The unusual tropism of H5N1 clade 2.3.4.4b for cattle mammary glands, causing necrotizing mastitis without major respiratory involvement, raises critical questions about its underlying mechanisms. We conducted glycomics and linkage-specific lectin histochemistry to characterize sialic acid (SA) receptor diversity and anatomical distribution, and virus binding assays and high-resolution electron microscopy (EM) to visualize virus-receptor interactions. Cattle mammary gland exhibited an abundance of N- and O-linked SA glycans, showing a stronger binding affinity to clade 2.3.4.4b H5 than clade 2.2 H5. In contrast, the cattle trachea contained only O-linked but not N-linked SAs and showed no detectable H5 binding, indicating limited compatible influenza A virus (IAV) receptor availability in the tracheal epithelium. EM of virus-bound tissues further validated the receptor basis of H5N1 infection in cattle. As H5N1 continues infecting unusual hosts, as evidenced by the first case in sheep, our study offers a methodological framework for assessing IAV susceptibility.

The H antigen (O blood group), the precursor to A and B blood groups, is expressed on human erythrocytes and other cells (e.g., endothelial cells). Remarkably, the specific expression of the H antigens is uncertain due to the lack of specific reagents for its detection. Here, we describe two monoclonal antibodies (mAbs), Tn4-31L and OmcFL3-02, generated by immunizing sea lampreys with human cells. Both mAbs are highly specific in comparison with the lectin Ulex europaeus agglutinin-I (UEA-I). We identified expression of H antigens in mammalian glycoproteins, human cells, and normal and malignant human tissues, discovering that different types of human carcinomas exhibit unexpected H antigen expression. Interestingly, H antigen detection in many cases was distinctly elevated by enzymatic desialylation, as well as being elevated in cells engineered to lack sialic acid. These highly specific mAbs will be valuable reagents in blood testing and for exploring the expression and function of H antigens.

The binding of the SARS-CoV-2 spike (S) glycoprotein to human host receptors, including ACE2, NRP1, and AXL, is essential for viral entry. Glycosylation of both the spike protein and its host receptors can significantly influence these interactions. While NRP1 is recognized as a key host receptor, the role of its glycosylation in spike binding and viral infectivity has not been fully elucidated. In this study, we developed a spike-NRP1 binding assay using recombinant proteins in HEK293 cells and performed LC-MS-based glycoproteomic analysis to characterize NRP1 glycosylation. We identified three N-linked glycosylation sites (N150, N261, N522) and four O-linked glycosylation sites (S612, S637, T638, S641) on NRP1. Mutational analysis revealed that glycosylation at these specific sites is critical for spike binding, as single-site mutants showed significantly reduced interaction. Further characterization of terminal sialic acid linkages, bisecting GlcNAc, and fucosylation patterns provided insight into the complexity of NRP1 glycosylation. Functional assays demonstrated that loss of glycosylation impaired SARS-CoV-2 pseudovirus entry and altered IL-6 secretion, indicating a broader role in host immune modulation. These findings demonstrate that site-specific NRP1 glycosylation modulates spike binding and viral entry in a cell-based model, providing a foundation for future studies to explore the potential of targeting glycosylation-dependent mechanisms in SARS-CoV-2 infection.

Glycoproteomics is a rapidly developing field, and data analysis has been stimulated by several technological innovations. As a result, there are many software tools from which to choose; and each comes with unique features that can be difficult to compare. This work presents a head-to-head comparison of five modern analytical software: Byonic, Protein Prospector, MSFraggerGlyco, pGlyco3, and GlycoDecipher. To enable a meaningful comparison, parameter variables were minimized. One potential confounding variable is the glycan database that informs glycoproteomic searches. We performed glycomic profiling of the samples and used the output to construct matched glycan databases for each software. Up to 17,000 glycopeptide spectra were identified across three replicates of wild-type SH-SY5Y cells. There was overlap among all software for glycoproteins identified, locations of glycosites, and glycans; but there was no clear winner. Incorporation of several comparative criteria was critically important for learning the most information in this study and should be used more broadly when assessing software. A single criterion, such as number of glycopeptide spectra found, is not sufficient. We present evidence that suggests Byonic reports many spurious results at the glycoprotein and glycosite level. Overall, our results indicate that glycoproteomic searches should involve more than one software, excluding the current version of Byonic, to generate confidence by consensus. It may be useful to consider software with peptide-first approaches and with glycan-first approaches.

