Recent Advances in Carbohydrate Chemistry and Application

Recent advances in carbohydrate chemistry and glycobiology have led to a better understanding of carbohydrates and the associated biological glycosylation in biology. This report describes new advances in carbohydrate synthesis and its application to the understanding of glycosylation in protein folding, cancer progression, influenza and SARS-CoV-2 infection and the development of carbohydrate-based medicines. © The AIC 2023. All rights reserved.

Visualization and functional annotation of coronavirus spike protein glycoshields

Spike proteins of coronaviruses are highly glycosylated and responsible for host recognition and viral entry. The glycans provide a camouflaging shield to help coronaviruses evade host immunity and, in some cases, modulate functional domain structures and dynamics pertinent to host recognition. However, the glycans are chemically and conformationally heterogeneous, making it challenging to determine the chemical compositions and conformations quantitatively. Combining cryo-electron microscopy, mass spectrometry, and molecular modeling, we systematically characterize a panel of spike protein variants of human and animal coronaviruses, including those of the variants of concern of SARS-CoV-2. We have established a robust workflow to quantify the heterogeneity of individual N-glycans by mass spectrometry. We also demonstrated the ability to visualize long glycan structures directly in regions where the dynamics are restricted. In places where the N-glycans are too dynamic, their structural information is generally lost after extended cryo-EM data processing that aims to achieve high resolution. To address this issue, we developed a computational tool called GlycoSHIELD to generate ensembles of glycan conformers to recapitulate the fuzzy structures that are in quantitative agreement with the experimental cryo-EM data. The ability to generate fully glycosylated spike protein models enables the prediction of hitherto unknown receptor and antibody binding sites. This work was supported by Academia Sinica intramural fund, an Academia Sinica Career Development Award, Academia Sinica to STDH (AS-CDA-109- L08), an Infectious Disease Research Supporting Grant to STDH (AS-IDR- 110-08), and the Ministry of Science and Technology (MOST), Taiwan (MOST 109-3114-Y-001-001, MOST 110-2113-M-001- 050-MY3 and MOST 110-2311-B-001-013-MY3) to STDH.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Targeting Spike Glycans to Inhibit SARS-CoV-2 Viral Entry

SARS-CoV-2 Spike harbors glycans which function as ligands for lectins. Therefore, it should be possible to exploit lectins to target SARS-CoV-2 and inhibit cellular entry by binding glycans on the Spike protein. Burkholderia oklahomensis agglutinin (BOA) is an antiviral lectin that interacts with viral glycoproteins via N-linked high mannose glycans. Here, we show that BOA binds to the Spike protein and is a potent inhibitor of SARS-CoV-2 viral entry at nanomolar concentrations. Using a variety of biophysical tools such as SEC chromatography, dynamics light scattering, fluorescence binding assays, and electron microscopy, we demonstrate that the interaction is avidity driven and that BOA crosslinks the Spike protein into soluble aggregates. Furthermore, using virus neutralization assays, we demonstrate that BOA effectively inhibits all tested variants of concern as well as SARS-CoV-1, establishing that glycan-targeting molecules have the potential to be pan-coronavirus inhibitors.Copyright © 2023 The American Society for Biochemistry and Molecular Biology, Inc.

Recent Progress in Development and Application of DNA, Protein, Peptide, Glycan, Antibody, and Aptamer Microarrays.

Microarrays are one of the trailblazing technologies of the last two decades and have displayed their importance in all the associated fields of biology. They are widely explored to screen, identify, and gain insights on the characteristics traits of biomolecules (individually or in complex solutions). A wide variety of biomolecule-based microarrays (DNA microarrays, protein microarrays, glycan microarrays, antibody microarrays, peptide microarrays, and aptamer microarrays) are either commercially available or fabricated in-house by researchers to explore diverse substrates, surface coating, immobilization techniques, and detection strategies. The aim of this review is to explore the development of biomolecule-based microarray applications since 2018 onwards. Here, we have covered a different array of printing strategies, substrate surface modification, biomolecule immobilization strategies, detection techniques, and biomolecule-based microarray applications. The period of 2018-2022 focused on using biomolecule-based microarrays for the identification of biomarkers, detection of viruses, differentiation of multiple pathogens, etc. A few potential future applications of microarrays could be for personalized medicine, vaccine candidate screening, toxin screening, pathogen identification, and posttranslational modifications.

