Pleurocybella porrigens is a mushroom that grows widely around the temperate northern hemisphere, and was once considered edible, especially in Japan. Following a number of deaths in 2004, investigations revealed the presence of various toxins, including a lectin (PPL) that apparently survives cooking and enters the bloodstream via the stomach. We have cloned PPL and solved its structure by X-ray crystallography and cryo-EM. We report the sugar binding properties of this β-trefoil lectin, which has a novel hexameric structure.

GalNAc-Ts are a large family of glycosyltransferases that regulate numerous cellular processes by initiating the post-translational modification mucin-type O-glycosylation. Disruptions in GalNAc-T expression and function are associated with congenital diseases, metabolic disorders, and cancer. The substrates and acceptor sites affected by the inactivation or over-activation of each specific family member are often not known due to acceptor site and substrate redundancies among the isoenzymes that are present within a cell type. However, substantial progress has been made in disentangling the enzyme-substrate conundrum by showing that each isoenzyme follows a unique set of substrate recognition rules. This review summarizes biochemical and structural findings that have advanced our understanding of the distinct substrate specificities of individual GalNAc-Ts.

The glycan distribution on cells is governed by the activities of different families of enzymes that are together called "glycoEnzymes." These include ~400 gene products or 2% of the proteome, that have recently been curated in an ontology called GlycoEnzOnto. With the goal of making this ontology more accessible to the larger biomedical and biotechnology community, we organized a web resource called GlycoEnzDB, presenting this enzyme classification in terms of enzyme function, the pathways that they participate in and their EC numbers. This information is linked to i) Figures from the "Essentials of Glycobiology" textbook, ii) General gene, enzyme and pathway data appearing in external databases, iii) Manual and generative-artificial intelligence (AI) based text describing the function and pathways regulated by these entities, iv) Single-cell expression data across cell lines, normal human cell-types and tissue, and v) CRISPR-knockout/activation/inactivation and Transcription factor activity predictions. Whereas these data are curated for human glycoEnzymes, the knowledge framework may be extended to other species also. The user-friendly web interface is accessible at www.virtualglycome.org/glycoenzdb.

O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a unique type of protein glycosylation that intricately links cellular metabolism to various signaling pathways. This reversible, nutrient-sensitive modification dynamically regulates a wide range of biological processes, including apoptosis, cell proliferation, and differentiation. Recent studies have made substantial progress in elucidating the pivotal roles of O-GlcNAcylation in modulating key oncogenes and signaling cascades. Aberrant O-GlcNAc cycling has been associated with a variety of pathological conditions, including cancer, metabolic disorders, and neurodegenerative diseases, underscoring its critical influence on cell fate decisions. In this review, we will highlight recent advances in understanding how O-GlcNAcylation modulates major cell fate regulating pathways, including nuclear factor kappaB (NF-κB), Notch, G protein-coupled receptor (GPCR) signaling, and transforming growth factor beta (TGF-β). We propose that O-GlcNAcylation integrates extracellular signals with intracellular metabolic states, functioning as an essential "Glyco-Switch" sensor that modulates cell fate decisions in both physiological and pathological contexts.

Glycoconjugate vaccines, also known as polysaccharide protein conjugate vaccines, consist of bacterial polysaccharides covalently linked to immunogenic carrier proteins. Bioconjugate vaccines are a type of glycoconjugate produced by oligosaccharyltransferases that catalyze the en bloc transfer of polysaccharides to specific amino acid motifs, called sequons, engineered into carrier proteins. Designing carrier proteins that are highly glycosylated by a specific oligosaccharyltransferase is critical for scalable bioconjugation platforms. Here, we describe the development of improved Pseudomonas aeruginosa exotoxin A (EPA) carrier proteins for glycosylation by the Acinetobacter baylyi ADP1 O-linking oligosaccharyltransferase PglS. Using a structure-guided approach, we integrated sequons at the termini or on surface-exposed loops of EPA and quantified the glycosylation of each site. Most sequons were 50% glycosylated on average, but glycosylation ranged from 20-75% suggesting a preference by PglS for certain sites. We then combined the best-glycosylated sites to design 3- and 6-sequon-containing EPA carriers and used capillary immunoassay electrophoresis to quantify EPA glycoforms. Using E. coli and Streptococcus glycans, we show that EPA carriers containing six sequons (EPA6) exhibit 1.5- to 5-fold higher glycosylation than carriers with fewer sequons. Furthermore, EPA6 could be comparably glycosylated with Klebsiella O2β O-antigen when secreted to the periplasm in an unfolded state via either the Sec or SRP pathways. However, no conjugates were produced when EPA6 was routed through the Tat pathway that secretes folded protein. Our results lay the groundwork for a general glycoengineering strategy for developing future bioconjugate vaccine carrier proteins as well as methods to evaluate such proteins.

