This is GlycoQL.

MOTIVATION: We have previously designed and implemented a tree-based ontology to represent glycan structures with the aim of searching these structures with a glyco-driven syntax. This resulted in creating the GlySTreeM knowledge-base as a linchpin of the structural matching procedure and we now introduce a query language, called GlycoQL, for the actual implementation of a glycan structure search. RESULTS: The methodology is described and illustrated with a use-case focused on Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) spike protein glycosylation. We show how to enhance site annotation with federated queries involving UniProt and GlyConnect, our glycoprotein database. AVAILABILITY AND IMPLEMENTATION: https://glyconnect.expasy.org/glycoql/.

The Sea Cucumber Thyonella gemmata Contains a Low Anticoagulant Sulfated Fucan with High Anti-SARS-CoV-2 Actions against Wild-Type and Delta Variants.

In this work, we isolated two new sulfated glycans from the body wall of the sea cucumber Thyonella gemmata: one fucosylated chondroitin sulfate (TgFucCS) (17.5 ± 3.5% kDa) and one sulfated fucan (TgSF) (383.3 ± 2.1% kDa). NMR results showed the TgFucCS backbone composed of [→3)-ß-N-acetylgalactosamine-(1→4)-ß-glucuronic acid-(1→] with 70% 4-sulfated and 30% 4,6-disulfated GalNAc units and one-third of the GlcA units decorated at the C3 position with branching α-fucose (Fuc) units either 4-sulfated (65%) or 2,4-disulfated (35%) and the TgSF structure composed of a tetrasaccharide repeating unit of [→3)-α-Fuc2,4S-(1→2)-α-Fuc4S-(1→3)-α-Fuc2S-(1→3)-α-Fuc2S-(1→]n. Inhibitory properties of TgFucCS and TgSF were investigated using SARS-CoV-2 pseudovirus coated with S-proteins of the wild-type (Wuhan-Hu-1) or the delta (B.1.617.2) strains and in four different anticoagulant assays, comparatively with unfractionated heparin. Molecular binding to coagulation (co)-factors and S-proteins was investigated by competitive surface plasmon resonance spectroscopy. Among the two sulfated glycans tested, TgSF showed significant anti-SARS-CoV-2 activity against both strains together with low anticoagulant properties, indicating a good candidate for future studies in drug development.

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.

Purification and characterisation of heparin-like sulfated polysaccharides with potent anti-SARS-CoV-2 activity from snail mucus of Achatina fulica.

Heparin-like sulfated polysaccharide, acharan sulfate, was purified from the mucus of an African giant snail with unique sulfated glycosaminoglycans (GAGs). This study reported on finding novel and safe heparin resources from Achatina fulica for further use as well as easy isolation and purification of the active fraction from the initial raw material. Its structure was characterised by a strong-anion exchange combined with high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the potential acharan sulfate fraction is a glycosaminoglycan composed of several repeating disaccharide units, namely, of →4)-α-IdoA(2S)(1→4)-α-GlcNAc/GlcNAc(6S)/GlcNSO3(6S)(1→, and hence, presents heterogeneity regarding negative net charge density. Furthermore, the heparinase digests inhibit the binding of SARS-CoV-2 spike protein to the ACE2 receptor. In summary, the acharan sulfate presented in this work has shown its great potential for application in the preparation of sulfated polysaccharides as an alternative to heparin with important biological activity.

Antiviral activity of marine sulfated glycans against pathogenic human coronaviruses.

