Glycobiology最新文献

Glycosaminoglycans (GAGs) interact with numerous proteins to regulate key biological processes such as coagulation, cell migration, and growth factor signaling. Despite their biological importance, mapping these interactions and identifying the structural determinants that govern GAG-protein recognition remains analytically challenging, particularly in complex or polydisperse systems. Here, we introduce a simple, label-free carbohydrate polyacrylamide gel electrophoresis (C-PAGE) approach that visualizes GAG-protein complex formation through binding-induced signal suppression of GAG bands. Instead of tracking the mobility of protein-GAG complexes, as it is classically performed in gel-shift assays, C-PAGE directly monitors the disappearance of the free GAG bands stained with Stains-All. Using model high-affinity heparin-binding proteins, the chemokine stromal cell-derived factor 1 α (SDF-1α) and the basic fibroblast growth factor (FGF-2), we demonstrate that C-PAGE clearly distinguishes specific interactions with heparin oligosaccharides from non-binding controls. The method was successfully extended to a micromolar-affinity heparin-binding protein, the interleukin 8 (IL-8), and to complex mixtures such as low-molecular-weight heparins (LMWH), revealing the preferential disappearance of highly sulfated and/or longer GAG species. Competitive assays further enabled qualitative ranking of GAG binding affinities. Finally, the selective interaction of antithrombin III with 3-O-sulfated motifs was unambiguously detected, underscoring the remarkable sensitivity of C-PAGE to fine structural modifications. Altogether, C-PAGE provides a rapid, visual, and cost-effective screening tool to assess GAG-protein binding specificity and structure-activity relationships, complementing advanced biophysical and structural methods in fundamental and applied glycobiology.

Microbial carbohydrate-active enzymes frequently have tandem carbohydrate-binding modules that enable enhanced enzyme activity on carbohydrates via targeting and proximity effects. CBM47 is a family of L-fucose-binding F-type Lectin Domains (FLDs) found in proteins with diverse domain architectures and possible roles in directing biological functions to fucosylated niches. In one such FLD-containing protein, Streptosporangium roseum α-L-fucosidase (SrFucNaFLD), the FLD enhances the enzyme activity of the tandem-positioned α-L-fucosidase domain for small, aqueous, freely diffusible, fucosylated oligosaccharides. Here, we performed domain engineering experiments on SrFucNaFLD to dissect and understand the spatial role of the domains on α-L-fucosidase activity. We found that the central NPCBM-associated (Na) domain was dispensable for optimal enhancement of α-L-fucosidase activity; however, the N-terminal to C-terminal domain order was critical, perhaps because it altered the relative spatial orientation of the α-L-fucosidase domain and the FLD, thereby affecting substrate access. We also explored the effect of replacing the native FLD on this protein with non-native FLDs (with different α-L-fucoside binding profiles) from different organisms. We found no enhancement of α-L-fucosidase activity towards the natural oligosaccharide substrates, Lewis a tetraose, H type-2 triaose, and H type-2 tetraose, by non-native FLDs other than Actinomyces turicensis FLD, which incidentally also exists in tandem with an α-L-fucosidase domain in the native context. Our results suggest that the S. roseum α-L-fucosidase is a well-optimized system with fine-tuned substrate channelling from the FLD to the tandem α-L-fucosidase domain. Our study has implications for future engineering studies of carbohydrate-active enzymes.

Accurate prediction of structure of protein-carbohydrate complexes remains a significant challenge in structural glycobiology, largely due to the flexibility of glycans and the shallow, hydrophilic nature of their binding sites. To address this issue, we developed a guided docking protocol that leverages Crystallographic Water Sites (CWS) to enhance glycan pose prediction using AutoDock Vina (ADV). By defining Waters Ideal Interactions (WII)-interaction hotspots derived from water molecules in apo structures-the protocol systematically rewards chemically meaningful receptor-ligand contacts during docking simulations. The WII Guided Approach (WIIGA) was benchmarked against a curated dataset of 30 high-quality protein-oligosaccharide complexes, which included ligands ranging from tetra- to nonasaccharides. Performance evaluation demonstrated that the guided protocol consistently outperformed conventional methods (ADV, Vina Carb (VC), Vina Carb with CH-π (VC CH-π) and GlycoTorch Vina (GTV)), delivering improved pose prediction accuracy. Our method proved robust even in the absence of holo structures and was effective in cross-docking drug-like glycomimetics. The protocol is easy to implement and broadly applicable to a wide range of glycan-binding proteins. These findings underscore the value of solvent-derived information for improving docking accuracy and support the use of guided approaches as a versatile tool for glyco-ligand modeling and structure-based design.

