scholarly journals A Chemoenzymatic Method for Glycoproteomic N-glycan Type Quantitation

2019 ◽  
Author(s):  
Henghui Li ◽  
Leyuan Li ◽  
Kai Cheng ◽  
Zhibin Ning ◽  
Janice Mayne ◽  
...  
Keyword(s):  
Vaccine Development ◽  
Cancer Cell Line ◽  
The Novel ◽  
Post Translational Modifications ◽  
Glycosylation Sites ◽  
Type Composition ◽  
Dimethyl Labeling ◽  
High Mannose ◽  
Biomarker Research ◽  
Glycan Type

ABSTRACTGlycosylation is one of the most important post-translational modifications in biological systems. Current glycoproteome methods mainly focus on qualitative identification of glycosylation sites or intact glycopeptides. However, the systematic quantitation of glycoproteins has remained largely unexplored. Here, we developed a chemoenzymatic method to quantitatively investigate N-glycoproteome based on the N-glycan types. Taking advantage of the specificity of different endoglycosidases and isotope dimethyl labeling, six N-glycan types of structures linked on each glycopeptide, including high-mannose/hybrid, bi-antennary and tri-antennary with/without core fucose, were quantified. As a proof of principle, the glycoproteomic N-glycan type quantitative (glyco-TQ) method was first used to determine the N-glycan type composition of immunoglobulin G1 (IgG1) Fc fragment. Then we applied the method to analyze the glycan type profile of proteins in the breast cancer cell line MCF7, and quantitatively revealed the N-glycan type micro-heterogeneity at both the glycopeptide and glycoprotein levels. The novel quantitative strategy to evaluate the relative intensity of the six states of N-glycan type glycosylation on each site provides a new avenue to investigate function of glycoproteins in broad areas, such as cancer biomarker research, pharmaceuticals characterization and anti-glycan vaccine development.

scholarly journals SARS-CoV-2 Nucleocapsid protein is decorated with multiple N- and O-glycans

2020 ◽  
Author(s):  
Nitin T. Supekar ◽  
Asif Shajahan ◽  
Anne S. Gleinich ◽  
Daniel Rouhani ◽  
Christian Heiss ◽  
...  
Keyword(s):  
Nucleocapsid Protein ◽  
Vaccine Development ◽  
Point Of Care ◽  
Variable Mass ◽  
Phosphorylation Site ◽  
Post Translational Modifications ◽  
N Protein ◽  
Point Of Care Diagnostics ◽  
Glycosylation Sites

AbstractSevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease (COVID-19) started at the end of 2019 in Wuhan, China has spread rapidly and became a pandemic. Since there is no therapy available that is proven as fully protective against COVID-19, a vaccine to protect against deadly COVID-19 is urgently needed. Nucleocapsid protein (N protein), is one of the most abundant proteins in coronaviruses and is a potential target for both vaccine development and point of care diagnostics. The variable mass of N protein (45 to 60 kDa), suggests the presence of post-translational modifications (PTMs), and it is critical to clearly define these PTMs to gain the structural understanding necessary for further vaccine research. There have been several reports suggesting that the N protein is phosphorylated but lacks glycosylation. Our comprehensive glycomics and glycoproteomics experiments confirm that the N protein is highly O-glycosylated and also contains significant levels of N-glycosylation. We were able to confirm the presence of O-glycans on seven sites with substantial glycan occupancy, in addition to less abundant O-glycans on four sites. We also detected N-glycans on two out of five potential N-glycosylation sites. Moreover, we were able to confirm one phosphorylation site. Recent studies have indicated that the N protein can serve as an important diagnostic marker for coronavirus disease and a major immunogen by priming protective immune responses. Thus, detailed structural characterization of the N protein may provide useful insights for understanding the roles of glycosylation on viral pathogenesis and also in vaccine design and development.

