Search Results (27)

Mannose-specific lectins are carbohydrate-binding proteins known for their antiviral potential. This study uses a bioinformatic approach to investigate the possibility of lectins from Allium sativum (garlic) and Allium ursinum (wild garlic) as inhibitors of SARS-CoV-2 entry. The information spectrum method (ISM) identified key interaction frequencies between the SARS-CoV-2 spike protein and these lectins, explicitly targeting the receptor-binding domain (RBD) and glycosylated asparagine residues, including N234. Lectins from Allium species showed a high affinity for oligomannose-type glycans on the spike protein, potentially blocking virus entry by preventing the spike-ACE2 receptor interaction. We propose that Allium lectins are promising candidates for further experimental validation as SARS-CoV-2 inhibitors, offering potential therapeutic applications in managing viral infections. Full article

Proteins found within the fungal cell wall usually contain both N- and O-oligosaccharides. N-glycosylation is the process where these oligosaccharides (hereinafter: glycans) are attached to asparagine residues, while in O-glycosylation the glycans are covalently bound to serine or threonine residues. The PMT family is grouped into PMT1, PMT2, and PMT4 subfamilies. Using bioinformatics analysis within the Botrytis cinerea genome database, an ortholog to Saccharomyces cerevisiae Pmt4 and other fungal species was identified. The aim of this study was to assess the relevance of the bcpmt4 gene in B. cinerea glycosylation. For this purpose, the bcpmt4 gene was disrupted by homologous recombination in the B05.10 strain using a hygromycin B resistance cassette. Expression of bcpmt4 in S. cerevisiae ΔScpmt4 or ΔScpmt3 null mutants restored glycan levels like those observed in the parental strain. The phenotypic analysis showed that Δbcpmt4 null mutants exhibited significant changes in hyphal cell wall composition, including reduced mannan levels and increased amounts of chitin and glucan. Furthermore, the loss of bcpmt4 led to decreased glycosylation of glycoproteins in the B. cinerea cell wall. The null mutant lacking PMT4 was hypersensitive to a range of cell wall perturbing agents, antifungal drugs, and high hydrostatic pressure. Thus, in addition to their role in glycosylation, the PMT4 is required to virulence, biofilm formation, and membrane integrity. This study adds to our knowledge of the role of the B. cinerea bcpmt4 gene, which is involved in glycosylation and cell biology, cell wall formation, and antifungal response. Full article

The hypoxic tumor microenvironment significantly impacts cellular behavior and intercellular communication, with extracellular vesicles (EVs) playing a crucial role in promoting angiogenesis, metastasis, and host immunosuppression, and presumed cancer progression and metastasis are closely associated with the aberrant surface N-glycan expression in EVs. We hypothesize that hypoxic tumors synthesize specific hypoxia-induced N-glycans in response to or as a consequence of hypoxia. This study utilized nano-LC–MS/MS to integrate quantitative proteomic and N-glycomic analyses of both cells and EVs derived from the MDA-MB-231 breast cancer cell line cultured under normoxic and hypoxic conditions. Whole N-glycome and proteome profiling revealed that hypoxia has an impact on the asparagine N-linked glycosylation patterns and on the glycolysis/gluconeogenesis proteins in cells in terms of altered N-glycosylation for their adaptation to low-oxygen conditions. Distinct N-glycan types, high-mannose glycans like Man3 and Man9, were highly abundant in the hypoxic cells. On the other hand, alterations in the sialylation and fucosylation patterns were observed in the hypoxic cells. Furthermore, hypoxia-induced EVs exhibit a signature consisting of mono-antennary structures and specific N-glycans (H4N3F1S2, H3N3F1S0, and H7N4F3S2; H8N4F1S0 and H8N6F1S2), which are significantly associated with poor prognoses for breast tumors, presumably altering the interactions within the tumor microenvironment to promote tumorigenesis and metastasis. Our findings provide an overview of the N-glycan profiles, particularly under hypoxic conditions, and offer insights into the potential biomarkers for tracking tumor microenvironment dynamics and for developing precision medicine approaches in oncology. Full article