Plasma markers of Covid-19 severity: A pilot study

SARS-CoV-2 infected patients show heterogeneous clinical presentations ranging from mild symptoms to severe respiratory failure and death. Consequently, various markers reflect certain disease presentations. Our cohort included moderate (n = 10) and severe (n = 10) COVID-19 patients, and 10 healthy controls. We determined plasma levels of nine acute phase proteins by nephelometry, full-length (M65), caspase-cleaved (M30) cytokeratin 18, and ADAMTS13 (a disintegrin-like and metalloprotease with thrombospondin type-1 motif 13) by ELISA. In addition, we examined whole plasma N-glycosylation by capillary gel electrophoresis coupled to laser-induced fluorescence detection. When compared to healthy controls, COVID-19 patients had significantly lower concentrations of ADAMTS13 and albumin (ALB) but higher M30, M65, alpha-1-acid glycoprotein, alpha1-antitrypsin (AAT), ceruloplasmin, haptoglobin, and highsensitivity C-reactive protein. The concentrations of alpha1-antichymotrypsin, alpha2-macroglobulin and serum amyloid A proteins did not differ. We found significantly higher levels of AAT and M65 but lower ALB in severe compared to moderate COVID-19 patients. N-glycan analysis of the serum proteome revealed increased levels of oligomannose and sialylated di-antennary glycans, while the non-sialylated di-antennary glycan A2G2 significantly decreased in COVID-19 patients compared to controls. COVID-19-associated changes in levels and N-glycosylation of specific plasma proteins highlight involvement of different pathophysiological mechanisms and grant further investigations.

Glycan masking in vaccine design: Targets, immunogens and applications.

Glycan masking is a novel technique in reverse vaccinology in which sugar chains (glycans) are added on the surface of immunogen candidates to hide regions of low interest and thus focus the immune system on highly therapeutic epitopes. This shielding strategy is inspired by viruses such as influenza and HIV, which are able to escape the immune system by incorporating additional glycosylation and preventing the binding of therapeutic antibodies. Interestingly, the glycan masking technique is mainly used in vaccine design to fight the same viruses that naturally use glycans to evade the immune system. In this review we report the major successes obtained with the glycan masking technique in epitope-focused vaccine design. We focus on the choice of the target antigen, the strategy for immunogen design and the relevance of the carrier vector to induce a strong immune response. Moreover, we will elucidate the different applications that can be accomplished with glycan masking, such as shifting the immune response from hyper-variable epitopes to more conserved ones, focusing the response on known therapeutic epitopes, broadening the response to different viral strains/sub-types and altering the antigen immunogenicity to elicit higher or lower immune response, as desired.

COVID-19 Pandemic: An Overview of its Origin, Current Status, and Ongoing Clinical Trials

The COVID-19 pandemic is raging across the globe, with the total active cases increas-ing each day. Globally over 63 million COVID-19cases and more than 1.4 million deaths have been reported to WHO. Throughout the world, academicians, clinicians and scientists are working tirelessly on developing a treatment to combat this pandemic. The origin of novel SARS-CoV-2 virus still remains foggy but is believed to have originated from a bat coronavirus RaTG13 with which it shares approximately 96% sequence similarity. In the present review, the authors have pro-vided an overview of the COVID-19 pandemic, epidemiology, transmission, developments related to diagnosis, drugs and vaccines, along with the genetic diversity and lifecycle of the SARS-CoV-2 based on the current studies and information available.Copyright © 2022 Bentham Science Publishers.

Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Studying the Effect of Glycosylation towards SARS-CoV-2 Spike RBD and ACE2 Interaction by Native Mass Spectrometry