Great interest exists towards the discovery and development of broad-spectrum antivirals. This occurs due to the frequent emergence of new viruses which can also eventually lead to pandemics. A reasonable and efficient strategy to develop new broad-spectrum antivirals relies on targeting a common molecular player of various viruses. Heparan sulfate is a sulfated glycosaminoglycan present on the surface of cells which plays a key role as co-receptor in many virus infections. In previous work, marine sulfated glycans (MSGs) were identified as having antiviral activities. Their mechanism of action relies primarily on competitive inhibition of virion binding to heparan sulfate, preventing virus attachment to the cell surface prior to entry. In the current work we used pseudotyped lentivirus particles to investigate in a comparative fashion the inhibitory properties of five structurally defined MSGs against SARS-CoV-1, SARS-CoV-2, MERS-CoV, and influenza A virus (IAV). MSGs include the disaccharide-repeating sulfated galactan from the red alga Botryocladia occidentalis, the tetrasaccharide-repeating sulfated fucans from the sea urchin Lytechinus variegatus and from the sea cucumber Isostichopus badionotus, and the two marine fucosylated chondroitin sulfates from the sea cucumbers I. badionotus and Pentacta pygmaea. Results indicate specificity of action against SARS-CoV-1 and SARS-CoV-2. Curiously, the MSGs showed decreased inhibitory potencies against MERS-CoV and negligible action against IAV. Among the five MSGs, the two sulfated fucans here studied deserve further attention since they have the lowest anticoagulant effects but still present potent and selective antiviral properties.

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.

[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.

Heterogeneity in IgG-CD16 signaling in infectious disease outcomes.

In this review, we discuss how IgG antibodies can modulate inflammatory signaling during viral infections with a focus on CD16a-mediated functions. We describe the structural heterogeneity of IgG antibody ligands, including subclass and glycosylation that impact binding by and downstream activity of CD16a, as well as the heterogeneity of CD16a itself, including allele and expression density. While inflammation is a mechanism required for immune homeostasis and resolution of acute infections, we focus here on two infectious diseases that are driven by pathogenic inflammatory responses during infection. Specifically, we review and discuss the evolving body of literature showing that afucosylated IgG immune complex signaling through CD16a contributes to the overwhelming inflammatory response that is central to the pathogenesis of severe forms of dengue disease and coronavirus disease 2019 (COVID-19).

In-depth characterization of protein N-glycosylation for a COVID-19 variant-design vaccine spike protein.

COVID-19 is caused by SARS-CoV-2 infection and remains one of the biggest pandemics around the world since 2019. Vaccination has proved to be an effective way of preventing SARS-CoV-2 infection and alleviating the hospitalization burden. Among different forms of COVID-19 vaccine design, the spike protein of SARS-CoV-2 virus is widely used as a candidate vaccine antigen. As a surface protein on the virus envelop, the spike was reported to be heavily N-glycosylated and glycosylation had a great impact on its immunogenicity and efficacy. Besides, N-glycosylation might vary greatly on different expression systems and sequence variant designs. Therefore, comprehensive analysis of spike N-glycosylation is of great significance for better vaccine understanding and quality control. In this study, full characterization of N-glycosylation was performed for a Chinese Hamster Ovary (CHO) cell expressed variant-designed spike protein. The spike protein featured the latest six-proline substitution design together with the incorporation of a combination of mutation sites. Trypsin and Glu-C digestion coupled with PNGase F strategies were adopted, and effective LC-MS/MS methods were applied to analyze samples. As a result, a total of 19 N-glycosites were identified in the recombinant pike protein at intact N-glycopeptide level. Quantitative analysis of released glycan by LC-MS/MS was also performed, and 31 high-abundance N-glycans were identified. Sequencing analysis of glycan was further provided to assist glycan structure confirmation. Moreover, all of the analyses were performed on three consecutive manufactured batches and the glycosylation results on both glycosite and glycans showed good batch-to-batch consistency. Thus, the reported analytical strategy and N-glycosylation information may well facilitate studies on SARS-CoV-2 spike protein analysis and quality studies.

Variations within the Glycan Shield of SARS-CoV-2 Impact Viral Spike Dynamics.

The emergence of SARS-CoV-2 variants alters the efficacy of existing immunity, whether arisen naturally or through vaccination. Understanding the structure of the viral spike assists in determining the impact of mutations on the antigenic surface. One class of mutation impacts glycosylation attachment sites, which have the capacity to influence the antigenic structure beyond the immediate site of attachment. Here, we compare the site-specific glycosylation of recombinant viral spike mimetics of B.1.351 (Beta), P.1 (Gamma), B.1.617.2 (Delta), B.1.1.529 (Omicron). The P.1 strain exhibits two additional N-linked glycan sites compared to the other variants analyzed and we investigate the impact of these glycans by molecular dynamics. The acquired N188 site is shown to exhibit very limited glycan maturation, consistent with limited enzyme accessibility. Structural modeling and molecular dynamics reveal that N188 is located within a cavity by the receptor binding domain, which influences the dynamics of these attachment domains. These observations suggest a mechanism whereby mutations affecting viral glycosylation sites have a structural impact across the protein surface.