X-ray footprinting coupled with mass spectrometry (XFMS) presents a novel approach in structural biology, offering insights into protein conformation and dynamics in the solution state. The interaction of the cancer-immunotherapy monoclonal antibody nivolumab with its antigen target PD-1 was used to showcase the utility of XFMS against the previously published crystal structure of the complex. Changes in side-chain solvent accessibility, as determined by the oxidative footprint of free PD-1 versus PD-1 bound to nivolumab, agree with the binding interface side-chain interactions reported from the crystal structure of the complex. The N-linked glycosylation sites of PD-1 were confirmed through an LC-MS/MS-based deglycosylation analysis of asparagine deamidation. In addition, subtle changes in side-chain solvent accessibility were observed in the C′D loop region of PD-1 upon complex formation with nivolumab. Full article

Glioma stem cells (GSCs) contribute to the pathogenesis of glioblastoma (GBM), which is the most malignant form of glioma. The implications and underlying mechanisms of protein glycosylation in GSC phenotypes and GBM malignancy are not fully understood. The implication of protein glycosylation and the corresponding candidate genes on the stem cell properties of GSCs and poor clinical outcomes in GBM were investigated, using datasets from the Gene Expression Omnibus, The Cancer Genome Atlas, and the Chinese Glioma Genome Atlas, accompanied by biological validation in vitro. N-linked glycosylation was significantly associated with GSC properties and the prognosis of GBM in the integrated bioinformatics analyses of clinical specimens. N-linked glycosylation was associated with the glioma grade, molecular biomarkers, and molecular subtypes. The expression levels of the asparagine-linked glycosylation (ALG) enzyme family, which is essential for the early steps in the biosynthesis of N-glycans, were prominently associated with GSC properties and poor survival in patients with GBM with high stem-cell properties. Finally, the oxidative phosphorylation pathway was primarily enriched in GSCs with a high expression of the ALG enzyme family. These findings suggest the role of N-linked glycosylation in the regulation of GSC phenotypes and GBM malignancy. Full article

Predatory innovations impose reciprocal selection pressures upon prey. The evolution of snake venom alpha-neurotoxins has triggered the corresponding evolution of resistance in the post-synaptic nicotinic acetylcholine receptors of prey in a complex chemical arms race. All other things being equal, animals like caecilians (an Order of legless amphibians) are quite vulnerable to predation by fossorial elapid snakes and their powerful alpha-neurotoxic venoms; thus, they are under strong selective pressure. Here, we sequenced the nicotinic acetylcholine receptor alpha-1 subunit of 37 caecilian species, representing all currently known families of caecilians from across the Americas, Africa, and Asia, including species endemic to the Seychelles. Three types of resistance were identified: (1) steric hindrance from N-glycosylated asparagines; (2) secondary structural changes due to the replacement of proline by another amino acid; and (3) electrostatic charge repulsion of the positively charged neurotoxins, through the introduction of a positively charged amino acid into the toxin-binding site. We demonstrated that resistance to alpha-neurotoxins convergently evolved at least fifteen times across the caecilian tree (three times in Africa, seven times in the Americas, and five times in Asia). Additionally, as several species were shown to possess multiple resistance modifications acting synergistically, caecilians must have undergone at least 20 separate events involving the origin of toxin resistance. On the other hand, resistance in non-caecilian amphibians was found to be limited to five origins. Together, the mutations underlying resistance in caecilians constitute a robust signature of positive selection which strongly correlates with elapid presence through both space (sympatry with caecilian-eating elapids) and time (Cenozoic radiation of elapids). Our study demonstrates the extent of convergent evolution that can be expected when a single widespread predatory adaptation triggers parallel evolutionary arms races at a global scale. Full article