The interactions between environmental glycans and glycan-binding proteins modulate a host of processes across biological systems. The Siglec/sialic acid axis has gained increasing attention as an immunologic checkpoint due to its involvement with reducing inflammatory processes and promoting tumor growth. Siglec-2, or CD22, has been extensively characterized as a co-receptor for the B cell receptor (BCR) and is critical for the prevention of self-reactive B cell responses through its recognition of α2,6-linked sialic acids. More recently, CD22 has emerged as an important receptor for macrophage biology. Here, we investigate the consequences of genetic ablation of CD22 in murine macrophages (CD22KO). Aged CD22KO mice developed a fatty liver phenotype similar to that seen in aged animals lacking hepatocyte α2,6-sialylation (HcKO). CD22KO bone marrow-derived macrophages (BMDMs) exhibited few differences in canonical markers of M1-like and M2-like polarization, but M2-like CD22KO BMDMs showed a pro-inflammatory shift in transcriptome and a reduction in endocytic and efferocytotic capacity. These data suggest that CD22 in murine M2-like macrophages is strongly associated with a homeostatic transcriptional profile and directly participates in immunologically silent housekeeping functions such as clearance of sialylated-self debris through the Siglec-sialic acid axis.

Human milk oligosaccharides (HMOs) are complex sugars. These sugars possess prebiotic, antibiotic and immunomodulatory properties and therefore are important for the health and well-being of newborn babies. The backbones of HMOs are terminated either with Type I LacNAc (Gal-β1,3-GlcNAc) or Type II LacNAc (Gal-β1,4-GlcNAc) that can be further fucosylated and sialylated. To detect all these HMOs including their fucosylated and sialylated versions, we explored enzymatic incorporation of azido-fucose (N3-Fuc) by FUT2 or FUT3 directly, or through a replacement approach where existing fucose and sialic acid are removed with a specific glycosidase and replaced with an N3-Fuc. Specifically, AfcA, an α1,2-linkage specific fucosidase cloned from Aspergillus oryzae, was used to remove existing α1,2-Fuc. The substrate specificities and relative efficiencies of AfcA, FUT2 and FUT3 in terms of the usage of N3-Fuc were demonstrated on standard HMOs. FUT2 was finally selected for labeling and validated on HMOs isolated from human milk samples. Furthermore, using Cy5-labeled antibody glycan G2 as a gel control, the relative gel separation of an N3-Fuc labeled HMO was established, which could aid identification of the oligosaccharide. This strategy by N3-Fuc labeling and glycan electrophoresis expands the ability to profile HMOs and is complementary to traditional methods for HMO study.

Mucin-type O-glycans are abundant protein modifications that regulate cell signalling, adhesion, and immune interactions. In cancer, their biosynthetic pathways are frequently disrupted, leading to the accumulation of truncated structures, such as Tn antigen and sialyl-Tn (STn). These aberrant glycans remodel the glycocalyx, alter receptor clustering, and drive key hallmarks of malignancy, including immune evasion, invasion, and therapy resistance. Over the past decade, increasing evidence has linked short O-glycans to poor prognosis across multiple tumour types, highlighting their potential as diagnostic and prognostic biomarkers. Moreover, their restricted expression in normal tissues positions them as attractive targets for therapeutic intervention, including monoclonal antibodies, antibody-drug conjugates, and CAR-T cell strategies. However, clinical translation remains limited by major analytical challenges. The structural diversity of O-glycans, their low abundance, and the lack of broadly specific enzymes for glycan release hinder comprehensive characterization. Recent advances in glycoproteomics, glycomics, and antibody engineering are beginning to overcome these barriers, enabling site-specific mapping and improved detection of cancer-associated glycoforms. This review summarizes current knowledge on the biosynthetic origins, biological roles, and clinical relevance of truncated O-glycans in cancer, while critically discussing emerging technologies and future directions for their integration into precision oncology.

