Mining the “glycocode”–exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy
- FASEB journal : official publication of the Federation of American Societies for Experimental Biology
- Published over 5 years ago
Mucins are the main components of the gastrointestinal mucus layer. Mucin glycosylation is critical to most intermolecular and intercellular interactions. However, due to the highly complex and heterogeneous mucin glycan structures, the encoded biological information remains largely encrypted. Here we have developed a methodology based on force spectroscopy to identify biologically accessible glycoepitopes in purified porcine gastric mucin (pPGM) and purified porcine jejunal mucin (pPJM). The binding specificity of lectins Ricinus communis agglutinin I (RCA), peanut (Arachis hypogaea) agglutinin (PNA), Maackia amurensis lectin II (MALII), and Ulex europaeus agglutinin I (UEA) was utilized in force spectroscopy measurements to quantify the affinity and spatial distribution of their cognate sugars at the molecular scale. Binding energy of 4, 1.6, and 26 aJ was determined on pPGM for RCA, PNA, and UEA. Binding was abolished by competition with free ligands, demonstrating the validity of the affinity data. The distributions of the nearest binding site separations estimated the number of binding sites in a 200-nm mucin segment to be 4 for RCA, PNA, and UEA, and 1.8 for MALII. Binding site separations were affected by partial defucosylation of pPGM. Furthermore, we showed that this new approach can resolve differences between gastric and jejunum mucins.-Gunning, A. P., Kirby, A. R., Fuell, C., Pin, C., Tailford L. E., Juge, N. Mining the “glycocode”-exploring the spatial distribution of glycans in gastrointestinal mucin using force spectroscopy.
Thermostable ricin vaccine protects rhesus macaques against aerosolized ricin: Epitope-specific neutralizing antibodies correlate with protection
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 3 years ago
Ricin toxin (RT) is the second most lethal toxin known; it has been designated by the CDC as a select agent. RT is made by the castor bean plant; an estimated 50,000 tons of RT are produced annually as a by-product of castor oil. RT has two subunits, a ribotoxic A chain (RTA) and galactose-binding B chain (RTB). RT binds to all mammalian cells and once internalized, a single RTA catalytically inactivates all of the ribosomes in a cell. Administered as an aerosol, RT causes rapid lung damage and fibrosis followed by death. There are no Food and Drug Administration-approved vaccines and treatments are only effective in the first few hours after exposure. We have developed a recombinant RTA vaccine that has two mutations V76M/Y80A (RiVax). The protein is expressed in Escherichia coli and is nontoxic and immunogenic in mice, rabbits, and humans. When vaccinated mice are challenged with injected, aerosolized, or orally administered (gavaged) RT, they are completely protected. We have now developed a thermostable, aluminum-adjuvant-containing formulation of RiVax and tested it in rhesus macaques. After three injections, the animals developed antibodies that completely protected them from a lethal dose of aerosolized RT. These antibodies neutralized RT and competed to varying degrees with a panel of neutralizing and nonneutralizing mouse monoclonal antibodies known to recognize specific epitopes on native RTA. The resulting antibody competition profile could represent an immunologic signature of protection. Importantly, the same signature was observed using sera from RiVax-immunized humans.
Bioenergy production combined with phytoremediation has been suggested to help in solving two critical world problems: the gradual reduction of fossil fuels and soil contamination. The aim of this research was to investigate the potential for the use of Ricinus communis L. (castor oil plant) as an energy crop and plant species to remediate metal-polluted sites. This study was performed in mine tailings containing high concentrations of Cu, Zn, Mn, Pb and Cd. Physico-chemical characterization, total, DTPA-extractable and water-soluble metals in rhizospheric tailings heap samples were carried. Metal concentrations in plant tissues and translocation factors (TFs) were also determined. The Ricinus seed-oil content was high between 41 and 64%, seeds from San Francisco site 6 had the highest oil content, while these from site 7 had the lowest. No trend between oil yield vs seed origin site was observed. Seed-oil content was negatively correlated with root concentration of Cu, Zn, Pb and Cd, but no correlation was observed with the extractable-metals. According to its shoot metal concentrations and TFs, castor bean is not a metal accumulator plant. This primary colonizing plant is well suited to cope with the local toxic conditions and can be useful for the stabilization of these residues, and for then decreasing metal bioavailability, dispersion and human health risks on these barren tailings heaps and in the surrounding area. Our work is the first report regarding combined oil production and a phytostabilization role for Ricinus plants in metal mine tailings and may give a new value to suitable metal-polluted areas.