Specific recognition of glycans by proteins is important in many biological processes and immune responses. Here we present a general approach for derivatizing free glycans with a novel linker MTZ (3-(methoxyamino)-propylamine added to a bioorthogonal-functional tetrazine tag) that exploits click chemistry to generate multiple platforms of glycan coupling. This derivatization preserves glycan integrity, is reversible and quantifiable, and incorporates a bioorthogonal tetrazine tag for click coupling. A library of ABO-(H) blood group MTZ-glycans was efficiently conjugated to avidin Luminex beads through a Biotin-PEG11-TCO (trans-cyclooctene) spacer, generating a multiplex array that was reproducibly interrogated in a high-throughput Luminex approach with multiple lectins and antibodies. We also rapidly profiled antiglycan IgG, IgM, and IgA antibodies in multiple, serially diluted human serum samples, revealing unique repertoires of antiglycan responses in each sera. Glycans were efficiently coupled to bovine serum albumin (BSA) at a high density (∼19-24 glycans/BSA) to generate a neoglycoprotein library that was useful in microarray formats that provided results equivalent to those obtained from the Luminex approach. Neoglycoproteins have many uses, including serving as acceptors for glycosyltransferases, as we demonstrate for assays of ST6Gal1 sialyltransferase. These facile and efficient technologies significantly expand the toolbox available to explore glycan-GBP interactions.

Many pathogenic bacteria form biofilms as a protective measure against environmental and host hazards. The underlying structure of the biofilm matrix consists of secreted macromolecules, often including exopolysaccharides. To escape the biofilm, bacteria may produce a number of matrix-degrading enzymes, including glycosidic enzymes that digest exopolysaccharide scaffolds. The human pathogen Vibrio cholerae assembles and secretes an exopolysaccharide called VPS (Vibrio polysaccharide) which is essential in most cases for the formation of biofilms and consists of a repeating tetrasaccharide unit. Previous studies have indicated that a secreted glycosidase called RbmB is involved in V. cholerae biofilm dispersal, although the mechanism by which this occurs is not understood. To approach the question of RbmB function, we recombinantly expressed and purified RbmB and tested its activity against purified VPS. Using a fluorescence-based biochemical assay, we show that RbmB specifically cleaves VPS in vitro under physiological conditions. Analysis of the cleavage process using mass spectrometry, solid-state NMR, and solution NMR indicates that RbmB cleaves VPS at a specific site (at the α-1,4 linkage between D-galactose and a modified L-gulose) into a mixture of tetramers and octamers. We demonstrate that the product of the cleavage contains a double bond in the modified guluronic acid ring, strongly suggesting that RbmB is cleaving using a glycoside lyase mechanism. Finally, we show that recombinant RbmB from V. cholerae and the related aquatic species Vibrio coralliilyticus are both able to disrupt living V. cholerae biofilms. Our results support the role of RbmB as a polysaccharide lyase involved in biofilm dispersal, as well as an additional glycolytic enzyme to add to the toolbox of potential therapeutic antibacterial enzymes.

Aberrant protein glycosylation is a hallmark alteration of cancer and is highly associated with cancer progression. Papillary thyroid cancer (PTC) is the most common type of thyroid cancer, but the N-glycosylation of its glycoproteins has not been well characterized. In this work, we analyzed multiple freshly prepared PTC specimens along with paired normal tissue obtained from thyroidectomies. Glycomic analyses focused on Asn-linked (N)-glycans and employed mass spectrometry (MS), along with Western blot approaches of total solubilized materials that were examined for binding by specific lectins and a monoclonal antibody (mAb) O6, specific for 3-O-sulfated galactose residues. We observed major differences in PTC versus paired normal specimens, as PTC specimens exhibited higher levels of N-glycan branching and bisection with N-acetylglucosamine residues, consistent with RNAseq data. We also found that 3-O-sulfated galactose was present in N-glycans of multiple glycoproteins from both PTC and control specimens, as recognized by the O6 mAb and as confirmed by MS analyses. These results provide new insights into the N-glycans present in glycoproteins of thyroid cancer and context for further studies of these altered glycans as biomarkers and targets for therapeutics.