Skp1 is an essential adaptor within the Skp1/Cul1/F-box (SCF) class of E3 polyubiquitin ligases that regulate protein degradation in all eukaryotes. Skp1 is also a target of a 5-enzyme glycosylation pathway in parasites and other unicellular eukaryotes. Glycosylation of Skp1 is contingent upon oxygen-dependent hydroxylation of a critical Pro residue by a homolog of the HIFα PHD2 oxygen sensor of animals. The resulting hydroxyproline is modified by a series of soluble, cytoplasmic, sugar nucleotide-dependent glycosyltransferases that vary among branches of protist evolution, and are evolutionarily related to counterparts in the Golgi and the cytoplasm of prokaryotes. Pair-wise gene fusions of the six enzymes occur in various protists, suggesting processing efficiency. The terminal glycosyltransferases exhibit a second site interaction with Skp1 that may modulate its function irrespective of glycosylation status. The pentasaccharide adopts a constrained fold that in turn promotes Skp1 conformations that inhibit sequestration by homodimerization and encourage binding to select F-box protein substrate receptors with varied effects on their expression levels. The occurrence of a second Skp1 copy in some protists that is resistant to modification indicates a mechanism to bypass glycoregulation. This review details evidence from the social amoeba Dictyostelium discoideum and the pathogens Toxoplasma gondii and Pythium ultimum for the specificity of the enzymes for Skp1 and their regulation, as support for a role in regulating protein turnover via E3(SCF) ubiquitin ligases, and in turn sensing oxygen at the cellular level.

Helicobacter pylori is a prevalent gastric pathogen that modulates host cell signaling pathways and represents a major risk factor for chronic gastritis, peptic ulcers, and gastric cancer. Galectin-3 is a host factor that contributes to immune regulation and cell death responses. However, its precise role in epithelial cell fate during H. pylori infection remains unclear. In this study, we demonstrate in AGS epithelial cells that H. pylori infection induces the accumulation of cytosolic galectin-3 around lysosomes damaged by the infection, detectable as puncta formation, and this process requires the presence of O-glycan. Using galectin-3 knockout cells, we show that galectin-3 expression correlates with the extent of apoptosis triggered by infection, which proceeds independently of VacA (vacuolating cytotoxin A). Pharmacological inhibition of galectin-3 glycan binding prevents lysosomal puncta formation but does not diminish apoptosis, indicating that galectin-3 promotes cell death through glycan-independent protein-protein interactions. Moreover, galectin-3 puncta co-localize with LC3-positive autophagosomal structures, and functional assays reveal that the initiation of autophagy facilitates apoptosis. Collectively, these findings identify galectin-3 as a pro-apoptotic factor involved in the epithelial response to H. pylori infection.

Layilin, an understudied C-type lectin receptor for hyaluronan, was initially hypothesized to regulate cell motility due to its binding partner, talin. Subsequent studies identified layilin as a receptor for hyaluronan with roles in regulating cell motility through interactions with key regulatory molecules upstream of cytoskeletal rearrangement: radixin, merlin, focal adhesion kinase (FAK), F-actin, and small GTPases such as RAC1, RAP1, and RhoA. Layilin is also associated with cell-cell interactions, co-localizing with integrins in both T-cells and platelets contributing to epithelial cell junction integrity. Recent studies have found that layilin also plays a role in inflammation, dependent on tissue and disease. In the context of cancer, multiple cancer cell types displaying increased layilin expression contributes to enhanced metastasis. Exhausted CD8+ T cells residing in the tumors exhibit high expression of layilin, with the receptor contributing to increased tissue anchoring and co-expressing with immune checkpoint resistance markers. In other contexts, such as inflammatory bowel disease and atherosclerosis, reduction of layilin results in worsened disease and inflammation. Transcriptomic and epigenetic studies have explored layilin as a prognostic marker, as layilin expression is elevated in multiple cancers, deep vein thrombosis, diabetes, and Alzheimer's. However, the mechanistic role of layilin in most of these studies remains unexplored. This review outlines current insights into Layilin as a molecular hub that links hyaluronan signaling with integrin activity and cytoskeletal dynamics, highlighting its roles in homeostasis, pathogenesis, disease prognosis, and therapeutic intervention across diverse conditions.