OBJECTIVE: In immunotherapy of HPV-16-associated cervical cancers, the E7 protein is considered as a prime candidate. However, it is a poor inducer of a cytotoxic T-cell response when used as a singular antigen in protein vaccination. Therefore, to design effective cancer vaccines, the best tumor antigens should be combined with the most effective immunogens or drug delivery tools to achieve positive clinical results. In this study, we fused HPV-16 E7 with the lectin subunit of ricin toxin (RTB) from castor plant as a vaccine adjuvant/carrier. MATERIALS AND METHODS: After reaching the soluble form of the recombinant protein, we designed 2 preventive and inhibition tumor models for investigation of the prevention and rejection of TC-1 cell growth in female C57BL/6 mice, respectively. In each model, mice were immunized with the recombinant protein of E7-RTB or E7 without any adjuvant. RESULTS: We demonstrated that prophylactic immunization of E7-RTB protected mice against challenge from TC-1 cells. Also in the therapeutic model, E7-RTB could inhibit TC-1 tumor growth in the lung. The results were significant compared with the immunization of E7 singularly. CONCLUSIONS: We concluded that immunization with E7-RTB protein without any adjuvant could generate antitumor effects in mice challenged with TC-1 cells. This research verifies the clinical applications and the future prospects for development of HPV-16 E7 therapeutic vaccines fused to immunoadjuvants.
In this study, we used a mass spectrometry-based quantification approach employing isotopic (ICPL) and isobaric (iTRAQ) labeling to investigate the pattern of protein deposition during castor oil seed (Ricinus communis L.) development, including that of proteins involved in fatty acid metabolism, seed storage proteins, toxins and allergens. Additionally, we have used an off-line Hydrophilic Interaction Chromatography (HILIC) as a step of peptide fractionation preceding the Reverse Phase nanoLC coupled with a LTQ Orbitrap. We were able to identify a total of 1875 proteins and from these 1748 could be mapped to extant castor gene models, expanding considerably the number proteins so far identified from developing castor seeds. Cluster validation and statistical analysis resulted in 975 protein trend patterns and the relative abundance of 618 proteins. The results presented in this work give important insights into certain aspects of the biology of castor oil seed development such as carbon flow, anabolism and catabolism of fatty acid and the pattern of deposition of reserve proteins, toxins and allergens such as ricin and 2S albumins. We also found, for the first time, some genes of reserve proteins that are differentially expressed during seed development.