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has serious consequences on global public health and social development. The binding of receptor binding domain (RBD) of spike protein to angiotensin converting enzyme 2 (ACE2) on the surface of SARS-CoV-2 host cell initiates the infection progress. Spike and ACE2 are both glycoproteins, the impact of glycosylation on protein structures and protein-protein interactions remains largely elusive. Characterizing the structural and dynamics of protein-protein binding progress will improve mechanism understanding of viral infection and facilitate targeted drug design. Structural mass spectrometry (MS) method is widely used in protein structural studies, providing complementary information to conventional biophysical methods, such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (cryo-EM). Native mass spectrometry (native MS) is an emerging technology that enables the study of intact protein, non-covalent protein-protein, and protein-ligand complexes in their biological state, which can provide structural stability, binding stoichiometry, and spatial arrangement information. Here, native MS was used to examine the interaction between RBD and ACE2 as well as the impact of deglycosylation on the interaction stability of the RBD-ACE2 complex. The results revealed that both RBD and ACE2 are highly glycosylated, ACE2 presents as a dimer while RBD as a monomer, and they form a (RBD-ACE2)2 complex. The conditions of using PNGasc F to remove the N-glycan were optimized. At least two Oglycans including NcuAc(2) and GalNAcC 1) Gal( 1) NcuAc(2) or GlcNAcd ) Gal(l) NeuAc(2) were observed for the N-glycan removed RBD. Furthermore, the stability of the complexes formed by glycosylated and deglycosylated RBD with ACE2 was compared, and the results showed that the removal of N-glycan significantly drops the interaction stability of the RBD-ACE2 complex. Therefore, we recommend that glycosyla-tion should not be removed for structural and functional studies. Additional glycosyla-tion, structural and dynamics studies on Spike (including separated RBD) and ACE2 complexes would help us to understand the process of viral infection, advance drug design and vaccine developments. Nowadays, a comprehensive MS-based toolbox has been developed for the analysis of protein structure, function, and dynamics, including hydrogen-deuterium exchange MS (HDX-MS), native top-down (nTD) MS, cross-linking MS (XL-MS), and covalent labelling MS (CL-MS), etc. Through integrating structural MS methods, more detailed and comprehensive structural information about glycoproteins and their complexes will be uncovered. © 2022 Chinese Society for Mass Spectrometry. All rights reserved.

Relapses or de-novo IgA nephropathy following COVID-19 vaccination, a narrative review

Immunoglobulin A (IgA) nephropathy is the most common type of glomerulonephritis worldwide characterized by excessive serum levels of glycosylated which triggers the generation of glycan-specific IgG and IgA autoantibodies. This pathological condition results in the formation of circulatory IgA immune complexes, which are essential for the development of glomerular inflammation, especially IgA nephropathy. The serum galactosylated IgA1, IgG, and IgA autoantibodies are suggested as the biomarkers of IgA nephropathy since IgA antibodies are early markers for disease activity too. Serum IgA antibodies emerged as the early COVID-19-specific antibody response about two days after initial symptoms of COVID-19 in comparison with IgG and IgM antibody concentrations, which appeared after five days. IgA nephropathy is frequently presented as microscopic or macroscopic hematuria and proteinuria with a male predominance. COVID-19 infection can include several organs aside from the lungs, such as kidneys through different mechanisms. It is demonstrated in most cases that short-lasting symptoms such as gross hematuria resolve either spontaneously or following a short course of steroids. This review summarized the reported cases of relapses or denovo reported cases of relapses or de-novo IgA nephropathy and IgA vasculitis following COVID-19 vaccination.Copyright © 2023 The Author(s);Published by Society of Diabetic Nephropathy Prevention. This is an open-access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Enhanced Interfacial H-Bond Networks Promote Glycan-Glycan Recognition and Interaction.

H-bond networks at heterogeneous interfaces play crucial roles in bioseparation, biocatalysis, biochip array profiling, and functional nanosystem self-assembly, but their precise modulation and enhancement remain challenging. In this study, we have discovered that interfacial hydrophobic hydration significantly enhances H-bond networks at the interface between a glycan-modified adsorbent and a methanol-water-acetonitrile ternary solution. The enhanced H-bond networks greatly promote the adsorbent-solution heterogeneous glycan-glycan recognition and interaction. This novel hydrophobic hydration-enhanced hydrophilic interaction (HEHI) strategy improves the affinity and efficiency of intact glycopeptide enrichment. Compared with the commonly used hydrophilic-interaction enrichment strategy, 23.5 and 48.5% more intact N- and O-glycopeptides are identified, and the enrichment recoveries of half of the glycopeptides are increased >100%. Further, in-depth profiling of both N- and O-glycosylation occurring on SARS-CoV-2 S1 and hACE2 proteins has been achieved with more glycan types and novel O-glycosylation information involved. Interfacial hydrophobic hydration provides a powerful tool for the modulation of hydrophilic interactions in biological systems.

DeGlyPHER: Highly sensitive site-specific analysis of N-linked glycans on proteins.

Traditional mass spectrometry-based glycoproteomic approaches have been widely used for site-specific N-glycoform analysis, but a large amount of starting material is needed to obtain sampling that is representative of the vast diversity of N-glycans on glycoproteins. These methods also often include a complicated workflow and very challenging data analysis. These limitations have prevented glycoproteomics from being adapted to high-throughput platforms, and the sensitivity of the analysis is currently inadequate for elucidating N-glycan heterogeneity in clinical samples. Heavily glycosylated spike proteins of enveloped viruses, recombinantly expressed as potential vaccines, are prime targets for glycoproteomic analysis. Since the immunogenicity of spike proteins may be impacted by their glycosylation patterns, site-specific analysis of N-glycoforms provides critical information for vaccine design. Using recombinantly expressed soluble HIV Env trimer, we describe DeGlyPHER, a modification of our previously reported sequential deglycosylation strategy to yield a "single-pot" process. DeGlyPHER is an ultrasensitive, simple, rapid, robust, and efficient approach for site-specific analysis of protein N-glycoforms, that we developed for analysis of limited quantities of glycoproteins.

