formation and direct cell-to-cell spread of the infection.

scholarly journals CMAS and ST3GAL4 Play an Important Role in the Adsorption of Influenza Virus by Affecting the Synthesis of Sialic Acid Receptors

2021 ◽  
Vol 22 (11) ◽  
pp. 6081
Author(s):  
Yaxin Zhao ◽  
Jiahui Zou ◽  
Qingxia Gao ◽  
Shengsong Xie ◽  
Jiyue Cao ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Sialic Acid ◽  
Cell Surface ◽  
Influenza A ◽  
Viral Envelope ◽  
Host Gene ◽  
Influenza A Viruses ◽  
Strong Affinity ◽  
Sialic Acid Receptors ◽  
The Impact

Influenza A viruses (IAVs) initiate infection by attaching Hemagglutinin (HA) on the viral envelope to sialic acid (SA) receptors on the cell surface. Importantly, HA of human IAVs has a higher affinity for α-2,6-linked SA receptors, and avian strains prefer α-2,3-linked SA receptors, whereas swine strains have a strong affinity for both SA receptors. Host gene CMAS and ST3GAL4 were found to be essential for IAV attachment and entry. Loss of CMAS and ST3GAL4 hindered the synthesis of sialic acid receptors, which in turn prevented the adsorption of IAV. Further, the knockout of CMAS had an effect on the adsorption of swine, avian and human IAVs. However, ST3GAL4 knockout prevented the adsorption of swine and avian IAV and the impact on avian IAV was more distinct, whereas it had no effect on the adsorption of human IAV. Collectively, our findings demonstrate that knocking out CMAS and ST3GAL4 negatively regulated IAV replication by inhibiting the synthesis of SA receptors, which also provides new insights into the production of gene-edited animals in the future.

Sign in / Sign up

Export Citation Format

Share Document