The coronavirus disease 2019 (COVID-19) pandemic is an ongoing global public health crisis. The causative agent, the SARS-CoV-2 virus, enters host cells via molecular interactions between the viral spike protein and the host cell ACE2 surface protein. The SARS-CoV-2 spike protein is extensively decorated with up to 66 N-linked glycans. Glycosylation of viral proteins is known to function in immune evasion strategies but may also function in the molecular events of viral entry into host cells. Here, we show that N-glycosylation at Asn331 and Asn343 of SARS-CoV-2 spike protein is required for it to bind to ACE2 and for the entry of pseudovirus harboring the SARS-CoV-2 spike protein into cells. Interestingly, high-content glycan binding screening data have shown that N-glycosylation of Asn331 and Asn343 of the RBD is important for binding to the specific glycan molecule G4GN (Gal beta-1,4 GlcNAc), which is critical for spike-RBD-ACE2 binding. Furthermore, IL-6 was identified through antibody array analysis of conditioned media of the corresponding pseudovirus assay. Mutation of N-glycosylation of Asn331 and Asn343 sites of the spike receptor-binding domain (RBD) significantly reduced the transcriptional upregulation of pro-inflammatory signaling molecule IL-6. In addition, IL-6 levels correlated with spike protein levels in COVID-19 patients' serum. These findings establish the importance of RBD glycosylation in SARS-CoV-2 pathogenesis, which can be exploited for the development of novel therapeutics for COVID-19.

At the end of 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a novel human coronavirus, emerged and rapidly caused a global pandemic. SARS-CoV-2 is the causative agent of coronavirus disease 2019 (COVID-19), which affects the respiratory tract and lungs of infected individuals. Due to the increased transmissibility of the SARS-CoV-2 virus compared to its previous versions, determining as fully as possible the various structural aspects of the virus became critical for the development of therapeutics and vaccines to combat this virus. Knowing the structures of viral proteins and their glycosylation is an essential foundation for the understanding of the mechanism of the disease. Glycopeptide analysis has been used to map the glycosylation of viral glycoproteins, including those of influenza and HIV. Thanks to the developments in the field over the last few decades, scientists were able to quickly develop therapeutics against SARS-CoV-2. This chapter discusses the four structural proteins of SARS-CoV-2, their glycosylation and modifications, and the techniques used to map SARS-CoV-2 glycosylation.

To capture prey, onychophorans (velvet worms) expel a slime that forms stiff fibers upon shearing and dehydration. The high quantities of phosphorus previously found in the slime of the velvet worm Euperipatoides rowelli were ascribed to protein phosphorylation. We provide clear evidence, instead, that it is primarily present as phosphonate moieties in the slime of representative from both major onychophoran subgroups which diverged ~380 MYA. Advanced NMR and mass spectrometry demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with high molecular weight slime proteins as phosphonoglycans. Biogenic phosphonates are a substantial component of the organophosphorus cycle in marine environments but were not previously reported in terrestrial invertebrates. The evolutionary conservation of this rare protein modification suggests a potential role in the formation and function of these biological adhesive fibers with implications for bio-inspiration.

The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both the N-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved both N- and O- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.