Inhibitory effects of fucoidan from Laminaria japonica against some pathogenic bacteria and SARS-CoV-2 depend on its large molecular weight.

Fucoidan is a highly sulfated polysaccharide with a wide range of bioactivities, including anti-pathogenic activity. However, the relationship between structure and activity of fucoidan in inhibiting pathogen infections remains unclear. Here, different-molecular-weight fucoidans were prepared by photocatalytic degradation followed by membrane ultrafiltration, and their chemical structures and anti-pathogenic microbiota activity were compared. Results showed that photocatalytic degradation could effectively degrade fucoidan while its structure block and sulfate groups were not destroyed obviously. Fucoidan (90.8 kDa) of 5 mg/mL could inhibit the growth of S. aureus, S. typhimurium and E. coli, but its degradation products, Dfuc1 (19.2 kDa) and Dfuc2 (5.5 kDa), demonstrated lower inhibitory effect. In addition, compared to Dfuc1 and Dfuc2, fucoidan showed stronger capability to prevent the adhesion of S. aureus, L. monocytogenes, V. parahaemolyticus and S. typhimurium to HT-29 cells. Moreover, the inhibitory effect against SARS-CoV-2 and the binding activity to S protein were also positively correlated to molecular weight. These results indicate that natural fucoidan with higher molecular weight are more effective to inhibit these pathogenic bacteria and SARS-CoV-2, providing a better understanding of the relationship between structure and activity of fucoidan against pathogenic microbiota.

Molecular modifications, biological activities, and applications of chitosan and derivatives: A recent update.

Polysaccharides arouse great interest due to their structure and unique properties, such as biocompatibility, biodegradability, and absence of toxicity. Polysaccharides from marine sources are particularly useful due to the wide variety of applications and biological activities. Chitosan, a deacetylated derivative of chitin, is an example of an interesting bioactive marine-derived polysaccharide. Moreover, a wide variety of chemical modifications and conjugation of chitosan with other bioactive molecules are responsible for improvements in physicochemical properties and biological activities, expanding the range of applications. An overview of the synthetic approaches for preparing chitosan, chitosan derivatives, and conjugates is described and discussed. A recent update of the biological activities and applications in different research fields, mainly focused on the last 5 years, is presented, highlighting current trends.

Antiviral Compounds Based on Natural Astragalus polysaccharides (APS): Research and Foresight in the Strategies for Combating SARS-CoV-2 (COVID-19).

Today, finding natural polymers with desirable properties for use in various industries is one of the critical axes of research in the world. Polysaccharides are a group of natural polymers that have various applications in the pharmaceutical industry. The attachment of monosaccharides forms polysaccharides through glycosidic bonds that are widely found in various sources, including plants. Genus Astragalus belongs to the Fabaceae family. Plants belonging to this genus have different polysaccharides. Astragalus polysaccharides (APS) have attracted a great deal of attention among natural polymers because they are non-toxic, biodegradable, and biocompatible. Currently, APS have great drug potential for curing or treating various diseases. Due to the different biological activities of polysaccharides, including Astragalus, this study has investigated the chemical structure of APS, reporting on the antiviral and anti-inflammatory activities as well as stimulation of cytokine secretion by these polysaccharides. Also, in this study, the pharmaceutical approaches of APS compounds, as a natural, new and inexpensive source, have been discussed as suitable candidates for use in pharmaceutical formulations and preparation of new drugs to control COVID-19 infection.

Structural and Biochemical Analysis Reveals Catalytic Mechanism of Fucoidan Lyase from Flavobacterium sp. SA-0082.