Cytosolic peptide:N-glycanase (PNGase/NGLY1 in mammals), a widely conserved amidase in eukaryotes, catalyzes the removal of N-glycans from glycoproteins and contributes to the quality control system for nascent glycoproteins. Since the first report of a patient with an autosomal recessive genetic disorder caused by NGLY1 deficiency in 2012, over 150 cases have been identified globally. Among the potential biomarkers for NGLY1 deficiency, Asn-linked mono/oligosaccharides-Asn-GlcNAc and Asn-HexNAc-Hex-NeuAc-have emerged as the most consistently and markedly elevated molecules in the plasma or urine of affected patients. This study examined the Asn-GlcNAc biosynthetic pathway, demonstrating that cytosolic endo-β-N-acetylglucosaminidase (ENGase), the proteasome, and peptidases are essential for its generation. NGLY1-deficient models and patients exhibited accumulation of novel elongated forms of Asn-GlcNAc, including Asn-GlcNAc-GalNAc, Asn-GlcNAc-Gal, and Asn-GlcNAc-Gal-NeuAc, in cells, culture supernatant, plasma, and urine. Our findings indicate that Asn-GlcNAc and Asn-oligosaccharides (Asn-OSs) may serve as promising diagnostic tools for NGLY1 deficiency.

Glycans found on the ErbB family of receptors (HER1, HER2, and HER3) represent promising targets for cancer treatment. Characterization and full quantification of the bivalent kinetic interactions of therapeutic antibodies against the ErbB family of receptors directly in their native cancer cellular environment represent a unique strategy to help overcome cancer drug resistance and to the development of more effective therapeutic drugs. In this study, surface plasmon resonance microscopy (SPRM) was implemented in a unique and innovative manner to quantify the bivalent kinetic interactions of monoclonal antibodies targeting HER1 (EFGR), HER2 and HER3 directly on whole BXPC3 pancreatic cancer cells under a glycosylated (native) and deglycosylated cellular environment. Results revealed in unprecedented detail that both the single-arm affinity and double-arm stronger avidity modes of binding interaction could be observed. For bivalent Cetuximab (anti-HER1) KDs of 151 nM and 4.6 nM were observed, for bivalent Herceptin (anti-HER2) KDs of 2 nM and 0.1 nM were observed, and for bivalent anti-HER3 KDs of 13 nM and 1.3 nM were observed. However, upon enzymatic N-deglycosylation of BXPC3 cells, HER1 and HER3 demonstrated significant increase in affinity of 1000-fold and 21-fold, respectively. In contrast, HER2 kinetic interactions were negligibly influenced by cellular N-deglycosylation of BXPC3 cells. This study highlights for the first time SPRM's unique ability to characterize the bivalent heterogeneous kinetic interactions of monoclonal antibodies with ErbB receptors on whole cancer cells, and to quantify the shielding influence of pancreatic cancer cell surface N-glycosylation on these interactions.