Intestinal mucin glycan-degrading bacteria are important for mucus turnover, stimulating mucus production, and producing beneficial metabolites. The mucin-degrading bacteria require various enzymes to break down mucin O-glycans. In this study, mucin glycan-degrading bacteria Akkermansia muciniphila, Ruminococcus torques, and Bacteroides thetaiotaomicron, were grown on porcine gastric mucin in monocultures, co-cultures, and a synthetic bacterial community. Enzyme extracts from these cultures were incubated with a selection of glycans, varying in sugar and linkage composition, to investigate enzyme specificities. Proteomics identified β-galactosidases, α-N-acetylgalactosaminidases, β-N-acetylglucosaminidases, α-fucosidases, α-sialidases, sulphatases, carbohydrate esterases, and polysaccharide lyases involved in O-glycan degradation. Enzymes produced by A. muciniphila and R. torques efficiently cleaved β-linked galactose and N-acetylgalactosamine. B. thetaiotaomicron enzymes minimally cleaved mucin glycans although multiple β-galactosidases and β-N-acetylglucosaminidases were produced. A. muciniphila favoured removal of fucose linked to non-terminal sugars whereas R. torques and B. thetaiotaomicron favoured removal of fucose linked to terminal sugars. A. muciniphila enzymes favoured cleavage of fucose α1-2 linked over α1-3 linked and cleavage of N-acetylglucosamine β1-3 linked over β1-4 linked. Both A. muciniphila and B. thetaiotaomicron favoured cleavage of galactose β1-4 linked over β1-3 linked and sialic acid α2-3 linked over α2-6 linked. Removal of sulphate from mucin structures was only observed by B. thetaiotaomicron. Bacterial co-cultures and the synthetic community produced all enzymes identified in the monocultures resulting in efficient mucin O-glycan degradation. Combining proteomics and glycan linkage cleavage by bacterial enzymes, showed differences in glycan degradation by the bacteria. This highlighted the importance of intestinal bacterial composition in mucin glycan degradation.

Gangliosides GD3 and GD2 are glycosphingolipids involved in key cellular processes and are overexpressed in various cancers, including T-cells leukemias. In this study, we investigated the temporal conversion of GD3 to GD2 in the human T lymphoblast cell line MOLT-4 by using flow cytometry and proton nuclear magnetic resonance (1H NMR) spectroscopy. Flow cytometry analysis revealed that at day 4 of culture, GD3 predominated on the cell surface, while GD2 showed low expression. By day 6, GD2 expression markedly increased, accompanied by a decrease in GD3, indicating a dynamic shift in ganglioside composition. Complementary 1H NMR analysis directly applied to cellular extracts identified diagnostic anomeric proton signals corresponding to GD3 at day 4 and GD2 at day 6, confirming the structural transition. This dual-platform approach demonstrates, for the first time, the NMR-mediated discrimination of GD3 and GD2 in unpurified human cell extracts, providing robust evidence of ganglioside remodeling during MOLT-4 cell proliferation and establishing a valuable methodology for functional and therapeutic studies involving gangliosides.

A complex extracellular matrix (ECM) assembles around the mammalian oocyte during maturation and ovulation, comprising hyaluronan as well as proteoglycans. These proteoglycans are hypothesised to carry heparan and chondroitin sulfate side chains. This matrix is essential for ovulation, mediates signalling, and regulates solute diffusion to control the oocyte environment. In vivo, ECM formation is initiated by epidermal growth factor-like peptides released within the follicle, acting together with oocyte-derived growth factors. Although proteoglycans are known components, the specific glycosaminoglycan (GAG) composition remains poorly understood. Here, we characterised GAG abundance in murine cumulus-oocyte-complexes and assessed differences between in vivo and in vitro maturation. The latter is an assisted reproductive technology that requires less drugs than IVF, but oocytes have reduced developmental potential. We found that sulfated GAG abundance increased significantly in vivo but not during in vitro oocyte maturation. We also employed high performance liquid-chromatography to measure the abundance of specific GAGs-hyaluronan, chondroitin sulfate, and heparan sulfate-in this matrix at different stages of maturation. These were enriched within the ECM during in vivo maturation but reduced or undetectable in vitro. Reduced GAG abundance following in vitro maturation was associated with poorer oocyte developmental potential. GAG deficiency following in vitro maturation likely arises from the failure of in vitro conditions to replicate the signalling milieu that occurs in vivo. Altered GAG abundance during in vitro maturation may impair functions of the ECM, including growth factor binding and activity or the regulated diffusion of solutes, potentially contributing to decreased oocyte developmental potential.

Oligosaccharyltransferase (OST), which is a multi-membrane protein complex that catalyzes asparagine-linked glycosylation (N-glycosylation) in the endoplasmic reticulum (ER), is a potential target to eradicate refractory cancer. Mammals express two distinct OST isoforms (OST-A and OST-B) that exhibit different acceptor site specificity to maximize N-glycosylation efficiency; however, the role of individual OST isoforms in tumor progression is not fully understood. Here, using mouse melanoma model, we showed that gene-edited knockout of either one of the OST isoforms did not compromise subcutaneous tumor growth, while their co-expression was required for efficient experimental lung metastasis. We further showed that the cytosolic N-terminal region of Stt3a, which is the catalytic subunit of OST-A, was critical for the N-glycosylation reaction and lung metastasis. This study opens a novel avenue for selective manipulation of OST-A activity, which might offer potential therapeutic strategies for metastatic cancers.