Glycobiology最新文献

Corticosteroid-binding globulin (CBG) is a serum glycoprotein that binds and delivers anti-inflammatory cortisol to inflammatory sites through neutrophil elastase-mediated proteolysis of an exposed reactive Centre loop (RCL) on CBG. Timely and tissue-specific delivery of cortisol is critical to alleviate inflammation including in life-threatening septic shock conditions. In this two-part communication, we firstly summarise our recently published report of functional RCL O- and N-glycosylation events of serum CBG (Chernykh, J biol Chem, 2023). A key finding of that published work was the LC-MS/MS-based discovery of RCL O-glycans at Thr342 and Thr345 of serum CBG and their inhibitory roles in neutrophil elastase-mediated RCL proteolysis. While these observations are of significance as they implicate RCL O-glycosylation as a potential regulator of cortisol delivery, the link to septic shock remains unexplored. In the second part of this communication, we therefore used a similar LC-MS/MS approach to profile the RCL O-glycosylation of CBG purified from serum of twelve septic shock patients. Serum CBG from all patients exhibited RCL O-glycosylation comprising (di)sialyl T (NeuAc1-2Gal1GalNAc1) O-glycan structures decorating exclusively the Thr342 site. Importantly, relative to less severe cases, individuals presenting with the most severe illness displayed elevated RCL O-glycosylation upon ICU admission, suggesting a previously unknown link to septic shock severity. Overall, we have elucidated the coordinated RCL N- and O-glycosylation events of serum CBG, which improve our understanding of molecular mechanisms governing the timely and tissue-specific delivery of cortisol to inflammatory sites. This work provides clues to molecular aberrations and disease mechanisms underpinning septic shock.

Heparan sulfate 6-endosulfatases (SULFs) remove 6-O-sulfo groups from heparan sulfate polysaccharide chains. SULFs modify the functions of heparan sulfate and contribute to the development of cancers, organ development and endothelial inflammatory responses. However, direct measurement of the activity of SULFs from human and mouse plasma is not currently possible. Here, we report a liquid chromatography coupled with tandem mass spectrometry (LS-MS/MS) assay to measure the activity of SULFs. The method uses a structurally homogeneous heparan sulfate dodecasaccharide (12-mer) in which the glucuronic and iduronic acid residues are labeled with both 13C- and 2H-atoms. The 12-mers desulfated by the SULFs is subjected to degradation with heparin lyases to yield disaccharides, which is followed by LC-MS/MS. The amount of two specific disaccharides, ΔIIIS and ΔIVS, quantified by LC-MS/MS reports the activity of the SULFs with high sensitivity and specificity. This method allows for the determination of the activity from conditioned cell media and mouse plasma. Our findings offer an essential novel tool to delineate many roles of SULFs in biological processes.

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 selectivity 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 synthetic glycomimetic GalNAc cluster ligand that targets siRNA molecules to hepatocytes through the asialoglycoprotein receptor. Additional small oligosaccharides that could 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, potential 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 selectivity 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 such as measuring clearance in animals.

Accumulating evidences have shown that unfolded protein response (UPR) contributes to the increased survival of tumor cells under endoplasmic reticulum (ER) stress conditions. Malectin is an ER-resident lectin that selectively traps misfolded glycoproteins in ER for degradation, and its expression is upregulated upon ER stress. However, contribution of malectin to malignant behavior of tumor has not been reported. Here, we revealed that malectin expression is aberrantly up-regulated in human hepatocellular carcinoma (HCC) tissues and HCC cell lines compared to their matched normal tissues and cell lines. Knockout of malectin in two HCC cell lines HepG2 and QGY-7703 using CRISPR-Cas9 technology had no obvious effects on cell proliferation, but significantly suppressed cell colony formation, migration and invasion. Consistently, subcutaneously implanted malectin-deficient HCC cells in nude mice also showed an obvious decrease in tumor growth. These results indicate that malectin might play an oncogenic role in HCC tumorigenesis and development.

The MIRAGE (Minimum Information Required for a Glycomics Experiment) project has been established by experts in glycobiology, glycoanalytics, and glycoinformatics under the auspieces of the Beilstein-Institut. The working group aims to develop guidelines for reporting results from various experiments and analyses conducted in structural and functional studies of glycans in the scientific literature. Previous guidelines have been established for glycomic analytics, including mass spectrometry and glycan microarrays. Lectin microarrays are used worldwide for glycan profiling of various biological samples, but there are often insufficient reports on information about experimental methods such as sample preparation and fluorescence labeling. Here, we propose guidelines specifically designed to improve the standards for reporting data from lectin microarray analyses. For each of the seven areas in the workflow of a lectin microarray experiment, we provide recommendations for the minimum information that should be included when reporting results. When adopted by the scientific community the MIRAGE lectin microarray guidelines are expected to enhance data interpretation, facilitate comparison of data between laboratories and encourage the deposition of lectin microarray data in international databases.

Approximately 300,000 American men were diagnosed with prostate cancer in 2024. Existing screening approaches based on measuring levels of prostate-specific antigen in the blood lack specificity for prostate cancer. Studying the glycans attached to proteins has the potential to generate new biomarker candidates and/or increase the specificity of existing protein biomarkers, and studying protein glycosylation changes in prostate cancer could also add new information to our understanding of prostate cancer biology. Here, we present the analysis of N-glycoproteins in clinical prostate cancer tissue and patient-matched, non-cancerous adjacent tissue using LC-MS/MS-based intact N-linked glycopeptide analysis. This analysis allowed us to characterize protein N-linked glycosylation changes in prostate cancer at the glycoprotein, glycopeptide, and glycosite levels. Overall, 1894 unique N-glycosites on 7022 unique N-glycopeptides from 1354 unique glycoproteins were identified. Importantly, we observed an overall increase in glycoprotein, glycopeptide, and glycosite counts in prostate cancer tissue than non-cancerous tissue. We identified biological functions enriched in prostate cancer that relate to cancer development. Additionally, we characterized N-glycosite-specific changes in prostate cancer, demonstrating significant meta- and micro-heterogeneity in N-glycan composition in prostate cancer in comparison to non-cancerous tissue. Our findings support the idea that protein glycosylation is heavily impacted and aberrant in prostate cancer and provide examples of N-glycosite-specific changes that could be exploited for more specific markers of prostate cancer.

Abdala is a COVID-19 vaccine produced in Pichia pastoris and is based on the receptor-binding domain (RBD) of the SARS-CoV-2 spike. Abdala is currently approved for use in multiple countries with clinical trials confirming its safety and efficacy in preventing severe illness and death. Although P. pastoris is used as an expression system for protein-based vaccines, yeast glycosylation remains largely uncharacterised across immunogens. Here, we characterise N-glycan structures and their site of attachment on Abdala and show how yeast-specific glycosylation decreases binding to the ACE2 receptor and a receptor-binding motif (RBM) targeting antibody compared to the equivalent mammalian-derived RBD. Reduced receptor and antibody binding is attributed to changes in conformational dynamics resulting from N-glycosylation. These data highlight the critical importance of glycosylation in vaccine design and demonstrate how individual glycans can influence host interactions and immune recognition via protein structural dynamics.