Glycobiology

Human lectins are critical carbohydrate-binding proteins that recognize diverse glycoconjugates from microorganisms and can play a key role in host-microbe interactions. Despite their importance in immune recognition and pathogen binding, the specific glycan ligands and functions of many human lectins remain poorly understood. Using previous proof-of-concept studies on selected lectins as the foundation for this work, we present ten additional glycan analysis probes (GAPs) from a diverse set of human soluble lectins, offering robust tools to investigate glycan-mediated interactions. We describe a protein engineering platform that enables scalable production of GAPs that maintain native-like conformations and oligomerization states, equipped with functional reporter tags for targeted glycan profiling. We demonstrate that the soluble GAP reagents can be used in various applications, including glycan array analysis, mucin- binding assays, tissue staining, and microbe binding in complex populations. These capabilities make GAPs valuable for dissecting interactions relevant to understanding host responses to microbes. The tools can be used to distinguish microbial from mammalian glycans, which is crucial for understanding the cross-target interactions of lectins in a physiological environment where both glycan types exist. GAPs have potential as diagnostic and prognostic tools for detecting glycan alterations in chronic diseases, microbial dysbiosis, and immune-related conditions.

Glycosylation is the most common post-translational modification and has myriad biological functions. However, glycan analysis and research has always been a challenge. Here, we would like to present new techniques of glycan fingerprinting based on enzymatic fluorescent labeling and gel electrophoresis. The method is illustrated on SARS-2 spike (S) glycoproteins. SARS-2, a novel coronavirus and the causative agent of COVID-19 pandemic, has devastated the world since the end of 2019. To obtain the N-glycan fingerprint of a S protein, glycans released from the protein are first labeled through enzymatic incorporation of fluorophore-conjugated sialic acid or fucose, and then separated on acrylamide gel through electrophoresis, and finally visualized with a fluorescent imager. To identify the labeled glycans of a fingerprint, glycan standards and glycan ladders that are enzymatically generated are run alongside the samples as references. By comparing the mobility of a labeled glycan to that of a glycan standard, the identity of glycans maybe determined. Due to lack of enzyme for broad O-glycans releasing, O-glycans on the RBD protein are labeled with fluorescent sialic acid and digested with trypsin to obtain labeled glycan peptides that are then separated on gel. Glycan fingerprinting could serve as a quick way for global assessment of the glycosylation of a glycoprotein.

Melanoma, the most dangerous form of skin cancer, is characterized by its high potential to spread or metastasize. Tumor progression is associated with changes in glycosylation that occur early in carcinogenesis and evolve as the cancer develops and spreads. To identify new melanoma transformation markers, we analyzed N-glycans from melanocytes and melanoma cell lines at different progression stages. We performed analyses using matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry (MS) and hydrophilic interaction liquid chromatography (HILIC)-high performance liquid chromatography (HPLC) techniques on N-glycans without and after digestion with exoglycosidase arrays. Our results showed that, unlike melanocytes, melanoma cells express higher levels of type 1 LacNAc units and triantennary complex type glycans instead of high-mannose type glycans. Our results revealed that the N-glycomes of all analyzed cell lines possess Lewis X/A epitopes and, for the first time, demonstrate the presence of Gal{beta}1-4Gal{beta}1-4GlcNAc and Gal{beta}1-3Gal{beta}1-3GlcNAc units. The characteristic feature of melanoma cells is the presence of LacdiNAc structures. Our study provides the first comprehensive characterization of the N-glycome of melanocytes and suggests novel glyco-biomarkers of melanoma progression.