Carbohydrate-Binding Agents: Potential of Repurposing for COVID-19 Therapy

2020 ◽  
Vol 21 (11) ◽  
pp. 1085-1096 ◽  
Author(s):  
Rajesh Kumar Gupta ◽  
Girish R. Apte ◽  
Kiran Bharat Lokhande ◽  
Satyendra Mishra ◽  
Jayanta K. Pal
Keyword(s):  
Vaccine Development ◽  
Host Cells ◽  
Carbohydrate Binding ◽  
Atypical Pneumonia ◽  
The Novel ◽  
High Infection ◽  
Binding Agents ◽  
Current Scenario ◽  
Antiviral Activities ◽  
Novel Drug

: With the emergence of the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the whole world is suffering from atypical pneumonia, which resulted in more than 559,047 deaths worldwide. In this time of crisis and urgency, the only hope comes from new candidate vaccines and potential antivirals. However, formulating new vaccines and synthesizing new antivirals are a laborious task. Therefore, considering the high infection rate and mortality due to COVID-19, utilization of previous information, and repurposing of existing drugs against valid viral targets have emerged as a novel drug discovery approach in this challenging time. The transmembrane spike (S) glycoprotein of coronaviruses (CoVs), which facilitates the virus’s entry into the host cells, exists in a homotrimeric form and is covered with N-linked glycans. S glycoprotein is known as the main target of antibodies having neutralizing potency and is also considered as an attractive target for therapeutic or vaccine development. Similarly, targeting of N-linked glycans of S glycoprotein envelope of CoV via carbohydrate-binding agents (CBAs) could serve as an attractive therapeutic approach for developing novel antivirals. CBAs from natural sources like lectins from plants, marine algae and prokaryotes and lectin mimics like Pradimicin-A (PRM-A) have shown antiviral activities against CoV and other enveloped viruses. However, the potential use of CBAs specifically lectins was limited due to unfavorable responses like immunogenicity, mitogenicity, hemagglutination, inflammatory activity, cellular toxicity, etc. Here, we reviewed the current scenario of CBAs as antivirals against CoVs, presented strategies to improve the efficacy of CBAs against CoVs; and studied the molecular interactions between CBAs (lectins and PRM-A) with Man9 by molecular docking for potential repurposing against CoVs in general, and SARSCoV- 2, in particular.

scholarly journals Strategies for Vaccination: Conventional Vaccine Approaches Versus New-Generation Strategies in Combination with Adjuvants

Pharmaceutics ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 140
Author(s):  
Abdellatif Bouazzaoui ◽  
Ahmed A. H. Abdellatif ◽  
Faisal A. Al-Allaf ◽  
Neda M. Bogari ◽  
Saied Al-Dehlawi ◽  
...  
Keyword(s):  
Viral Vectors ◽  
Protein Production ◽  
Vaccine Development ◽  
Effective Vaccine ◽  
The Novel ◽  
Research Teams ◽  
Advantages And Disadvantages ◽  
Rapid Spread ◽  
New Generation ◽  
Conventional Vaccine

The current COVID-19 pandemic, caused by severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2), has raised significant economic, social, and psychological concerns. The rapid spread of the virus, coupled with the absence of vaccines and antiviral treatments for SARS-CoV-2, has galvanized a major global endeavor to develop effective vaccines. Within a matter of just a few months of the initial outbreak, research teams worldwide, adopting a range of different strategies, embarked on a quest to develop effective vaccine that could be effectively used to suppress this virulent pathogen. In this review, we describe conventional approaches to vaccine development, including strategies employing proteins, peptides, and attenuated or inactivated pathogens in combination with adjuvants (including genetic adjuvants). We also present details of the novel strategies that were adopted by different research groups to successfully transfer recombinantly expressed antigens while using viral vectors (adenoviral and retroviral) and non-viral delivery systems, and how recently developed methods have been applied in order to produce vaccines that are based on mRNA, self-amplifying RNA (saRNA), and trans-amplifying RNA (taRNA). Moreover, we discuss the methods that are being used to enhance mRNA stability and protein production, the advantages and disadvantages of different methods, and the challenges that are encountered during the development of effective vaccines.

