Euonymus Europaeus Lectin Research Articles

Roles of glycoprotein glycosylation in the pathogenesis of an endemic osteoarthritis, Kashin–Beck disease, and effectiveness evaluation of sodium hyaluronate treatment

Background/aim We aimed to explore the roles of glycoprotein glycosylation in the pathogenesis of Kashin–Beck disease (KBD), and evaluated the effectiveness of sodium hyaluronate treatment. Materials and methods Blood and saliva were collected from KBD patients before and after the injection of sodium hyaluronate. Normal healthy subjects were included as controls. Saliva and serum lectin microarrays and saliva and serum microarray verifications were used to screen and confirm the differences in lectin levels among the three groups. Results In saliva lectin microarray, bindings to Sophora japonica agglutinin (SJA), Griffonia (Bandeiraea) simplicifolia lectin I (GSL-I), Euonymus europaeus lectin (EEL), Maackia amurensis lectin II (MAL-II), Sambucus nigra lectin (SNA), Hippeastrum hybrid lectin (HHL), and Aleuria aurantia lectin (AAL) were higher in the untreated KBD patients than in the control group. Increased levels of HHL, MAL-II, and GSL-I in the untreated KBD patients discriminated them in particular from the treated ones. Jacalin was lower in the untreated KBD patients compared to the treated KBD and control groups. In serum lectin microarray, HHL and peanut agglutinin (PNA) were increased in the untreated KBD group in comparison to the control one. AAL, Phaseolus vulgaris agglutinin (E+L) (PHA-E+L), and Psophocarpus tetragonolobus lectin I (PTL-I) were lower in the untreated KBD patients compared to the treated KBD and control groups. Hyaluronate treatment appeared to normalize SNA, AAL, and MAL-II levels in saliva, and HHL, PNA, AAL, PTL-I, and PHA-E+L levels in serum. Saliva reversed microarray verification confirmed significant differences between the groups in SNA and Jacalin, in particular for GSL-I levels, while serum reversed microarray verification indicated that HHL, PNA, and AAL levels returned to normal levels after the hyaluronate treatment. Lectin blot confirmed significant differences in HHL, AAL, and Jacalin in saliva, and HHL, PNA, PHA-E+L, and AAL in serum. Conclusion HHL in saliva and serum may be a valuable diagnostic biomarker of KBD, and it may be used as follow-up for the hyaluronate treatment.

Subtle Differences in Symbiont Cell Surface Glycan Profiles Do Not Explain Species-Specific Colonization Rates in a Model Cnidarian-Algal Symbiosis.

Mutualisms between cnidarian hosts and dinoflagellate endosymbionts are foundational to coral reef ecosystems. These symbioses are often re-established every generation with high specificity, but gaps remain in our understanding of the cellular mechanisms that control symbiont recognition and uptake dynamics. Here, we tested whether differences in glycan profiles among different symbiont species account for the different rates at which they initially colonize aposymbiotic polyps of the model sea anemone Aiptasia (Exaiptasia pallida). First, we used a lectin array to characterize the glycan profiles of colonizing Symbiodinium minutum (ITS2 type B1) and noncolonizing Symbiodinium pilosum (ITS2 type A2), finding subtle differences in the binding of lectins Euonymus europaeus lectin (EEL) and Urtica dioica agglutinin lectin (UDA) that distinguish between high-mannoside and hybrid-type protein linked glycans. Next, we enzymatically cleaved glycans from the surfaces of S. minutum cultures and followed their recovery using flow cytometry, establishing a 48–72 h glycan turnover rate for this species. Finally, we exposed aposymbiotic host polyps to cultured S. minutum cells masked by EEL or UDA lectins for 48 h, then measured cell densities the following day. We found no effect of glycan masking on symbiont density, providing further support to the hypothesis that glycan-lectin interactions are more important for post-phagocytic persistence of specific symbionts than they are for initial uptake. We also identified several methodological and biological factors that may limit the utility of studying glycan masking in the Aiptasia system.

Sorafenib induced alteration of protein glycosylation in hepatocellular carcinoma cells.