Enveloped viruses co-opt host glycosylation pathways to decorate their surface proteins. As viruses evolve, emerging strains can modify their glycosylation patterns to influence host interactions and subvert immune recognition. Still, changes in viral glycosylation or their impact on antibody protection cannot be predicted from genomic sequences alone. Using the highly glycosylated SARS-CoV-2 Spike protein as a model system, we present a lectin fingerprinting method that rapidly reports on changes in variant glycosylation state, which are linked to antibody neutralization. In the presence of antibodies or convalescent and vaccinated patient sera, unique lectin fingerprints emerge that distinguish neutralizing versus non-neutralizing antibodies. This information could not be inferred from direct binding interactions between antibodies and the Spike receptor-binding domain (RBD) binding data alone. Comparative glycoproteomics of the Spike RBD of wild-type (Wuhan-Hu-1) and Delta (B.1.617.2) variants reveal O-glycosylation differences as a key determinant of immune recognition differences. These data underscore the interplay between viral glycosylation and immune recognition and reveal lectin fingerprinting to be a rapid, sensitive, and high-throughput assay to distinguish the neutralization potential of antibodies that target critical viral glycoproteins.

Nature is rich with examples of highly specialized biological materials produced by organisms for functions, including defense, hunting, and protection. Along these lines, velvet worms (Onychophora) expel a protein-based slime used for hunting and defense that upon shearing and dehydration forms fibers as stiff as thermoplastics. These fibers can dissolve back into their precursor proteins in water, after which they can be drawn into new fibers, providing biological inspiration to design recyclable materials. Elevated phosphorus content in velvet worm slime was previously observed and putatively ascribed to protein phosphorylation. Here, we show instead that phosphorus is primarily present as phosphonate moieties in the slime of distantly related velvet worm species. Using high-resolution nuclear magnetic resonance (NMR), natural abundance dynamic nuclear polarization (DNP), and mass spectrometry (MS), we demonstrate that 2-aminoethyl phosphonate (2-AEP) is associated with glycans linked to large slime proteins, while transcriptomic analyses confirm the expression of 2-AEP synthesizing enzymes in slime glands. The evolutionary conservation of this rare protein modification suggests an essential functional role of phosphonates in velvet worm slime and should stimulate further study of the function of this unusual chemical modification in nature.

Influenza A viruses (IAV) utilize sialic acid (Sia) containing cell surface glycoconjugates for host cell infection, and IAV strains from different host species show preferences for structurally distinct Sia at the termini of glycoconjugates. Various types of cell surface glycoconjugates (N-glycans, O-glycans, glycolipids) display significant diversity in both structure and carbohydrate composition. To define the types of sialylglycoconjugates that facilitate IAV infection, we utilized the CRISPR/Cas9 technique to truncate the three major types of glycoconjugates, either individually or in combination, by targeting glycosyltransferases essential to glycan biosynthesis in a human lung epithelial cell line. Our studies show that both human and avian IAV strains do not display strict preferences for a specific type of glycoconjugate. Interestingly, truncation of the three major types of glycoconjugates significantly decreased replication of human IAV strains, yet did not impact replication of avian IAV strains. Unlike human IAV strains, avian IAV strains were able to efficiently utilize other less prevalent shorter glycoconjugates such as sialyl Tn and sialyl T antigens. Taken together, our studies demonstrate that avian IAV strains utilize a broader repertoire of glycoconjugates for host cell infection as compared to human IAV strains. IMPORTANCE It is well known that influenza A viruses (IAV) initiate host cell infection by binding to sialic acid, a sugar molecule present at the ends of various sugar chains called glycoconjugates. These sugar chains can vary in chain length, structure, and composition. However, it remains unknown if IAV strains preferentially bind to sialic acid on specific glycoconjugate type(s) for host cell infection. Here, we utilized CRISPR gene editing to abolish sialic acid on different glycoconjugate types in human lung cells, and evaluated human versus avian IAV infections. Our studies show that both human and avian IAV strains can infect human lung cells by utilizing any of the three major sialic acid-containing glycoconjugate types, specifically N-glycans, O-glycans, and glycolipids. Interestingly, simultaneous elimination of sialic acid on all three major glycoconjugate types in human lung cells dramatically decreased human IAV infection, yet had little effect on avian IAV infection. These studies show that avian IAV strains effectively utilize other less prevalent glycoconjugates for infection, whereas human IAV strains rely on a limited repertoire of glycoconjugate types. The remarkable ability of avian IAV strains to utilize diverse glycoconjugate types may allow for easy transmission into new host species.