scholarly journals Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Aleksandar Antanasijevic ◽  
Leigh M. Sewall ◽  
Christopher A. Cottrell ◽  
Diane G. Carnathan ◽  
Luis E. Jimenez ◽  
...  
Keyword(s):  
High Resolution ◽  
Polyclonal Antibody ◽  
Vaccine Development ◽  
Mutational Analysis ◽  
Rhesus Macaques ◽  
Antibody Responses ◽  
Glycosylation Sites ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Resolution Mapping

AbstractEngineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, we used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. We applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. We demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, our analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

scholarly journals ACE2 Nascence, trafficking, and SARS-CoV-2 pathogenesis: the saga continues

2021 ◽  
Vol 15 (1) ◽  
Author(s):  
Sally Badawi ◽  
Bassam R. Ali
Keyword(s):  
Cell Surface ◽  
Angiotensin Converting Enzyme ◽  
The Novel ◽  
Converting Enzyme ◽  
Post Translational Modifications ◽  
Angiotensin Converting Enzyme 2 ◽  
Viral Attachment ◽  
Novel Coronavirus ◽  
Effective Medication ◽  
Potential Use

AbstractWith the emergence of the novel coronavirus SARS-CoV-2 since December 2019, more than 65 million cases have been reported worldwide. This virus has shown high infectivity and severe symptoms in some cases, leading to over 1.5 million deaths globally. Despite the collaborative and concerted research efforts that have been made, no effective medication for COVID-19 (coronavirus disease-2019) is currently available. SARS-CoV-2 uses the angiotensin-converting enzyme 2 (ACE2) as an initial mediator for viral attachment and host cell invasion. ACE2 is widely distributed in the human tissues including the cell surface of lung cells which represent the primary site of the infection. Inhibiting or reducing cell surface availability of ACE2 represents a promising therapy for tackling COVID-19. In this context, most ACE2–based therapeutic strategies have aimed to tackle the virus through the use of angiotensin-converting enzyme (ACE) inhibitors or neutralizing the virus by exogenous administration of ACE2, which does not directly aim to reduce its membrane availability. However, through this review, we present a different perspective focusing on the subcellular localization and trafficking of ACE2. Membrane targeting of ACE2, and shedding and cellular trafficking pathways including the internalization are not well elucidated in literature. Therefore, we hereby present an overview of the fate of newly synthesized ACE2, its post translational modifications, and what is known of its trafficking pathways. In addition, we highlight the possibility that some of the identified ACE2 missense variants might affect its trafficking efficiency and localization and hence may explain some of the observed variable severity of SARS-CoV-2 infections. Moreover, an extensive understanding of these processes is necessarily required to evaluate the potential use of ACE2 as a credible therapeutic target.

scholarly journals Tumor Suppressor SMAR1 Activates and Stabilizes p53 through Its Arginine-Serine-rich Motif

2005 ◽  
Vol 280 (16) ◽  
pp. 16019-16029 ◽  
Author(s):  
Archana Jalota ◽  
Kamini Singh ◽  
Lakshminarasimhan Pavithra ◽  
Ruchika Kaul-Ghanekar ◽  
Shahid Jameel ◽  
...  
Keyword(s):  
Cell Fate ◽  
The Novel ◽  
Nuclear Retention ◽  
Post Translational Modifications ◽  
Rich Domain ◽  
Dna Damaging Agents ◽  
Proliferation And Apoptosis ◽  
Interfering Rna ◽  
The Guardian

Various stresses and DNA-damaging agents trigger transcriptional activity of p53 by post-translational modifications, making it a global regulatory switch that controls cell proliferation and apoptosis. Earlier we have shown that the novel MAR-associated protein SMAR1 interacts with p53. Here we delineate the minimal domain of SMAR1 (the arginine-serine-rich domain) that is phosphorylated by protein kinase C family proteins and is responsible for p53 interaction, activation, and stabilization within the nucleus. SMAR1-mediated stabilization of p53 is brought about by inhibiting Mdm2-mediated degradation of p53. We also demonstrate that this arginine-serine (RS)-rich domain triggers the various cell cycle modulating proteins that decide cell fate. Furthermore, phenotypic knock-down experiments using small interfering RNA showed that SMAR1 is required for activation and nuclear retention of p53. The level of phosphorylated p53 was significantly increased in the thymus of SMAR1 transgenic mice, showingin vivosignificance of SMAR1 expression. This is the first report that demonstrates the mechanism of action of the MAR-binding protein SMAR1 in modulating the activity of p53, often referred to as the “guardian of the genome.”