The O-GlcNAc modification of Notch receptors regulates Notch ligand interactions in a manner distinct from other forms of O-glycans on epidermal growth factor-like (EGF) repeats of Notch receptors. Although many proteins, besides Notch receptors, are expected to be O-GlcNAcylated by EGF domain-specific O-GlcNAc transferase (EOGT), only a small number of proteins have been reported to be modified in vivo, and elongated O-GlcNAc glycans have not been extensively explored. To extend our view of the specificity and variety of the glycan modification, we conducted a comprehensive analysis of O-GlcNAc glycans on NOTCH1 in mammals. Mass spectrometric analysis of NOTCH1 fragments expressed in HEK293T cells revealed that several EGF domains with putative O-GlcNAcylation sites were hardly modified with O-GlcNAc. Although amino acid residues before the modification site are preferentially occupied with aromatic residues, Phe and Tyr are preferable to Trp for the apparent modification with O-GlcNAc. Furthermore, a minor form of fucosylated O-GlcNAc glycans was detected in a subset of EGF domains. Fucosylation of O-GlcNAc glycans was enhanced by FUT1, FUT2, or FUT9 expression. The FUT9-dependent Lewis X epitope was confirmed by immunoblotting using an anti-Lewis X antibody. As expected from the similarity in the glycan structures, the Lexis X antigen was detected on O-fucose glycans. Our results refined the putative consensus sequence for the EOGT-dependent extracellular O-GlcNAc modification in mammals and revealed the structural diversity of functional Notch O-glycans.

IntroductionMultiple previous studies have shown the monoclonal antibody Das-1 (formerly called 7E12H12) specifically recognizes metaplastic and carcinomatous lesions in multiple organs of the gastrointestinal system (e.g. Barretts esophagus, intestinal-type metaplasia of the stomach, gastric adenocarcinoma, high-grade pancreatic intraepithelial neoplasm, and pancreatic ductal adenocarcinoma) as well as in other organs (bladder and lung carcinomas). Beyond being a useful biomarker in tissue, mAb Das-1 has recently proven to be more accurate than current paradigms for identifying cysts harboring advanced neoplasia. Though this antibody has been used extensively for clinical, basic science, and translational applications for decades, its epitope has remained elusive. MethodsIn this study, we chemically deglycosylated a standard source of antigen, which resulted in near complete loss of the signal as measured by western blot analysis. The epitope recognized by mAb Das-1 was determined by affinity to a comprehensive glycan array and validated by inhibition of a direct ELISA. ResultsThe epitope recognized by mAb Das-1 is 3-Sulfo-Lewis A (3-Sulfo-LeA). 3-Sulfo-LeA is broadly reexpressed across numerous GI epithelia and elsewhere only after metaplastic and carcinomatous transformation. Discussion3-Sulfo-LeA is a clinically important antigen that can be detected both intracellularly in tissue using immunohistochemistry and extracellularly in cyst fluid and serum by ELISA. The results open new avenues for tumorigenic risk stratification of various gastrointestinal lesions.

Monoclonal antibody (mAb) glycosylation is a critical quality attribute that is difficult to rationally engineer and rapidly assess during cell line development. Here, we investigate whether cell-surface glycosylation can serve as a predictive indicator of mAb product glycosylation following targeted glycogene engineering in CHO cells. Five key glycogenes (COSMC, FUT8, B4GALT1, ST3GAL4, ST6GAL1) were investigated in two mAb-producing CHO cell lines. Product glycan analysis revealed consistent, gene-specific effects across hosts, including loss of core fucosylation, and tuneable galactosylation and sialylation. Lectin-based surface profiling reliably reflected product outcomes for COSMC and FUT8 modifications but showed limited predictive power for galactosylation and 2,3-sialylation, highlighting glycosylation pathway redundancy and context dependence. This study provides the first systematic, cross-cell line evaluation of lectin-based cell-surface glycan profiling as a predictor of mAb product glycosylation, establishing its practical utility and inherent limitations for CHO glycoengineering workflows. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=118 SRC="FIGDIR/small/724788v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@6d5cfborg.highwire.dtl.DTLVardef@1f38e0aorg.highwire.dtl.DTLVardef@f25fa2org.highwire.dtl.DTLVardef@64a0dc_HPS_FORMAT_FIGEXP M_FIG C_FIG