Every animal secretes mucus, placing them among the most diverse biological materials. Mucus hydrogels are complex mixtures of water, ions, carbohydrates, and proteins. Uncertainty surrounding their composition and how interactions between components contribute to mucus function complicates efforts to exploit their properties. There is substantial interest in commercializing mucus from the garden snail, Cornu aspersum , for skincare, drug delivery, tissue engineering, and composite materials. C. aspersum secretes three mucus—one shielding the animal from environmental threats, one adhesive mucus from the pedal surface of the foot, and another pedal mucus that is lubricating. It remains a mystery how compositional differences account for their substantially different properties. Here, we characterize mucus proteins, glycosylation, ion content, and mechanical properties that could be used to provide insight into structure-function relationships through an integrative “mucomics” approach. We identify macromolecular components of these hydrogels, including a previously unreported protein class termed Conserved Anterior Mollusk Proteins (CAMPs). Revealing differences between C. aspersum mucus shows how considering structure at all levels can inform the design of mucus-inspired materials.

Glycosidic linkages in oligosaccharides play essential roles in determining their chemical properties and biological activities. MSn has been widely used to infer glycosidic linkages but requires a substantial amount of starting material, which limits its application. In addition, there is a lack of rigorous research on what MSn protocols are proper for characterizing glycosidic linkages. In this work, to deliver high-quality experimental data and analysis results, we propose a machine learning-based framework to establish appropriate MSn protocols and build effective data analysis methods. We demonstrate the proof-of-principle by applying our approach to elucidate sialic acid linkages (α2'-3' and α2'-6') in a set of sialyllactose standards and NIST sialic acid-containing N-glycans as well as identify several protocol configurations for producing high-quality experimental data. Our companion data analysis method achieves nearly 100% accuracy in classifying α2'-3' vs α2'-6' using MS5, MS4, MS3, or even MS2 spectra alone. The ability to determine glycosidic linkages using MS2 or MS3 is significant as it requires substantially less sample, enabling linkage analysis for quantity-limited natural glycans and synthesized materials, as well as shortens the overall experimental time. MS2 is also more amenable than MS3/4/5 to automation when coupled to direct infusion or LC-MS. Additionally, our method can predict the ratio of α2'-3' and α2'-6' in a mixture with 8.6% RMSE (root-mean-square error) across data sets using MS5 spectra. We anticipate that our framework will be generally applicable to analysis of other glycosidic linkages.

Root-knot nematodes (RKN) (Meloidogyne spp.) represent one of the most damaging groups of plant-parasitic nematodes. They secrete effector proteins through a protrusible stylet to manipulate host cells for their benefit. Stylet-secreted effector proteins are produced within specialized secretory esophageal gland cells, one dorsal gland (DG) and two subventral glands (SvG), whose activity differ throughout the nematode life cycle. Previous gland transcriptomic profiling studies identified dozens of candidate RKN effectors but were focused on the juvenile stages of the nematode, when the SvGs are most active. We developed a new approach to enrich for the active DGs of M. incognita adult female RKN for RNA and protein extraction. Female heads were manually cut from the body, and a combination of sonication and vortexing was used to dislodge contents inside the heads. DG-enriched fractions were collected by filtering, using cell strainers. Comparative transcriptome profiling of pre-parasitic second-stage juveniles, female heads, and DG-enriched samples was conducted using RNA sequencing. Application of an established effector mining pipeline led to the identification of 83 candidate effector genes upregulated in DG-enriched samples of adult females that code for proteins with a predicted signal peptide but lack transmembrane domains or homology to proteins in the free-living nematode Caenorhabditis elegans. In situ hybridization resulted in the identification of 14 new DG-specific candidate effectors expressed in adult females. Taken together, we have identified novel candidate Meloidogyne effector genes that may have essential roles during later stages of parasitism. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.