PTEN – A MOLECULAR MARKER FOR THE DIAGNOSIS OF GLIOMA BABUL REDDY TATIREDDY

2021 ◽  
pp. 45-48
Author(s):  
Babul Reddy Tatireddy
Keyword(s):  
Brain Tumor ◽  
Literature Search ◽  
Recipient Cell ◽  
Primary Brain Tumor ◽  
The Novel ◽  
Cellular Phenotype ◽  
Therapeutic Approaches ◽  
Downstream Signaling ◽  
Novel Biomarkers ◽  
Biomarker Research

Glioblastoma is an uncommon primary brain tumor accounts for upto 15.4% of all primary brain tumors. Although several modern therapies against glioma are discovered, management is still a critical concern. The existing therapies were relatively inconsistent; moreover, the procedures as well are difcult to treat. These ineffective therapeutic approaches led researchers to identify the novel biomarkers that can be implemented with the existing therapies for better management. In this view, along with biomarker research, a literature search for exosomal PTEN detection in glioblastoma was conducted and the recent studies observed that exosomes can transport tumor-suppressive proteins (PTEN) and oncogenic mRNAs, microRNAs to a recipient cell, which subsequently activates the downstream signaling pathways and inuences the cellular phenotype. These exosomes facilitate the transfer of PTEN released from tumor cells to receipt cells that leads to tumor progression. Similarly, glioma was also associated with a reduction or loss of PTEN expression. Hence, our present review aimed to provide a holistic picture of glioblastoma, its pathogenesis and novel biomarkers with an emphasis on PTEN detection in exosomes for the early identication of glioblastoma

scholarly journals Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants

2021 ◽  
Author(s):  
Satoshi Ikegame ◽  
Mohammed Siddiquey ◽  
Chuan-Tien Hung ◽  
Griffin Haas ◽  
Luca Brambilla ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Cell Line ◽  
Vesicular Stomatitis Virus ◽  
Neutralizing Antibodies ◽  
Vaccine Development ◽  
The Novel ◽  
Serum Samples ◽  
Egfp Reporter ◽  
Vesicular Stomatitis ◽  
Reduced Activity

Abstract The novel pandemic betacoronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected at least 120 million people since its identification as the cause of a December 2019 viral pneumonia outbreak in Wuhan, China1,2. Despite the unprecedented pace of vaccine development, with six vaccines already in use worldwide, the emergence of SARS-CoV-2 ‘variants of concern’ (VOC) across diverse geographic locales have prompted re-evaluation of strategies to achieve universal vaccination3. All three officially designated VOC carry Spike (S) polymorphisms thought to enable escape from neutralizing antibodies elicited during initial waves of the pandemic4–8. Here, we characterize the biological consequences of the ensemble of S mutations present in VOC lineages B.1.1.7 (501Y.V1) and B.1.351 (501Y.V2). Using a replication-competent EGFP-reporter vesicular stomatitis virus (VSV) system, rcVSV-CoV2-S, which encodes S from SARS coronavirus 2 in place of VSV-G, and coupled with a clonal HEK-293T ACE2 TMPRSS2 cell line optimized for highly efficient S-mediated infection, we determined that only 1 out of 12 serum samples from a cohort of recipients of the Gamaleya Sputnik V Ad26 / Ad5 vaccine showed effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralized S from B.1.1.7 and showed only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

scholarly journals Local Evolutionary Patterns of Human Respiratory Syncytial Virus Derived from Whole-Genome Sequencing