The N-glycome profiles purified from dietary bovine whey, egg white, pea, soy protein isolates and a recently commercialized animal-free whey is described. Purified glycoproteins resulting from centrifugation and ethanol precipitation of protein powder supplements were treated with peptide-N- glycosidase F (PNGase F) to release protein-bound N-glycans. Once released from the protein, N-glycans were labeled by procainamide labeling, purified via cotton-hydrophilic interaction liquid chromatography (HILIC), and analyzed using HILIC high performance liquid chromatography equipped with a fluorescence detector and a quadrupole time-of-flight tandem mass spectrometry (HILIC-FLD-QTOF-MS/MS). A total of 33, 33, 10, and 10 N-glycan structures were identified from bovine whey, egg, soy, and pea glycoprotein isolates, respectively. The type of N-glycans per glycoprotein source were highly predictable, likely attributed to differences in biosynthetic glycosylation pathways. Mammalian glycoprotein sources favored a combination of complex and hybrid glycan configurations while the plant proteins were dominated by oligomannosidic N-glycans. Bovine whey glycoprotein isolate contained the most diverse N-glycans by monosaccharide composition as well as structure, while plant sources such as pea and soy glycoprotein isolates contained an overlap of oligomannosidic N-glycans. The results suggest N-glycan structure and composition is dependent on the host organism rather than protein sequence homology, likely driven by the differences in N-glycan biosynthetic pathways.

A central tenet in the design of vaccines is the display of native-like antigens in the elicitation of protective immunity. The abundance of N-linked glycans across the SARS-CoV-2 spike protein is a potential source of heterogeneity between the many different vaccine candidates under investigation. Here, we investigate the glycosylation of recombinant SARS-CoV-2 spike proteins from five different laboratories and compare them against infectious virus S protein. We find patterns which are conserved across all samples and this can be associated with site-specific stalling of glycan maturation which act as a highly sensitive reporter of protein structure. Molecular dynamics (MD) simulations of a fully glycosylated spike support s a model of steric restrictions that shape enzymatic processing of the glycans. These results suggest that recombinant spike-based SARS-CoV-2 immunogen glycosylation reproducibly recapitulates signatures of viral glycosylation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein cytokine with therapeutic potential in cancer and neutropenia treatment. While glycosylation of GM-CSF reduces immunogenicity and enhances serum bioavailability, it can also diminish receptor binding and bioactivity. Based on transcriptomic analysis of human T lymphocytes reported previously, GM-CSF-producing cells exhibit elevated expression of Alpha-1,6-Mannosylglycoprotein 6-Beta-N-Acetylglucosaminyltransferase (MGAT5), which encodes N-acetylglucosaminyltransferase V, an enzyme involved in N-glycan branching. Given this role of MGAT5 in glycosylation, we produced GM-CSF variants using glycoengineered Chinese hamster ovary cells to generate diverse glycoforms and assessed their bioactivity. Testing their activity on TF-1 cell proliferation, we found that decreases in GM-CSF N-glycan branching significantly suppressed its activity. These findings underscore the importance of glycosylation in modulating the efficacy and safety of GM-CSF-based therapeutics, suggesting that precise glycoengineering may be key to optimizing GM-CSF performance in clinical applications.

Human parainfluenza virus type 3 (HPIV-3) remains a major cause of respiratory illness particularly among young children, the elderly and immunocompromised individuals. Despite significant efforts in therapeutic discovery research, there is neither an effective antiviral nor a vaccine available against HPIV-3. Host cell glycosylation is known to play a pivotal role in virus entry and replication. While some host glycan-based cellular receptors for HPIV-3 have been identified, the dynamics of the host glycome upon HPIV-3 infection has never been studied. Herein, we report the first mass spectrometry-based study that provides direct insight into the remodelling of the human lung adenocarcinoma cell (A549) glycome upon HPIV-3 infection. In this study we observed that HPIV-3 infection led to significant host-cell glycome changes in both oligomannose and sialylated complex-type N-glycans. Moreover, notable changes were also observed in both core 1 and core 2 type O-glycans, along with distinct glycosphingolipid remodelling in infected cells compared to their mock-infected counterparts. Our study presents the first evidence that hPIV-3 infection alters host-cell glycome, offering new insights into the viruss impact on host cellular processes.