Heparan sulfate is present on the cell surface and within the extracellular matrix of most animal species, and it regulates various physiological processes by binding to a wide variety of functional proteins. The diverse array of sulfation patterns on the heparan sulfate chain enables specific binding to each protein. Of particular interest is the 3-O-sulfated (3S) structure of glucosamine residues, which is considered a key structure in determining binding specificity to functional proteins. However, 3-O-sulfation is a rare modification, which makes it difficult to analyze and hinders elucidation of its physiological function. Establishing a general method for measuring the 3S structure is thus important for continued progress in this research field. We previously developed an HPLC method to separate and quantify 13 components of heparan sulfate, including five 3S components, as a complete heparin lyase digestion product. Application of this method in routine analysis required the development of a standard sample for quantitation that is simple to prepare in large amounts and exhibits good stability. A heparan sulfate standard composed of 13 components in known quantities, designated HS13, was prepared for this purpose. A standard mixture of the 13 components can be obtained by digesting HS13 with a heparin lyase. A compositional analysis of heparan sulfate derived from various rat organs was then conducted to test the newly developed standard. The organ-specific distribution of each 3S component was elucidated, and it was confirmed that the 3S components in biological samples can be quantified by routine HPLC analysis.

Although the interaction between β-1,3-glucans (BG) and β-1,3-glucan recognition protein (BGRP) derived from insects is well established, the binding interaction and recognition mechanism of BG when complexed with deoxyadenosine (dA) or CpG oligodeoxynucleotides (CpG) remain poorly understood. In this study, we investigated the binding properties of Schizophyllan and Curdlan to BGRP both in their native forms and in BG/DNA complexes. Our findings revealed that BG/dA complexes bind to BGRP via a canonical BG-BGRP binding mechanism, whereas BG/CpG complexes exhibited a recognition mechanism distinct from the BG-BGRP interaction. Structural alterations in BG upon complexation with CpGs appear to induce a unique mode of BGRP recognition. This study reveals a novel mode of BGRP recognition in CpG-containing complexes, offering insights into improved immune detection strategies.

Bacterial vaginosis (BV), a dysbiosis of the vaginal microbiome, affects approximately 30% of women worldwide (up to 50% in some regions) and is associated with several adverse health outcomes including preterm birth and increased incidence of sexually transmitted infections (STIs). BV-associated bacteria such as Gardnerella vaginalis and Prevotella timonensis damage the vaginal mucosa through the activity of sialidase enzymes that remodel the epithelial glycocalyx and degrade mucin glycoproteins. This damage may contribute to adverse health outcomes. However, whether BV-associated glycolytic enzymes also damage sperm has not yet been determined. Here, we show that sialidase-mediated glycocalyx remodeling of human sperm increases sperm susceptibility to damage and adversely affects their function in vitro. Specifically, we report that sperm motility was not adversely affected by sialidase treatment, but desialylated human sperm demonstrate increased susceptibility to agglutination and complement-mediated cytotoxicity as well as impaired transit through cervical mucus. Our results demonstrate mechanisms by which BV-associated sialidases affect sperm survival and function and potentially contribute to adverse reproductive outcomes such as infertility.

Glycomics and glycoproteomics represent the systematic exploration of glycan structures and glycoprotein compositions within biological systems, aiming to elucidate their roles in physiological and pathological processes, including cancer, inflammation and infectious diseases. To support this investigation, glycomics and glycoproteomics utilize a diverse array of methodologies from molecular biology, biochemistry, analytical chemistry and bioinformatics. In this study, we investigated the semantic representation experimental workflows in glycomics and glycoproteomics publications through graph-based annotation using combination of existing domain-relevant ontologies. Rather than adhering to evolving metadata standards, this investigation explored a broad spectrum of biomedical and analytical ontologies to identify optimal annotations for the generative (e.g. sample preparation and derivatization) and transformative (e.g. separation and detection) phases of the workflow. The results show that integrating several ontologies yields more precise annotations than relying on a single one. However, several challenges arose, particularly where methodological reporting lacked critical metadata, such as derivatization conditions or glycan release protocols. Furthermore, the annotations imply that methodologies in the glycomic and glycoproteomic fields are more complex, on average, than those in other scientific fields. The results suggests that, while some specific concepts are missing in the ontologies, a limited number of ontologies adequately encompass the majority of aspects related to glycomics and glycoproteomics experiments. These can serve as a foundation for community-wide metadata standards and direct future efforts to refine and expand the ontologies for glycoscience research.