Nanoparticles, especially gold nanoparticles (GNPs) have emerged as promising tools for biomedical applications due to their unique properties and ability to be functionalized. However, quantitative ...analysis of biomolecules conjugated to nanoparticles remains a challenge. Here we report a method to conjugate a synthetic hybrid‐type N‐glycan to GNPs and quantitatively analyze the glycan content. The glycan was first conjugated to N‐hydroxysuccinimide (NHS)‐ester activated GNPs and confirmed qualitatively by Fourier‐transform infrared spectroscopy and flow cytometry. The glycan conjugated‐GNPs were then treated with neuraminidase to release terminal sialic acid from the glycan, which was labeled with 1,2‐diamino‐4,5‐methylenedioxybenzene and quantitatively analyzed by Ultraperformance Liquid Chromatography (UPLC). The amount of the sialic acid released was equivalent to the glycan content on GNPs. This method enabled a precise quantification of glycans conjugated to gold nanoparticles and should facilitate its biomedical applications, including studies of multivalent receptor‐glycan interaction, cell targeting and sorting, and immunization. Overall, this work provides an effective approach to synthesize and characterize nanoparticles conjugates.
The influenza virus hemagglutinin (HA) and coronavirus spike (S) protein mediate virus entry. HA and S proteins are heavily glycosylated, making them potential targets for carbohydrate binding agents ...such as lectins. Here, we show that the lectin FRIL, isolated from hyacinth beans (Lablab purpureus), has anti-influenza and anti-SARS-CoV-2 activity. FRIL can neutralize 11 representative human and avian influenza strains at low nanomolar concentrations, and intranasal administration of FRIL is protective against lethal H1N1 infection in mice. FRIL binds preferentially to complex-type N-glycans and neutralizes viruses that possess complex-type N-glycans on their envelopes. As a homotetramer, FRIL is capable of aggregating influenza particles through multivalent binding and trapping influenza virions in cytoplasmic late endosomes, preventing their nuclear entry. Remarkably, FRIL also effectively neutralizes SARS-CoV-2, preventing viral protein production and cytopathic effect in host cells. These findings suggest a potential application of FRIL for the prevention and/or treatment of influenza and COVID-19.
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•FRIL is a plant lectin with potent anti-influenza and anti-SARS-CoV-2 activity•FRIL preferentially binds to complex-type N-glycans on viral glycoproteins•FRIL inhibits influenza virus entry by sequestering virions in late endosomes•Intranasal administration of FRIL protects against lethal H1N1 challenge in mice
Liu et al. demonstrate that FRIL, a plant lectin isolated from the hyacinth bean, has potent antiviral activity against SARS-CoV-2 and diverse influenza virus strains. FRIL is effective in vivo against H1N1. FRIL’s antiviral activity is mediated by binding to complex-type N-glycans on viral glycoproteins, interfering with viral entry.
Since the outbreak of Severe Acute Respiratory Syndrome Virus-2 (SARS-CoV-2) in 2019, more than 15 million spike protein sequences have been identified, raising a new challenge for the development of ...a broadly protective vaccine against the various emerging variants. We found that the virus, like most other human viruses, depends on host-made glycans to shield the conserved epitopes on spike protein from immune response and demonstrated that deletion of the glycan shields exposed highly conserved epitopes and elicited broadly protective immune responses. In this study, we identified 17 conserved epitopes from 14 million spike protein sequences and 11 of the conserved epitopes are in the S2 domain, including the six most conserved epitopes in the stem region. We also demonstrated that deletion of the glycosites in the spike messenger RNA (mRNA) S2 domain or the stem region exposed the highly conserved epitopes and elicited broadly protective immune responses, particularly CD-8
T cell response against various SARS-CoV-2 variants, and other human coronaviruses including MERS, SARS viruses, and those causing common cold.
Development of the messenger RNA (mRNA) vaccine has emerged as an effective and speedy strategy to control the spread of new pathogens. After vaccination, the mRNA is translated into the real protein ...vaccine, and there is no need to manufacture the protein in vitro. However, the fate of mRNA and its posttranslational modification inside the cell may affect immune response. Here, we showed that the mRNA vaccine of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with deletion of glycosites in the receptor-binding domain (RBD) or especially the subunit 2 (S2) domain to expose more conserved epitopes elicited stronger antibody and CD8
T cell responses with broader protection against the alpha, beta, gamma, delta, and omicron variants, compared to the unmodified mRNA. Immunization of such mRNA resulted in accumulation of misfolded spike protein in the endoplasmic reticulum, causing the up-regulation of BiP/GRP78, XBP1, and p-eIF2α to induce cell apoptosis and strong CD8
T cell response. In addition, dendritic cells (DCs) incubated with S2-glysosite deleted mRNA vaccine increased class I major histocompatibility complex (MHC I) expression. This study provides a direction for the development of broad-spectrum mRNA vaccines which may not be achieved with the use of expressed proteins as antigens.