Bacterial glycoconjugates are structurally diverse, with enormous variation in sugar identity, modifications and linkages. Glycoconjugates play key roles in numerous cell functions, acting as the primary interface with the environment. Asparagine (N)-linked glycosylation has been extensively studied in the pathogenic Campylobacter genus, due to the availability of numerous genome sequences and the highly conserved pathway logic, despite the final N-linked glycan product diversity. We recently reported on a partitioning of N-linked glycan structures between the Campylobacter species, focused on the inclusion of a C6-carboxyl-sugar in the third position of the growing glycan in Campylobacter concisus. However, at the time, the final glycan was not fully defined. Here, we identify the final glycan product in C. concisus, demonstrating surprising substrate promiscuity of the GT-A enzyme, PglI, which adds the penultimate sugar, and uncover a previously uncharacterized enzyme (GT-25) that unexpectedly adds the final sugar to complete the heptasaccharide product. Through a detailed study of these two C. concisus pathway enzymes, the intermediate and final glycans were defined, with determination of linkage positions of major and minor isomeric products following each glycan addition, through high-resolution electronic excitation dissociation tandem mass spectrometry. These findings on the Group II C. concisus N-linked glycan highlight the diversification of the glycan and the utilization, at the non-reducing end, of GlcNAc over GalNAc, which is dominant in the Group I species.

LipoPolySaccharides are a hallmark of Gram-negative bacteria and their presence at the cell surface is key for bacterial integrity. As surface exposed components, they are recognized by immunity C-type lectin receptors present on Antigen Presenting Cells. Human Macrophage Galactose Lectin binds E. coli surface that presents a specific glycan motif. Nevertheless, this high affinity interaction occurs regardless of the integrity of its canonical calcium-dependent glycan binding site. Nuclear Magnetic Resonance of MGL carbohydrate recognition domain and complete extracellular domain revealed a new glycan binding site opposite to the canonical site. A model of trimeric Macrophage Galactose Lectin was determined based on a combination of Small Angle X-ray scattering and Alphafold. A disulphide bond positions the Carbohydrate Recognition Domain perpendicular to the coiled-coil domain. This unique configuration for a C-type lectin orients the six glycan sites of MGL in an ideal position to bind LipoPolySaccharides at the bacterial surface with high avidity.

An increasing number of clinical applications employ oligosaccharides as tags to direct therapeutic proteins and RNA molecules to specific target cells. Current applications are focused on endocytic receptors that result in cellular uptake, but additional applications of sugar-based targeting in signaling and protein degradation are emerging. These approaches all require development of ligands that bind selectively to specific sugar-binding receptors, known as lectins. In the work reported here, a human lectin array has been employed as a predictor of targeting specificity of different oligosaccharide ligands and as a rapid in vitro screen to identify candidate targeting ligands. The approach has been validated with existing targeting ligands, such as a GalNAc cluster ligand that targets siRNA molecules to hepatocytes through the asialoglycoprotein receptor. Additional small oligosaccharides that can selectively target other classes of cells have also been identified and the potential of larger glycans derived from glycoproteins has been investigated. In initial screens, ligands for targeting either vascular or sinusoidal endothelial cells and plasmacytoid dendritic cells have been identified. Lectin array screening has also been used to characterize the specificity of glycolipid-containing liposomes that are used as carriers for targeted delivery. The availability of a rapid in vitro screening approach to characterizing natural oligosaccharides and glycomimetic compounds has the potential to facilitate selection of appropriate targeting tags before undertaking more complex in vivo studies.