Fucoidans represent a type of polyanionic fucose-containing sulfated polysaccharides (FCSPs) that are cleaved by fucoidan-degrading enzymes, producing low-molecular-weight fucoidans with multiple biological activities suitable for pharmacological use. Most of the reported fucoidan-degrading enzymes are glycoside hydrolases, which have been well studied for their structures and catalytic mechanisms. Little is known, however, about the rarer fucoidan lyases, primarily due to the lack of structural information. FdlA from Flavobacterium sp. SA-0082 is an endo-type fucoidan-degrading enzyme that cleaves the sulfated fuco-glucuronomannan (SFGM) through a lytic mechanism. Here, we report nine crystal structures of the catalytic N-terminal domain of FdlA (FdlA-NTD), in both its wild type (WT) and mutant forms, at resolutions ranging from 1.30 to 2.25 Å. We show that the FdlA-NTD adopts a right-handed parallel ß-helix fold, and possesses a substrate binding site composed of a long groove and a unique alkaline pocket. Our structural, biochemical, and enzymological analyses strongly suggest that FdlA-NTD utilizes catalytic residues different from other ß-helix polysaccharide lyases, potentially representing a novel polysaccharide lyase family.

Structural- and Site-Specific N-Glycosylation Characterization of COVID-19 Virus Spike with StrucGP.

The spike (S) protein plays a key role in COVID-19 (SARS-CoV-2) infection and host-cell entry. Previous studies have systematically analyzed site-specific glycan compositions as well as many important structural motifs of the S protein. Here, we further provide structural-clear N-glycosylation of the S protein at a site-specific level by using our recently developed structural- and site-specific N-glycoproteomics sequencing algorithm, StrucGP. In addition to the common N-glycans as detected in previous studies, many uncommon glycosylation structures such as LacdiNAc structures, Lewis structures, Mannose 6-phosphate (M6P) residues, and bisected core structures were unambiguously mapped at a total of 20 glycosites in the S protein trimer and protomer. These data further support the glycosylation structural-functional investigations of the COVID-19 virus spike.

Investigation of the N-Glycosylation of the SARS-CoV-2 S Protein Contained in VLPs Produced in Nicotiana benthamiana.

The emergence of the SARS-CoV-2 coronavirus pandemic in China in late 2019 led to the fast development of efficient therapeutics. Of the major structural proteins encoded by the SARS-CoV-2 genome, the SPIKE (S) protein has attracted considerable research interest because of the central role it plays in virus entry into host cells. Therefore, to date, most immunization strategies aim at inducing neutralizing antibodies against the surface viral S protein. The SARS-CoV-2 S protein is heavily glycosylated with 22 predicted N-glycosylation consensus sites as well as numerous mucin-type O-glycosylation sites. As a consequence, O- and N-glycosylations of this viral protein have received particular attention. Glycans N-linked to the S protein are mainly exposed at the surface and form a shield-masking specific epitope to escape the virus antigenic recognition. In this work, the N-glycosylation status of the S protein within virus-like particles (VLPs) produced in Nicotiana benthamiana (N. benthamiana) was investigated using a glycoproteomic approach. We show that 20 among the 22 predicted N-glycosylation sites are dominated by complex plant N-glycans and one carries oligomannoses. This suggests that the SARS-CoV-2 S protein produced in N. benthamiana adopts an overall 3D structure similar to that of recombinant homologues produced in mammalian cells.

Serum N-glycomic profiling may provide potential signatures for surveillance of COVID-19.

Disease development and progression are often associated with aberrant glycosylation, indicating that changes in biological fluid glycome may potentially serve as disease signatures. The corona virus disease-2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) represents a significant threat to global human health. However, the effect of SARS-CoV-2 infection on the overall serum N-glycomic profile has been largely unexplored. Here, we extended our 96-well-plate-based high-throughput, high-sensitivity N-glycan profiling platform further with the aim of elucidating potential COVID-19-associated serum N-glycomic alterations. Use of this platform revealed both similarities and differences between the serum N-glycomic fingerprints of COVID-19 positive and control cohorts. Although there were no specific glycan peaks exclusively present or absent in COVID-19 positive cohort, this cohort showed significantly higher levels of glycans and variability. On the contrary, the overall N-glycomic profiles for healthy controls were well-contained within a narrow range. From the serum glycomic analysis, we were able to deduce changes in different glycan subclasses sharing certain structural features. Of significance was the hyperbranched and hypersialylated glycans and their derived glycan subclass traits. T-distributed stochastic neighbor embedding and hierarchical heatmap clustering analysis were performed to identify 13 serum glycomic variables that potentially distinguished the COVID-19 positive from healthy controls. Such serum N-glycomic changes described herein may indicate or correlate to the changes in serum glycoproteins upon COVID-19 infection. Furthermore, mapping the serum N-glycome following SARS-CoV-2 infection may help us better understand the disease and enable "Long-COVID" surveillance to capture the full spectrum of persistent symptoms.