Sorafenib is a multikinase inhibitor and is effective in treating hepatocellular carcinoma (HCC). However, it remains unknown whether sorafenib induces the alteration of protein glycosylation. The present study treated HCC MHCC97L and MHCC97H cells with a 50% inhibitory concentration of sorafenib. Following this treatment, alteration of protein glycosylation was detected using a lectin microarray. Compared with the controls, the binding capacity of glycoproteins extracted from sorafenib-treated HCC cells to the lectins Bauhinia purpurea lectin, Dolichos biflorus agglutinin, Euonymus europaeus lectin, Helix aspersa lectin, Helix pomatia lectin, Jacalin, Maclura pomifera lectin and Vicia villosa lectin were enhanced; while, the binding capacities to the lectins Caragana arborescens lectin, Lycopersicon esculentum lectin, Limulus polyphemus lectin, Maackia amurensis lecin I, Phaseolus vulgaris leucoagglutinin, Ricinus communis agglutinin 60, Sambucus nigra lectin and Solanum tuberosum lectin were reduced (spot intensity median/background intensity median ≥2, P<0.05). This difference in glycoprotein binding capacity indicates that cells treated with sorafenib could increase α-1,3GalNAc/Gal, β-1,3 Gal, GalNAcα-Ser/Thr(Tn) and α-GalNAc structures and decrease GlcNAc, sialic acid, tetra-antennary complex-type N-glycan and β-1,4Gal structures. These results were additionally confirmed by lectin blotting. Expression levels of signaling molecules including erythroblastosis 26–1 (Ets-1), extracellular signal-related kinases (ERK) and phosphorylated-ERK were measured by western blotting. There was a reduction in the expression of Ets-1 and ERK phosphorylation in sorafenib or 1,4-Diamino-2,3-dicyano-1,4-bis (2-aminophenylthio) butadiene treated cells suggesting that sorafenib may reduce the expression levels of Ets-1 by blocking the Ras/Raf/mitogen activated protein kinase signaling pathway. In the present study, it was clear that sorafenib could inhibit the proliferation of HCC cells and alter protein glycosylation. The findings of this study may lead to providing a novel way of designing new anti-HCC drugs.

The carbohydrate-binding promiscuity of Euonymus europaeus lectin is predicted to involve a single binding site.

Euonymus europaeus lectin (EEL) is a carbohydrate-binding protein derived from the fruit of the European spindle tree. EEL was first identified for its erythrocyte agglutinating properties and specificity for B and H blood groups. However, a detailed molecular picture of the structural basis of carbohydrate recognition by EEL remains to be developed. In this study, we performed fluorescence titrations of a range of carbohydrates against EEL. Binding of EEL to a wide range of carbohydrates was observed, including a series of blood group-related carbohydrates, mannosides, chitotriose and sialic acid. Affinity was strongest for carbohydrates with H-related structures and the B trisaccharide. A homology model of EEL was produced from templates identified using the HHPred server, which employs hidden Markov models (HMMs) to identify templates. The HMM approach identified that the best templates for EEL were proteins featuring a ricin B-like (R-type) fold. Separate templates were used to model the core and binding site regions of the lectin. Through the use of constrained docking and spatial comparison with a template ligand, binding modes for the carbohydrate ligands were predicted. A relationship between the experimental binding energies and the computed binding energies of the selected docked poses was determined and optimized. Collectively, our results suggest that EEL utilizes a single site for recognition of carbohydrates terminating in a variety of monosaccharides.

Over-expression of ArathEULS3 confers ABA sensitivity and drought tolerance in Arabidopsis

Abscisic acid (ABA) is an important phytohormone involved in the regulation of plant growth, development and adaption to various environmental challenges. Regulatory component of ABA receptor 1 (RCAR1, also known as PYL9) acts as a newly discovered ABA receptor in Arabidopsis. To identify interacting partners of RCAR1, we have carried out a yeast two-hybrid screen. One protein was identified, ArathEULS3, which belongs to the Euonymus europaeus lectin (EUL) family of plant lectins. The interaction between RCAR1 and ArathEULS3 was confirmed by GST pull-down assay. Transient expression of RCAR1-EGFP and ArathEULS3-EGFP in Arabidopsis protoplasts revealed that both proteins were mainly expressed in cytoplasm and nucleus. Real time qRT-PCR analysis showed that over-expression of RCAR1 increased the expression of ArathEULS3. Furthermore, up-regulating ArathEULS3 in Arabidopsis conferred ABA hypersensitivity during post-germination growth and enhanced drought tolerance, but did not affect the expression of RD29B, RAB18 and RD29A (ABA- and drought-responsive genes). Previously, ArathEULS3 was shown as a carbohydrate-binding plant lectin. Thus, our results reveal a direct connection between abiotic stress responses and plant lectin.

