Glycobiology最新文献

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.

Protein O-GlcNAcylation is a dynamic post-translational modification with emerging roles in cardiac pathophysiology. The availability of different pan-specific antibodies to assess global O-GlcNAc levels, and variability in western blot results has hindered cross-study reproducibility and interpretation. In this study, we applied optimized immunoblotting protocols using both CTD110.6 and RL2 O-GlcNAc antibodies, alongside subcellular fractionation, to investigate temporal and sex-specific changes in cardiac O-GlcNAcylation during pressure overload hypertrophy (POH) from transverse aortic constriction (TAC) during early (1-week POH, 1wTAC) and chronic (6-weeks POH, 6wTAC) POH in mice. Global O-GlcNAc levels were elevated in early POH and returned to baseline in chronic POH, consistent across both antibodies and sexes. Subcellular fractionation revealed persistent O-GlcNAc elevations in cytoplasmic and membrane fractions in chronic POH for both sexes, which were not detected in unfractionated samples. Female mice exhibited significantly higher O-GlcNAc levels than males during POH, particularly at early POH, highlighting sex-specific regulation. OGT and OGA protein levels also varied by compartment and sex, suggesting differential enzymatic control. In conclusion, our findings underscore the importance of methodological rigor in O-GlcNAc detection and demonstrate that fractionation enhances sensitivity to subtle changes in cardiac O-GlcNAcylation. Our principal new findings are protein O-GlcNAcylation dysregulation continues from early POH (1wTAC) into chronic POH (6wTAC groups) along with showing differences in O-GlcNAc levels between males and females during POH. These results provide new insights into the temporal and sex-dependent dynamics of O-GlcNAc signaling in POH and support its potential as a therapeutic target in cardiovascular disease.

Glycosphingolipids (GSLs) from invertebrates often exhibit unique sugar chain structures distinct from those in vertebrates, reflecting evolutionary diversity in glycan biosynthesis. In this study, neutral GSLs were extracted and purified from the mantle skin of the jumbo flying squid Dosidicus gigas (Cephalopoda, Mollusca) using silicic acid (Iatrobeads) column chromatography. Their structures were characterized by gas-liquid chromatography, gas chromatography-mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and 1H-nuclear magnetic resonance spectroscopy. The purified GSLs contained oligosaccharide chains comprising one to eight sugar residues, including mollusk-specific mannose-containing cores (Mollu-series). Notably, the octasaccharide fractions included GSLs bearing human blood group A- and B-like epitopes, which reacted specifically with anti-A and anti-B sera in thin-layer chromatographic immunostaining. The principal structures were identified as GalNAcα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-2Manα1-3(Xylβ1-2)Manβ1-4Glcβ1-Cer (A-type) and Galα1-3(Fucα1-2)Galβ1-3GlcNAcβ1-2Manα1-3(Xylβ1-2)Manβ1-4Glcβ1-Cer (B-type). Ceramide analysis revealed fatty acids ranging from C14 to C24, with C16:0, C22:1, and C24:1 as dominant components. Odd-chain and hydroxy fatty acids were also detected. Sphingoids such as d16:1, d17:1, and d18:1 were present, with d16:1 being the most abundant. These findings highlight the structural complexity of molluscan GSLs and provide the first molecular evidence of both A- and B-type blood group-like glycosphingolipids in cephalopods, offering novel insights into the evolution and functional diversification of glycan biosynthesis across animal lineages.

Lung cancer remains the leading cause of cancer-related deaths globally, underscoring the need for novel therapeutic strategies. The relationship between high glucose levels, N-linked glycosylation, and cancer progression has been observed across various cancers, but its underlying mechanisms are not fully understood. Recent studies using CRISPR-Cas9 screens have highlighted the roles of glucose transporter 1 (GLUT1), UDP-N-acetylglucosamine pyrophosphorylase 1 (UAP1), and UDP-glucose pyrophosphorylase 2 (UGP2) in N-linked glycosylation. This study aims to elucidate how glucose influences lung cancer progression by examining its impact on aggressive phenotypes in A549 and PC-9 cell lines. The aggressive phenotypes-proliferation, colony formation, migration, and invasion- were investigated using MTT, and Boyden chamber assays. N-linked glycosylation status was monitored via lectin blot with Con A and PHA-E and molecular shift of GP130. Glucose dependently enhanced aggressive phenotypes in these cells through increased expression of GLUT1, UAP1, UGP2, and enhanced N-linked glycosylation. Conversely, inhibiting GLUT1 activity by selective inhibitors or knocking-out UAP1, and UGP2 expression using CRISPR-Cas9 significantly reduced aggressive behaviors and glycosylation levels. These observations were modulated through mediators of cell cycle (cyclin D1, p21, XIAP) and EMT (E-cadherin, vimentin, slug, snail). Notably, high expression of either GLUT1, UAP1, or UGP2, and the coordinated expression of these genes in tumor tissues correlated with poor survival outcomes in lung cancer patients. Our findings highlight the roles of GLUT1-UAP1-UGP2 axis and N-linked glycosylation in high glucose-induced progression of lung cancer cells. GLUT1, UAP1, and UGP2 may serve as prognostic markers and potential targets for future lung cancer treatments.

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.

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.

Alpha-1,6-fucosyltransferase (FUT8) transfers fucose to the innermost GlcNAc residue of N-glycans, forming the core fucose structure. Core fucose critically regulates the functions of various glycoproteins and is associated with several diseases, including chronic obstructive pulmonary disease and cancer. However, the regulatory mechanisms of its enzymatic activity and the intracellular localization of FUT8 remain largely unknown. We previously demonstrated that ribophorin I (RPN1), a subunit of the oligosaccharyltransferase (OST) complex, interacts with FUT8 and positively regulates the enzymatic activity of FUT8; however, it remains unclear whether other OST subunits interact with FUT8 and regulate FUT8 activity. In this study, we assessed the enzymatic activity of FUT8 with knockdown of each OST subunit and showed that silencing ribophorin II (RPN2) as well as RPN1 significantly reduced FUT8 activity. By contrast, no significant effect on FUT8 activity was observed by depleting STT3A and STT3B, which are catalytic subunits of OST, suggesting that the regulation of FUT8 activity by OST is irrelevant to the N-glycosylation activity of OST. Furthermore, various OST subunits were detected in anti-FUT8-immunoprecipitates, and FUT8 was detected in STT3s-dependent high molecular weight complexes in native-PAGE, whereas FUT8 at steady state was mainly localized in the Golgi apparatus, distinct from the endoplasmic reticulum localization of OST. These results suggest that FUT8 transiently interacts with OST complexes during transport in cells. Our findings provide insights into both the intracellular regulatory mechanisms of FUT8 activity and some unexplored functions of OST subunits.

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.

Standalone AlphaFold 3 (AF3) models proteins, post-translational modifications, and ligands (including glycans) from a single JSON input file. Plausible glycan stereochemistry is more consistently achieved when monosaccharides are specified as Chemical Component Dictionary (CCD) entries and connected using bondedAtomPairs (BAP) syntax. Although this approach preserves glycan stereochemistry, assembling JSON input files manually is challenging due to the diversity of monosaccharides, linkages, branching, and structural complexity. To simplify this process, we developed JAAG (JSON input file Assembler for AlphaFold 3 with Glycan integration (https://biofgreat.org/JAAG and https://github.com/chinchc/JAAG), a web-based graphical interface that streamlines AF3 JSON file creation, reduces errors, and facilitates modeling of glycans and glycan-macromolecule interactions.

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.