The current SARS-CoV-2 pandemic has become one of the most challenging global health threats, with over 530 million reported infections by May 2022. In addition to vaccines, research and development have also been directed towards novel drugs. Since the highly glycosylated spike protein of SARS-CoV-2 is essential for infection, it constitutes a prime target for antiviral agents. The pineapple-derived jacalin-related lectin (AcmJRL) is present in the medication bromelain in significant quantities and has previously been described to bind mannosides. Here, we elucidated its ligand specificity by glycan array analysis, quantified the interaction with carbohydrates and validated high-mannose glycans as preferred ligands. Because the SARS-CoV-2 spike protein was previously reported to carry a high proportion of high-mannose N-glycans, we tested the binding of AcmJRL to recombinantly produced spike protein. We could demonstrate that AcmJRL binds the spike protein with a low micromolar KD in a carbohydrate-dependent fashion, suggesting its use as a potential SARS-CoV-2 neutralising agent.

Alpha-1-antitrypsin (A1AT) is a multifunctional, clinically important, high value therapeutic glycoprotein that can be used for the treatment of many diseases such as alpha-1-antitrypsin deficiency, diabetes, graft-versus-host-disease, cystic fibrosis and various viral infections. Currently, the only FDA-approved treatment for A1AT disorders is intravenous augmentation therapy with human plasma-derived A1AT. In addition to its limited supply, this approach poses a risk of infection transmission, since it uses therapeutic A1AT harvested from donors. To address these issues, we sought to generate recombinant human A1AT (rhA1AT) that is chemically and biologically indistinguishable from its plasma-derived counterpart using glycoengineered Chinese Hamster Ovary (geCHO-L) cells. By deleting nine key genes that are part of the CHO glycosylation machinery and expressing the human ST6GAL1 and A1AT genes, we obtained stable, high producing geCHO-L lines that produced rhA1AT having an identical glycoprofile to plasma-derived A1AT (pdA1AT). Additionally, the rhA1AT demonstrated in vitro activity and in vivo half-life comparable to commercial pdA1AT. Thus, we anticipate that this platform will help produce human-like recombinant plasma proteins, thereby providing a more sustainable and reliable source of therapeutics that are cost-effective and better-controlled with regard to purity, clinical safety and quality.

N-glycoproteomic analyses provide valuable resources for investigation of cancer mechanisms, biomarkers, and therapeutic targets. Here, we mapped and compared the site-specific N-glycoproteomes of colon cancer HCT116 cells and isogenic non-tumorigenic DNMT1/3b double knockout (DKO1) cells using Fbs1-GYR N-glycopeptide enrichment technology and trapped ion mobility spectrometry. Many significant changes in site-specific N-glycosylation were revealed, providing a molecular basis for further elucidation of the role of N-glycosylation in protein function. HCT116 cells display hypersialylation especially in cell surface membrane proteins. Both HCT116 and DKO1 show an abundance of paucimannose and 80% of paucimannose-rich proteins are annotated to reside in exosomes. The most striking N-glycosylation alteration was the degree of mannose-6-phosphate (M6P) modification. N-glycoproteomic analyses revealed that HCT116 display hyper-M6P modification, which was orthogonally validated by M6P immunodetection. Significant observed differences in N-glycosylation patterns of the major M6P receptor, CI-MPR in HCT116 and DKO1 may contribute to the hyper-M6P phenotype of HCT116 cells.

The carbohydrate 3D structure-prediction tools (builders) at GLYCAM-Web (glycam.org) are widely used for generating experimentally-consistent 3D structures of oligosaccharides suitable for data interpretation, hypothesis generation, simple visualization, and subsequent molecular dynamics (MD) simulation. The graphical user interface (GUI) enables users to create carbohydrate sequences (e.g. DGalpb1-4DGlcpb1-OH) that are converted to 3D models of the carbohydrate structures in multiple formats, including PDB and OFF (AMBER software format). The resulting structures are energy minimized prior to download and online visualization. There are advanced options for selecting which shapes (rotamers) of the oligosaccharide to generate, and for creating explicitly solvated structures for subsequent MD simulation. The GLYCAM-Web builders integrate known conformational preferences of oligosaccharides, summarized here, and employ the GLYCAM forcefield for energy minimization with algorithms tailored for speed and scalability. Even for large oligosaccharides (100 residues, ~2100 atoms) a 3D structure is typically returned to the user in less than a minute.