Asparagine (Asn, N)-linked glycosylation is a conserved process and an essential post-translational modification that occurs on the NXT/S motif of the nascent polypeptides in endoplasmic reticulum (ER). The mechanism of N-glycosylation and biological functions of key catalytic enzymes involved in this process are rarely documented for oomycetes. In this study, an N-glycosylation inhibitor tunicamycin (TM) hampered the mycelial growth, sporangial release, and zoospore production of Phytophthora capsici, indicating that N-glycosylation was crucial for oomycete growth development. Among the key catalytic enzymes involved in N-glycosylation, the PcSTT3B gene was characterized by its functions in P. capsici. As a core subunit of the oligosaccharyltransferase (OST) complex, the staurosporine and temperature sensive 3B (STT3B) subunit were critical for the catalytic activity of OST. The PcSTT3B gene has catalytic activity and is highly conservative in P. capsici. By using a CRISPR/Cas9-mediated gene replacement system to delete the PcSTT3B gene, the transformants impaired mycelial growth, sporangial release, zoospore production, and virulence. The PcSTT3B-deleted transformants were more sensitive to an ER stress inducer TM and display low glycoprotein content in the mycelia, suggesting that PcSTT3B was associated with ER stress responses and N-glycosylation. Therefore, PcSTT3B was involved in the development, pathogenicity, and N-glycosylation of P. capsici. Full article

To improve the efficacy and safety of chimeric antigen receptor (CAR)-expressing T cell therapeutics through enhanced CAR design, we analysed CAR structural factors that affect CAR-T cell function. We studied the effects of disulphide bonding at cysteine residues and glycosylation in the HD on CAR-T function. We used first-generation CAR[V/28/28/3z] and CAR[V/8a/8a/3z], consisting of a mouse vascular endothelial growth factor receptor 2 (VEGFR2)-specific single-chain variable fragment tandemly linked to CD28- or CD8α-derived HD, transmembrane domain (TMD) and a CD3ζ-derived signal transduction domain (STD). We constructed structural variants by substituting cysteine with alanine and asparagine (putative N-linked glycosylation sites) with aspartate. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] formed homodimers, the former through a single HD cysteine residue and the latter through the more TMD-proximal of the two cysteine residues. The absence of disulphide bonds did not affect membrane CAR expression but reduced antigen-specific cytokine production and cytotoxic activity. CAR[V/28/28/3z] and CAR[V/8a/8a/3z] harboured one N-linked glycosylation site, and CAR[V/8a/8a/3z] underwent considerable O-linked glycosylation at an unknown site. Thus, N-linked glycosylation of CAR[V/28/28/3z] promotes stable membrane CAR expression, while having no effect on the expression or CAR-T cell activity of CAR[V/8a/8a/3z]. Our findings demonstrate that post-translational modifications of the CAR HD influence CAR-T cell activity, establishing a basis for future CAR design. Full article

Breast cancer is an ancient disease recognized first by the Egyptians as early as 1600 BC. The first cancer-causing gene in a chicken tumor virus was found in 1970. The United States signed the National Cancer Act in 1971, authorizing federal funding for cancer research. Irrespective of multi-disciplinary approaches, diverting a great deal of public and private resources, breast cancer remains at the forefront of human diseases, affecting as many as one in eight women during their lifetime. Because of overarching challenges and changes in the breast cancer landscape, five-year disease-free survival is no longer considered adequate. The absence of a cure, and the presence of drug resistance, severe side effects, and destruction of the patient’s quality of life, as well as the fact that therapy is often expensive, making it unaffordable to many, have created anxiety among patients, families, and friends. One of the reasons for the failure of cancer therapeutics is that the approaches do not consider cancer holistically. Characteristically, all breast cancer cells and their microenvironmental capillary endothelial cells express asparagine-linked (N-linked) glycoproteins with diverse structures. We tested a small biological molecule, Tunicamycin, that blocks a specific step of the protein N-glycosylation pathway in the endoplasmic reticulum (ER), i.e., the catalytic activity of N-acetylglusosaminyl 1-phosphate transferase (GPT). The outcome was overwhelmingly exciting. Tunicamycin quantitatively inhibits angiogenesis in vitro and in vivo, and inhibits the breast tumor progression of multiple subtypes in pre-clinical mouse models with “zero” toxicity. Mechanistic details support ER stress-induced unfolded protein response (upr) signaling as the cause for the apoptotic death of both cancer and the microvascular endothelial cells. Additionally, it interferes with Wnt signaling. We therefore conclude that Tunicamycin can be expected to supersede the current therapeutics to become a glycotherapy for treating breast cancer of all subtypes. Full article