The diversity of the glycan shield of sarbecoviruses related to SARS-CoV-2.

Animal reservoirs of sarbecoviruses represent a significant risk of emergent pandemics, as evidenced by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Vaccines remain successful at limiting severe disease and death, but the potential for further coronavirus zoonosis motivates the search for pan-coronavirus vaccines. This necessitates a better understanding of the glycan shields of coronaviruses, which can occlude potential antibody epitopes on spike glycoproteins. Here, we compare the structure of 12 sarbecovirus glycan shields. Of the 22 N-linked glycan attachment sites present on SARS-CoV-2, 15 are shared by all 12 sarbecoviruses. However, there are significant differences in the processing state at glycan sites in the N-terminal domain, such as N165. Conversely, glycosylation sites in the S2 domain are highly conserved and contain a low abundance of oligomannose-type glycans, suggesting a low glycan shield density. The S2 domain may therefore provide a more attractive target for immunogen design efforts aiming to generate a pan-coronavirus antibody response.

[Glycosylation, glycan receptors recognition of SARS-CoV-2 and discoveries of glycan inhibitors against SARS-CoV-2].

COVID-19 represents the most serious public health event in the past few decades of the 21st century. The development of vaccines, neutralizing antibodies, and small molecule chemical agents have effectively prevented the rapid spread of COVID-19. However, the continued emergence of SARS-CoV-2 variants have weakened the efficiency of these vaccines and antibodies, which brought new challenges for searching novel anti-SARS-CoV-2 drugs and methods. In the process of SARS-CoV-2 infection, the virus firstly attaches to heparan sulphate on the cell surface of respiratory tract, then specifically binds to hACE2. The S protein of SARS-CoV-2 is a highly glycosylated protein, and glycosylation is also important for the binding of hACE2 to S protein. Furthermore, the S protein is recognized by a series of lectin receptors in host cells. These finding implies that glycosylation plays important roles in the invasion and infection of SARS-CoV-2. Based on the glycosylation pattern and glycan recognition mechanisms of SARS-CoV-2, it is possible to develop glycan inhibitors against COVID-19. Recent studies have shown that sulfated polysaccharides originated from marine sources, heparin and some other glycans display anti-SARS-CoV-2 activity. This review summarized the function of glycosylation of SARS-CoV-2, discoveries of glycan inhibitors and the underpinning molecular mechanisms, which will provide guidelines to develop glycan-based new drugs against SARS-CoV-2.

Glycan masking of a non-neutralising epitope enhances neutralising antibodies targeting the RBD of SARS-CoV-2 and its variants.

The accelerated development of the first generation COVID-19 vaccines has saved millions of lives, and potentially more from the long-term sequelae of SARS-CoV-2 infection. The most successful vaccine candidates have used the full-length SARS-CoV-2 spike protein as an immunogen. As expected of RNA viruses, new variants have evolved and quickly replaced the original wild-type SARS-CoV-2, leading to escape from natural infection or vaccine induced immunity provided by the original SARS-CoV-2 spike sequence. Next generation vaccines that confer specific and targeted immunity to broadly neutralising epitopes on the SARS-CoV-2 spike protein against different variants of concern (VOC) offer an advance on current booster shots of previously used vaccines. Here, we present a targeted approach to elicit antibodies that neutralise both the ancestral SARS-CoV-2, and the VOCs, by introducing a specific glycosylation site on a non-neutralising epitope of the RBD. The addition of a specific glycosylation site in the RBD based vaccine candidate focused the immune response towards other broadly neutralising epitopes on the RBD. We further observed enhanced cross-neutralisation and cross-binding using a DNA-MVA CR19 prime-boost regime, thus demonstrating the superiority of the glycan engineered RBD vaccine candidate across two platforms and a promising candidate as a broad variant booster vaccine.

Rational Development of Hypervalent Glycan Shield-Binding Nanoparticles with Broad-Spectrum Inhibition against Fatal Viruses Including SARS-CoV-2 Variants.