Glycosphingolipid glycosyltransferases (GGTs) can organize as multienzyme complexes localized along the Golgi complex. However, the influence of the relative presence of GGTs on the localization of their clients is unclear. Here, we determine that expression of certain full-length GGTs increases the levels of Golgi phosphoprotein 3 (GOLPH3), an adaptor oncoprotein involved in Golgi trafficking and organization. Furthermore, we demonstrate that expression of the N-terminal domain of GGTs, which lacks the catalytic domain, is sufficient to achieve this regulation on GOLPH3 in a cell type-dependent manner. We also identify the N-terminal domain of {beta}4GalT-VI GGT as an inhibitor of GOLPH3 expression and thus a potential therapeutic application, since GOLPH3 overexpression is associated with progression and poor prognosis of multiple tumor types. Our data further suggest that the cytoplasmic tail of {beta}4GalT-VI N-terminal domain interferes with the ability of GOLPH3 to interact with phosphatidylinositol 4-phosphate, which consequently reduces the levels of GOLPH3, thereby impairing its function in the acquisition of mesenchymal features.

Glycocins are a growing family of ribosomally synthesized and posttranslationally modified peptides that are O- and/or S-glycosylated. Using a sequence similarity network of putative glycosyltransferases, the tht biosynthetic gene cluster was identified in the genome of Thermoanaerobacterium thermosaccharolyticum. ThtA is the precursor peptide to a member of the glycocin F family of glycocins. Like other members of this family, the glycosyltransferase (ThtS) encoded in the biosynthetic gene cluster adds N-acetyl-glucosamine to both Ser and Cys residues of ThtA. S-linked glycosylation has been shown to be chemically and enzymatically resistant to cleavage and therefore ThtS may be a valuable starting point for engineering efforts. The glycocin derived from ThtA, which we name thermoglycocin, was structurally characterized. Thermoglycocin is unique in that in addition to two nested disulfide bonds, it contains an additional disulfide bond creating a C-terminal loop. Unexpectedly, ThtA lacks the common double glycine motif that denotes a C39-peptidase leader peptide cleavage site. Based on AlphaFold3 modeling, we postulate that cleavage between the leader and core peptide occurs instead at a GK motif. This study adds to the small number of characterized glycocins, employs AlphaFold3 to aid in predicting the structure of the mature peptide product, and suggests a common naming convention similar to that established for lanthipeptides. One sentence summaryThermoglycocin is a novel glycocin derived from the thermophile Thermoanaerobacterium thermosaccharolyticum, containing three disulfide bonds, O- and S-GlcNAcylation, and is postulated to have a unique C39 protease cut site. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/655019v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@17c74bforg.highwire.dtl.DTLVardef@1d4ddb3org.highwire.dtl.DTLVardef@27490dorg.highwire.dtl.DTLVardef@12cffd7_HPS_FORMAT_FIGEXP M_FIG C_FIG

The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control or influence crucial biological processes. Attempts to describe its structure-function relationships with HS binding proteins in a classical lock and key type manner, however, have been unsuccessful. HS chains are synthesized in a non-template driven process in the ER and Golgi apparatus, involving a large number of enzymes capable of fine-tuning structures. Changes in the localization of HS-modifying enzymes throughout the Golgi, rather than protein expression levels, were found to correlate with changes in the structure of HS. Following brefeldin A treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Further, shortly after treatment with heparin, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS trisulfated disaccharide content. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings highlight that the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking processes are critical prerequisite for the complete delineation of HS biosynthesis.