Tumor hypoxia induces angiogenesis, which is required for tumor cell survival. The aminopeptidase N receptor (APN/CD13) is an excellent marker of angiogenesis since it is overexpressed in angiogenic blood vessels and in tumor cells. Asparagine-glycine-arginine (NGR) peptide analogs bind selectively to the APN/CD13 recepto, therefore, they are important vector molecules in the development of a PET radiotracer which is capable of detecting APN-rich tumors. To investigate the effect of glycosylation and pegylation on in-vivo efficacy of an NGR-based radiotracer, two ⁶⁸Ga-labeled radioglycopeptides were synthesized. A lactosamine derivative was applied to glycosylation of the NGR derivative and PEG₄ moiety was used for pegylation. The receptor targeting potential and biodistribution of the radiopeptides were evaluated with in vivo PET imaging studies and ex vivo tissue distribution studies using B16-F10 melanoma tumor-bearing mice. According to these studies, all synthesized radiopeptides were capable of detecting APN expression in B16-F10 melanoma tumor. In addition, lower hepatic uptake, higher tumor-to background (T/M) ratio and prolonged circulation time were observed for the novel [⁶⁸Ga]-10 radiotracer due to pegylation and glycosylation, resulting in more contrasting PET imaging. These in vivo PET imaging results correlated well with the ex vivo tissue distribution data. Full article

Mutations in HBsAg, the surface antigen of the hepatitis B virus (HBV), might affect the serum HBV DNA level of HBV-infected patients, since the reverse transcriptase (RT) domain of HBV polymerase overlaps with the HBsAg-coding region. We previously identified a diagnostic escape mutant (W3S) HBV that produces massively glycosylated HBsAg. In this study, we constructed an HBV-producing vector that expresses W3S HBs (pHB-W3S) along with a wild-type HBV-producing plasmid (pHB-WT) in order to analyze the physicochemical properties, replication, and antiviral drug response of the mutant. Transfection of either pHB-WT or W3S into HepG2 cells yielded similar CsCl density profiles and eAg expression, as did transfection of a glycosylation defective mutant, pHB-W3S (N146G), in which a glycosylation site at the 146aa asparagine (N) site of HBs was mutated to glycine (G). Virion secretion, however, seemed to be severely impaired in cases of pHB-W3S and pHB-W3S (N146G), compared with pHB-WT, as determined by qPCR and Southern blot analysis. Furthermore, inhibition of glycosylation using tunicamycin^TM on wild-type HBV production also reduced the virion secretion. These results suggested that the HBV core and Dane particle could be formed either by massively glycosylated or glycosylation-defective HBsAg, but reduced and/or almost completely blocked the virion secretion efficiency, indicating that balanced glycosylation of HBsAg is required for efficient release of HBV, and mutations inducing an imbalanced glycosylation of HBs would cause the virion to become stuck in the cells, which might be associated with various pathogeneses due to HBV infection. Full article

Currently, diagnosing type 2 diabetes (T2D) is a great challenge. Thus, there is a need to find rapid, simple, and reliable analytical methods that can detect the disease at an early stage. The aim of this work was to shed light on the importance of sample collection options, sample preparation conditions, and the applied capillary electrophoresis bioanalytical technique, for a high-resolution determination of the N-glycan profile in human blood samples of patients with type 2 diabetes (T2D). To achieve the profile information of these complex oligosaccharides, linked by asparagine to hIgG in the blood, the glycoproteins of the samples needed to be cleaved, labelled, and purified with sufficient yield and selectivity. The resulting samples were analyzed by capillary electrophoresis, with laser-induced fluorescence detection. After separation parameter optimization, the capillary electrophoresis technique was implemented for efficient N-glycan profiling of whole blood samples from the diabetic patients. Our results revealed that there were subtle differences between the N-glycan profiles of the diabetic and control samples; in particular, two N-glycan structures were identified as potential glycobiomarkers that could reveal significant changes between the untreated/treated type 2 diabetic and control samples. By analyzing the resulting oligosaccharide profiles, clinically relevant information was obtained, revealing the differences between the untreated and HMG-CoA reductase-inhibitor-treated diabetic patients on changes in the N-glycan profile in the blood. In addition, the information from specific IgG N-glycosylation profiles in T2D could shed light on underlying inflammatory pathophysiological processes and lead to drug targets. Full article