Multifaceted Computational Modeling in Glycoscience.

Glycoscience assembles all the scientific disciplines involved in studying various molecules and macromolecules containing carbohydrates and complex glycans. Such an ensemble involves one of the most extensive sets of molecules in quantity and occurrence since they occur in all microorganisms and higher organisms. Once the compositions and sequences of these molecules are established, the determination of their three-dimensional structural and dynamical features is a step toward understanding the molecular basis underlying their properties and functions. The range of the relevant computational methods capable of addressing such issues is anchored by the specificity of stereoelectronic effects from quantum chemistry to mesoscale modeling throughout molecular dynamics and mechanics and coarse-grained and docking calculations. The Review leads the reader through the detailed presentations of the applications of computational modeling. The illustrations cover carbohydrate-carbohydrate interactions, glycolipids, and N- and O-linked glycans, emphasizing their role in SARS-CoV-2. The presentation continues with the structure of polysaccharides in solution and solid-state and lipopolysaccharides in membranes. The full range of protein-carbohydrate interactions is presented, as exemplified by carbohydrate-active enzymes, transporters, lectins, antibodies, and glycosaminoglycan binding proteins. A final section features a list of 150 tools and databases to help address the many issues of structural glycobioinformatics.

Pathogen-sugar interactions revealed by universal saturation transfer analysis.

Many pathogens exploit host cell-surface glycans. However, precise analyses of glycan ligands binding with heavily modified pathogen proteins can be confounded by overlapping sugar signals and/or compounded with known experimental constraints. Universal saturation transfer analysis (uSTA) builds on existing nuclear magnetic resonance spectroscopy to provide an automated workflow for quantitating protein-ligand interactions. uSTA reveals that early-pandemic, B-origin-lineage severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike trimer binds sialoside sugars in an "end-on" manner. uSTA-guided modeling and a high-resolution cryo-electron microscopy structure implicate the spike N-terminal domain (NTD) and confirm end-on binding. This finding rationalizes the effect of NTD mutations that abolish sugar binding in SARS-CoV-2 variants of concern. Together with genetic variance analyses in early pandemic patient cohorts, this binding implicates a sialylated polylactosamine motif found on tetraantennary N-linked glycoproteins deep in the human lung as potentially relevant to virulence and/or zoonosis.

Site-specific glycosylation of SARS-CoV-2: Big challenges in mass spectrometry analysis.

Glycosylation of viral proteins is required for the progeny formation and infectivity of virtually all viruses. It is increasingly clear that distinct glycans also play pivotal roles in the virus's ability to shield and evade the host's immune system. Recently, there has been a great advancement in structural identification and quantitation of viral glycosylation, especially spike proteins. Given the ongoing pandemic and the high demand for structure analysis of SARS-CoV-2 densely glycosylated spike protein, mass spectrometry methodologies have been employed to accurately determine glycosylation patterns. There are still many challenges in the determination of site-specific glycosylation of SARS-CoV-2 viral spike protein. This is compounded by some conflicting results regarding glycan site occupancy and glycan structural characterization. These are probably due to differences in the expression systems, form of expressed spike glycoprotein, MS methodologies, and analysis software. In this review, we recap the glycosylation of spike protein and compare among various studies. Also, we describe the most recent advancements in glycosylation analysis in greater detail and we explain some misinterpretation of previously observed data in recent publications. Our study provides a comprehensive view of the spike protein glycosylation and highlights the importance of consistent glycosylation determination.