2015 ◽  
Vol 89 (7) ◽  
pp. 3444-3454 ◽  
Author(s):  
Charles N. Agoti ◽  
James R. Otieno ◽  
Patrick K. Munywoki ◽  
Alexander G. Mwihuri ◽  
Patricia A. Cane ◽  
...  
Keyword(s):  
Respiratory Syncytial Virus ◽  
Large Scale ◽  
Vaccine Development ◽  
Human Respiratory Syncytial Virus ◽  
Genomic Variation ◽  
Genomic Sequences ◽  
The Novel ◽  
Sequencing Method ◽  
The World ◽  
Syncytial Virus

ABSTRACTHuman respiratory syncytial virus (RSV) is associated with severe childhood respiratory infections. A clear description of local RSV molecular epidemiology, evolution, and transmission requires detailed sequence data and can inform new strategies for virus control and vaccine development. We have generated 27 complete or nearly complete genomes of RSV from hospitalized children attending a rural coastal district hospital in Kilifi, Kenya, over a 10-year period using a novel full-genome deep-sequencing process. Phylogenetic analysis of the new genomes demonstrated the existence and cocirculation of multiple genotypes in both RSV A and B groups in Kilifi. Comparison of local versus global strains demonstrated that most RSV A variants observed locally in Kilifi were also seen in other parts of the world, while the Kilifi RSV B genomes encoded a high degree of variation that was not observed in other parts of the world. The nucleotide substitution rates for the individual open reading frames (ORFs) were highest in the regions encoding the attachment (G) glycoprotein and the NS2 protein. The analysis of RSV full genomes, compared to subgenomic regions, provided more precise estimates of the RSV sequence changes and revealed important patterns of RSV genomic variation and global movement. The novel sequencing method and the new RSV genomic sequences reported here expand our knowledge base for large-scale RSV epidemiological and transmission studies.IMPORTANCEThe new RSV genomic sequences and the novel sequencing method reported here provide important data for understanding RSV transmission and vaccine development. Given the complex interplay between RSV A and RSV B infections, the existence of local RSV B evolution is an important factor in vaccine deployment.

scholarly journals Chikungunya E2 Protein Produced in E. coli and HEK293-T Cells—Comparison of Their Performances in ELISA

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 939
Author(s):  
Flávia Fonseca Bagno ◽  
Lara Carvalho Godói ◽  
Maria Marta Figueiredo ◽  
Sarah Aparecida Rodrigues Sérgio ◽  
Thaís de Fátima Silva Moraes ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Indirect Elisa ◽  
Acute Onset ◽  
Diagnostic Tools ◽  
Igg Antibodies ◽  
Post Translational Modifications ◽  
E Coli ◽  
Glycosylation Sites ◽  
Igm Elisa ◽  
Antigen Antibody

Chikungunya virus (CHIKV) is a mosquito-borne pathogen that causes a disease characterized by the acute onset of fever accompanied by arthralgia and intense joint pain. Clinical similarities and cocirculation of this and other arboviruses in many tropical countries highlight the necessity for efficient and accessible diagnostic tools. CHIKV envelope proteins are highly conserved among alphaviruses and, particularly, the envelope 2 glycoprotein (CHIKV-E2) appears to be immunodominant and has a considerable serodiagnosis potential. Here, we investigate how glycosylation of CHIKV-E2 affects antigen/antibody interaction and how this affects the performance of CHIKV-E2-based Indirect ELISA tests. We compare two CHIKV-E2 recombinant antigens produced in different expression systems: prokaryotic-versus eukaryotic-made recombinant proteins. CHIKV-E2 antigens are expressed either in E. coli BL21(DE3)—a prokaryotic system unable to produce post-translational modifications—or in HEK-293T mammalian cells—a eukaryotic system able to add post-translational modifications, including glycosylation sites. Both prokaryotic and eukaryotic recombinant CHIKV-E2 react strongly to anti-CHIKV IgG antibodies, showing accuracy levels that are higher than 90%. However, the glycan-added viral antigen presents better sensitivity and specificity (85 and 98%) than the non-glycosylated antigen (81 and 71%, respectively) in anti-CHIKV IgM ELISA assays.

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