Infectious virus diseases, particularly coronavirus disease 2019, have posed a severe threat to public health, whereas the developed therapeutic and prophylactic strategies are seriously challenged by viral evolution and mutation. Therefore, broad-spectrum inhibitors of viruses are highly demanded. Herein, an unprecedented antiviral strategy is reported, targeting the viral glycan shields with hypervalent mannose-binding nanoparticles. The nanoparticles exhibit a unique double-punch mechanism, being capable of not only blocking the virus-receptor interaction but also inducing viral aggregation, thereby allowing for inhibiting the virus entry and facilitating the phagocytosis of viruses. The nanoparticles exhibit potent and broad-spectrum antiviral efficacy to multiple pseudoviruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its major variants (D614G, N501Y, N439K, Δ69-70, Delta, and Omicron; lentiviruses expressing only the spike proteins), as well as other vital viruses (human immunodeficiency virus 1 and Lassa virus), with apparent EC50 values around the 10-9  m level. Significantly, the broad-spectrum inhibition of authentic viruses of both wild-type SARS-CoV-2 and Delta variants is confirmed. Therefore, this hypervalent glycan-shield targeting strategy opens new access to broad-spectrum viral inhibition.

Core fucose-specific Pholiota squarrosa lectin (PhoSL) as a potent broad-spectrum inhibitor of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.

Investigating sialic acid usage in species D human adenoviruses

Human adenoviruses are phylogenetically divided across seven species, A-G, causing transient mild illnesses, except in immunocompromised individuals. Their double stranded DNA genome is amenable to genetic manipulations, enabling development of highly engineered virotherapies. Species D adenoviruses have naturally low seroprevalence rates, an important trait in avoiding neutralising anti-vector immunity. We previously demonstrated that HAdV-D26, the platform of the Janssen SARS-CoV2 vaccine, uses sialic acid as a primary cell entry receptor. Here, we structurally and biologically investigated sialic acid usage across species D. We solved multiple structures of species D adenovirus fiber knob proteins alone and in complex with sialic acid, identifying a conserved binding pocket common with known sialic acid binders HAdV-D26 and 37. Using fiber-knob pseudotyped viruses, we demonstrate significantly reduced transduction in cells treated with neuraminidase to remove sialic acid residues in HAdV-D26 and 53, with HAdV-D15, 24 and 29 also demonstrating non-significant reductions. IC50 data also showed highlighted binding to CAR, although at a significantly lower affinity compared to the CAR-binding HAdV-C5. Improved understanding of the usage of sialic acid as a receptor will enable better exploitation of the species D adenoviruses as therapeutic vectors. Our findings raise the possibility of a conserved sialic acid binding pocket within species D adenoviruses resulting in varying affinity levels. Further evaluation of specific glycan binding patterns used by these viruses, as observed between HAdV-D37 and GD1a glycan, will better inform the design of appropriate antivirals to contain adenovirus outbreaks as well as the engineering of targeted vectors for translational applications.

N-Glycosylation Network Construction and Analysis to Modify Glycans on the Spike (S) Glycoprotein of SARS-CoV-2.

Background: The N-glycan structure and composition of the spike (S) protein of SARS-CoV-2 are pertinent to vaccine development and efficacy. Methods: We reconstructed the glycosylation network based on previously published mass spectrometry data using GNAT, a glycosylation network analysis tool. Our compilation of the network tool had 26 glycosyltransferase and glucosidase enzymes and could infer the pathway of glycosylation machinery based on glycans in the virus spike protein. Once the glycan biosynthesis pathway was generated, we simulated the effect of blocking specific enzymes-swainsonine or deoxynojirimycin for blocking mannosidase-II and indolizidine for blocking alpha-1,6-fucosyltransferase-to see how they would affect the biosynthesis network and the glycans that were synthesized. Results: The N-glycan biosynthesis network of SARS-CoV-2 spike protein shows an elaborate enzymatic pathway with several intermediate glycans, along with the ones identified by mass spectrometric studies. Of the 26 enzymes, the following were involved-Man-Ia, MGAT1, MGAT2, MGAT4, MGAT5, B3GalT, B4GalT, Man-II, SiaT, ST3GalI, ST3GalVI, and FucT8. Blocking specific enzymes resulted in a substantially modified glycan profile of SARS-CoV-2. Conclusion: Variations in the final N-glycan profile of the virus, given its site-specific microheterogeneity, are factors in the host response to the infection, vaccines, and antibodies. Heterogeneity in the N-glycan profile of the spike (S) protein and its potential effect on vaccine efficacy or adverse reactions to the vaccines remain unexplored. Here, we provide all the resources we generated-the glycans in the glycoCT xml format and the biosynthesis network for future work.