Context. Ingestions of the seed of the castor bean plant (Ricinus communis) carries the risk of toxicity from ricin, a potent inhibitor of protein synthesis. Objective. We sought to describe characteristics of castor bean seed exposures reported to a state-wide poison control system. Methods. This was an observational case series. A state-wide poison control system’s database was reviewed for exposures to castor bean plant seeds from 2001 to 2011. Case notes were reviewed and data collected, when available, included age, gender, circumstances surrounding exposure, number of castor beans consumed, whether beans were chewed or crushed, symptoms described, laboratory values (aspartate aminotransferase [AST], alanine aminotransferase [ALT], prothrombin time [PT] and international normalized ratio [INR]), duration of follow-up, treatment, and patient outcomes. Results. Eighty-four cases were identified. Ingestions were unintentional in 50 cases (59%) cases and intentional in 34 (40%) cases. A median of 10 seeds (range: 1-20) were ingested in intentional cases versus 1 seed (range: 1-40) in unintentional cases. In 49 (58%) of cases the seeds were reported to have been chewed or crushed. Gastrointestinal symptoms were the most commonly reported symptoms. Vomiting (n = 39), nausea (n = 24), diarrhea (n = 17), and abdominal pain (n = 16) predominated. One patient developed hematochezia and vomiting after reportedly ingesting and intravenously injecting castor bean seeds. Laboratory values were documented in 17 (20%) cases. Only one abnormality was noted; an asymptomatic patient one week following ingestion had AST/ALT of 93 U/L and 164 U/L, respectively. Ricinine was confirmed in the urine of two patients. Twenty-three (27%) cases received activated charcoal. Seventy-two (86%) of cases were calls from health care facilities or referred to health care facilities by the poison control center. Twenty-two (26%) cases were admitted for a median of 2 days (range: 1-10). Admitted cases ingested a median of 8.5 seeds (range: 1-20). Intentional ingestions were followed for median of 37.5 h (range: 0.5-285.5) while unintentional cases were followed for 14 h (range: 1-182). No delayed symptoms, serious outcomes, or deaths were reported. Discussion. Due to the presence of ricin, there is concern for serious outcomes after ingestions of the seeds of the castor bean plant. In this study GI symptoms were most commonly reported but serious morbidity or mortality was not present. The true risk of castor bean plant seed ingestions should continue to be re-evaluated. Conclusion. In this retrospective review, gastrointestinal symptoms were the most common symptoms described after reported exposures to castor bean seeds. These exposures were not associated with serious morbidity, mortality, or delayed symptoms.
Plants store volatile compounds in specialized organs. The properties of these storage organs prevent precarious evaporation and protect neighbouring tissues from cytotoxicity. Metabolic engineering of plants is often done in tissues such as leaf mesophyll cells, which are abundant and easily accessible by engineering tools. However, these tissues are not suitable for the storage of volatile and hydrophobic compound such as sesquiterpenes and engineered volatiles are often lost into the headspace. In the present study we show that the seeds of Arabidopsis thaliana, which naturally contain lipid bodies, accumulate sesquiterpenes upon engineered expression. Subsequently, storage of volatile sesquiterpenes was achieved in Nicotiana benthamiana leaf tissue, by introducing oleosin-coated lipid bodies through metabolic engineering. Hereto, different combinations of genes encoding diacylglycerol acyltransferases (DGATs), transcription factors (WRINKL1) and oleosins (OLE1), from the oil-seed producing species Castor bean (Ricinus communis) and Arabidopsis, were assessed for their suitability to promote lipid body formation. Co-expression of α-bisabolol-synthase with Arabidopsis DGAT1 and WRINKL1 and OLE1 from Castor bean promoted storage of α-bisabolol in N. benthamiana mesophyll tissue more than 17 fold. A clear correlation between neutral lipids and storage of sesquiterpenes, using synthases for α-bisabolol, (E)-β-caryophyllene and α-barbatene. The co-localisation of neutral lipids and α-bisabolol was shown using microscopy. This work demonstrates that lipid bodies can be used as intracellular storage compartment for hydrophobic sesquiterpenes, also in the vegetative parts of plants, creating the possibility to improve yields of metabolic engineering strategies in plants. This article is protected by copyright. All rights reserved.