The globo-series glycosphingolipids (GSLs) SSEA3, SSEA4, and Globo-H specifically expressed on cancer cells are found to correlate with tumor progression and metastasis, but the functional roles of ...these GSLs and the key enzyme β1,3-galactosyltransferase V (β3GalT5) that converts Gb4 to SSEA3 remain largely unclear. Here we show that the expression of β3GalT5 significantly correlates with tumor progression and poor survival in patients, and the globoseries GSLs in breast cancer cells form a complex in membrane lipid raft with caveolin-1 (CAV1) and focal adhesion kinase (FAK) which then interact with AKT and receptor-interacting protein kinase (RIP), respectively. Knockdown of β3GalT5 disrupts the complex and induces apoptosis through dissociation of RIP from the complex to interact with the Fas death domain (FADD) and trigger the Fas-dependent pathway. This finding provides a link between SSEA3/SSEA4/Globo-H and the FAK/CAV1/AKT/RIP complex in tumor progression and apoptosis and suggests a direction for the treatment of breast cancer, as demonstrated by the combined use of antibodies against Globo-H and SSEA4.
causes pneumonia and liver abscesses in humans worldwide and contains virulence factor capsular polysaccharides and lipopolysaccharides linked to the cell wall. Although capsular polysaccharides are ...good antigens for vaccine production and capsular oligosaccharides conjugate vaccines are proven effective against infections caused by encapsulated pathogens, there is still no
vaccine available. One obstacle is that the capsular polysaccharide of a dominated
serotype K2 is difficult to synthesize chemically due to the three 1,2-
linkages in its structure. In this study, we successfully synthesized K2 capsular polysaccharides from tetra- to octasaccharides in highly a stereoselective manner. Subsequently, three synthesized glycans were conjugated to DT protein to provide glycoconjugate vaccine candidates (DT-Hexa, DT-Hepta, and DT-Octa) that were used in
immunization experiments in mice. The results of immunized studies showed all three glycoconjugates elicited antibodies that recognized all of the synthetic glycans at 1:200-fold dilution. Particularly, the DT-Hepta conjugate elicited a higher level of antibodies that can recognize longer glycan (octasaccharide) even at 1:12800-fold dilution and exhibited good bactericidal activity. Our results concluded that heptasaccharide is the minimal epitope and a potential candidate for the vaccine against the K2 sero group of
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is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum β-lactams and carbapenem. In recent years,
isolates have ...emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no
vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of
by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human
infections.
Abstract
A novel Gal-binding lectin from mussels (Crenomytilus grayanus, CGL) with 6 binding sites in the dimeric structure has been previously shown to have antifungal, anticancer, and antibacterial ...activities. In this study, a glycan array was used to confirm that CGL recognizes a range of non-reducing end α- or β-linked Gal glycans on normal cells but not sialic acid-capped glycans. This finding suggests that CGL has potential in the tumor detection due to the hyper-sialylation present in cell surface glycans from cancer cells. To evaluate the feasibility of this possibility, we labeled CGL with biotin and then mixed it with streptavidin-horseradish peroxidase (HRP) to create a CGL-biotin-SP complex as a probe for use in enzyme-linked lectin assays. CGL-biotin-SP successfully distinguished not only HeLa cells and de-sialylated HeLa cells that mimic normal cell surface glycans but also lung and breast cancer cells with different metastatic abilities. This work provides the insights into a new Gal-binding lectin by establishing its specificity and also demonstrates practical applications in cancer diagnosis greater than other reported lectins.
Deciphering signaling mechanisms critical for the extended pluripotent stem cell (EPSC) state and primed pluripotency is necessary for understanding embryonic development. Here, a membrane protein, ...podocalyxin‐like protein 1 (PODXL) as being essential for extended and primed pluripotency, is identified. Alteration of PODXL expression levels affects self‐renewal, protein expression of c‐MYC and telomerase, and induced pluripotent stem cell (iPSC) and EPSC colony formation. PODXL is the first membrane protein reported to regulate de novo cholesterol biosynthesis, and human pluripotent stem cells (hPSCs) are more sensitive to cholesterol depletion than fibroblasts. The addition of exogenous cholesterol fully restores PODXL knockdown‐mediated loss of pluripotency. PODXL affects lipid raft dynamics via the regulation of cholesterol. PODXL recruits the RAC1/CDC42/actin network to regulate SREBP1 and SREBP2 maturation and lipid raft dynamics. Single‐cell RNA sequencing reveals PODXL overexpression enhanced chimerism between human cells in mouse host embryos (hEPSCs 57%). Interestingly, in the human–mouse chimeras, laminin and collagen signaling‐related pathways are dominant in PODXL overexpressing cells. It is concluded that cholesterol regulation via PODXL signaling is critical for ESC/EPSC.
Cholesterol is important for membrane composition and cell division. Chen et al. show that PODXL regulates de novo cholesterol biosynthesis and further sustains the primed and extended renewal states in hPSCs through c‐MYC, TERT, and ITGA2. PODXL overexpression facilitates the extended pluripotency state in vitro and in vivo. These data highlight that PODXL links cellular metabolism to stem cell self‐renewal.
Pseudaminic acid (Pse) has been known for participating in crucial bacterial virulence and thus is an attractive target in the development of glycoconjugate vaccine. Particularly, this therapeutic ...alternative was suggested to be a potential solution against antibiotic resistant Acinetobacter baumannii that poses a serious global health threat. Also, Pse was found to be involved in the exopolysaccharide (EPS) of mild antibiotic resistant A. baumannii strain 54149 ( Ab-54149) of which specific glycosyl linkage can be depolymerized by phage ΦAB6 tailspike protein (ΦAB6TSP). In this study, we found that the antibodies induced by Ab-54149 EPS was capable of recognizing a range of EPS of other clinical A. baumannii strains, and deemed as a great potential material for vaccination. To efficiently acquire homogeneous EPS-derived oligosaccharide with significant immunogenic activity for the production of glycoconjugate, we used the ΦAB6TSP for the fragmentation of Ab-54149 EPS instead of chemical methods. Moreover, insight into the ligand binding characterization of ΦAB6TSP suggested the branched Pse on the Ab-54149 EPS served as a recognition site of ΦAB6TSP. The serum boosted by ΦAB6TSP-digested product and carrier protein CRM197 conjugate complex displayed specific sensitivity toward Ab-54149 EPS with bacterial killing activity. Strikingly, Pse is an ideal epitope with strong antigenicity, profiting the application of the probe for pathogen detection and glyco-based vaccine.
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