Influenza virus is a highly contagious virus that causes significant human mortality and morbidity annually. The most effective drugs for treating influenza are the neuraminidase inhibitors, but resistance to these inhibitors has emerged, and additional drug discovery research on neuraminidase and other targets is needed. Traditional methods of neuraminidase production from embryonated eggs are cumbersome, while insect cell derived protein is less reflective of neuraminidase produced during human infection. Herein we describe a method for producing neuraminidase from a human cell line, HEK293-6E, and demonstrate the method by producing the neuraminidase from the 1918 H1N1 pandemic influenza strain. This method produced high levels of soluble neuraminidase expression (>3000 EU/mL), was enhanced by including a secretion signal from a viral chemokine binding protein, and does not require co-expression of additional proteins. The neuraminidase produced was of sufficient quantity and purity to support high resolution crystal structure determination. The structure solved using this protein conformed to the previously reported structure. Notably the glycosylation at three asparagine residues was superior in quality to that from insect cell derived neuraminidase. This method of production of neuraminidase should prove useful in further studies, such as the characterisation of inhibitor binding. Full article

Members of the Degenerin/epithelial Na⁺ channel (ENaC) protein family and the extracellular cell matrix (ECM) form a mechanosensitive complex. A core feature of this complex are tethers, which connect the channel with the ECM, however, knowledge about the nature of these tethers is scarce. N-glycans of α ENaC were recently identified as potential tethers but whether N-glycans serve as a ubiquitous feature for mechanosensation processes remains unresolved. The purpose of this study was to reveal whether the addition of N-glycans to δ ENaC—which is less responsive to shear force (SF)—increases its SF-responsiveness and whether this relies on a linkage to the ECM. Therefore, N-glycosylation motifs were introduced via site-directed mutagenesis, the resulting proteins expressed with β and γ ENaC in Xenopus oocytes, and SF-activated currents measured by two-electrode voltage-clamp. The insertion of N-glycosylation motifs increases δ ENaC’s SF responsiveness. The inclusion of a glycosylated asparagine (N) at position 487 did increase the molecular mass and provided a channel whose SF response was abolished following ECM degradation via hyaluronidase. This indicates that the addition of N-glycans improves SF-responsiveness and that this effect relies on an intact ECM. These findings further support the role of N-glycans as tethers for mechanotransduction. Full article

Envelope glycoprotein (g)B is conserved throughout the Herpesviridae and mediates fusion of the viral envelope with cellular membranes for infectious entry and spread. Like all viral envelope fusion proteins, gB is modified by asparagine (N)-linked glycosylation. Glycans can contribute to protein function, intracellular transport, trafficking, structure and immune evasion. gB of the alphaherpesvirus pseudorabies virus (PrV) contains six consensus sites for N-linked glycosylation, but their functional relevance is unknown. Here, we investigated the occupancy and functional relevance of N-glycosylation sites in PrV gB. To this end, all predicted N-glycosylation sites were inactivated either singly or in combination by the introduction of conservative mutations (N➔Q). The resulting proteins were tested for expression, fusion activity in cell–cell fusion assays and complementation of a gB-deficient PrV mutant. Our results indicate that all six sites are indeed modified. However, while glycosylation at most sites was dispensable for gB expression and fusogenicity, inactivation of N154 and N700 affected gB processing by furin cleavage and surface localization. Although all single mutants were functional in cell–cell fusion and viral entry, simultaneous inactivation of all six N-glycosylation sites severely impaired fusion activity and viral entry, suggesting a critical role of N-glycans for maintaining gB structure and function. Full article