As an important glycosaminoglycan, keratan sulfate (KS) mainly exists in corneal and cartilage, possessing various biological activities. In this study, we purified KS from blue shark (Prionace glauca) cartilage and prepared KS oligosaccharides (KSO) through keratanase II-catalyzed hydrolysis. The structures of KS and KSO were characterized using multi-dimensional nuclear magnetic resonance (NMR) spectra and liquid chromatography-mass spectrometry (LC-MS). Shark cartilage KS was highly sulfated and modified with ~2.69% N-acetylneuraminic acid (NeuAc) through α(2,3)-linked to galactose. Additionally, KS exhibited binding affinity to Ricinus communis agglutinin I (RCA120) in a concentration-dependent manner, a highly toxic lectin from beans of the castor plant. Furthermore, KSO from dp2 to dp8 bound to RCA120 in the increasing trend while the binding affinity of dp8 was superior to polysaccharide. These results define novel structural features for KS from Prionace glauca cartilage and demonstrate the potential application on ricin-antidote exploitation.
In this study, a series of diblock glycopolymers, poly(6-O-methacryloyl-d-galactopyranose)-b-poly(6-cholesteryloxyhexyl methacrylate) (PMAgala-b-PMAChols), with cholesterol/galactose grafts were prepared through a sequential reversible addition-fragmentation chain transfer (RAFT) polymerization and deprotection process. The glycopolymers could self-assemble into aggregates with various morphologies depending on cholesterol/galactose-containing block weight ratios, as determined by transmission electronic microscopy (TEM) and dynamic laser light scattering (DLS). In addition, the lectin (Ricinus communis agglutinin II, RCA120) recognition and bovine serum albumin (BSA) adsorption of the PMAgala-b-PMAChol aggregates were evaluated. The SK-Hep-1 tumor cell inhibition properties of the PMAgala-b-PMAChol/doxorubicin (DOX) complex aggregates were further examined in vitro. Results indicate that the PMAgala-b-PMAChol aggregates with various morphologies showed different interaction/recognition features with RCA120and BSA. Spherical aggregates (d ≈ 92 nm) possessed the highest RCA120recognition ability and lowest BSA protein adsorption. In addition, the DOX-loaded spherical complex aggregates exhibited a better tumor cell inhibition property than those of nanofibrous complex aggregates. The morphology-variable aggregates derived from the amphiphilic glycopolymers may serve as multifunctional biomaterials with biomolecular recognition and drug delivery features.
Ricinus communis (castor plant) is a potent medicinal plant, which is commonly used in the treatment of various ailments. The present study was conducted to appraise the cytotoxicity and mutagenicity of R. communis along with antioxidant and antimicrobial activities. Cytotoxicity was evaluated by hemolytic and brine shrimp assays, whereas Ames test (TA98 and TA100) was used for mutagenicity evaluation. Plant different parts were extracted in methanol by shaking, sonication and Soxhlet extraction methods. The R. communis methanolic extracts showed promising antioxidant activity evaluated as through total phenolic contents (TPC), total flavonoid content (TFC), DPPH free radical inhibition, reducing power and inhibition of linoleic acid oxidation. R. communis seeds, stem, leaves, fruit and root methanolic extracts showed mild to moderate cytotoxicity against red blood cells (RBCs) of human and bovine. Brine shrimp lethality also revealed the cytotoxic nature of extracts with LC50 in the range of 0.22-3.70 (µg/mL) (shaking), 1.59-60.92 (µg/mL) (sonication) and 0.72-33.60 (µg/mL) (Soxhlet), whereas LC90 values were in the range of 345.42-1695.81, 660.50-14,794.40 and 641.62-15,047.80 µg/mL for shaking, sonication and Soxhlet extraction methods, respectively. R. communis methanolic extracts revealed mild mutagenicity against TA98 (range 1975 ± 67 to 2628 ± 79 revertant colonies) and TA100 (range 2773 ± 92 to 3461 ± 147 revertant colonies) strains and these values were 3267 ± 278 and 4720 ± 346 revertant colonies in case of TA98 and TA100 positive controls, respectively. R. communis methanolic extracts prevented the H2O2 and UV to Plasmid pBR322 DNA oxidative damage. Results revealed that R. communis is a potential source of bioactive compounds and in future studies the bioactive compounds will be identified by advanced spectroscopic techniques.