Lectin microarray analysis of pluripotent and multipotent stem cells

Stem cells have a capability to self-renew and differentiate into multiple types of cells; specific markers are available to identify particular stem cells for developmental biology research. In this study, we aimed to define the status of somatic stem cells and the pluripotency of human embryonic stem (hES) and induced pluripotent stem (iPS) cells using a novel molecular methodology, lectin microarray analysis. Our lectin microarray analysis successfully categorized murine somatic stem cells into the appropriate groups of differentiation potency. We then classified hES and iPS cells by the same approach. Undifferentiated hES cells were clearly distinguished from differentiated hES cells after embryoid formation. The pair-wise comparison means based on 'false discovery rate' revealed that three lectins -Euonymus europaeus lectin (EEL), Maackia amurensis lectin (MAL) and Phaseolus vulgaris leucoagglutinin [PHA(L)]- generated maximal values to define undifferentiated and differentiated hES cells. Furthermore, to define a pluripotent stem cell state, we generated a discriminant for the undifferentiated state with pluripotency. The discriminant function based on lectin reactivities was highly accurate for judgment of stem cell pluripotency. These results suggest that glycomic analysis of stem cells leads to a novel comprehensive approach for quality control in cell-based therapy and regenerative medicine.

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

BackgroundCloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases.ResultsComprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed.ConclusionThe identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family.

Impact of adsorbents selection on capture efficiency of cell culture derived human influenza viruses

The study aims on affinity matrix selection for a cell culture derived influenza virus capture step in downstream processing. Euonymus europaeus lectin (EEL) was used as an affinity ligand. Human influenza A/Puerto Rico/8/34 (H1N1) virus produced in MDCK cells was chosen as a model strain. The chromatographic separation characteristics of reinforced cellulose membranes and different matrices such as agarose, cellulose, polymer and glass particles with immobilized EEL have been determined. Results obtained were compared to affinity matrices, which are currently used in large-scale vaccine manufacturing. Mass balances for the viral membrane protein hemagglutinin showed that EEL affinity chromatography results in higher recoveries than conventional processes using Cellufine™ sulphate and heparinized agarose. The most efficient media, a polymer and a cellulose membrane, have been further characterized by protein and host cell DNA measurements. Separations based on the polymer matrix and the cellulose membrane removed contaminating DNA to 0.2 and 1%, respectively. Total protein contents were decreased to 50 and 31%, respectively. The EEL-membrane showed the highest influenza virus binding capacity. These characteristics demonstrate that EEL affinity chromatography is a promising candidate for capturing influenza viruses from MDCK cell culture broths in addition to currently applied chromatographic media.

Lectin-induced damage to the enterocyte brush border. An electron-microscopic study in rabbits.

The ultrastructural effects of 11 lectins on the intestinal brush border were examined by means of an in vitro rabbit ileal mucosal explant culture system. Five of the lectins that bind to oligosaccharides containing either N-acetylglucosamine (phytohaemagglutinin, Euonymus europaeus lectin, pokeweed mitogen, and wheat germ agglutinin) or N-acetylneuraminic acid (Mycoplasma gallisepticum lectin) all had a specific effect on microvilli. The effects varied in accordance with the lectin and included lengthening, distortion, and vesiculation of the microvilli. In contrast, lectins binding specifically to galactose, glucose, mannose, and N-acetylgalactosamine had no effect. Incubation of mucosal fragments with the divalent cation ionophore A23187 did not mimic the effect of the lectins. This apparent relationship between lectin damage and receptor specificity may reflect either accessibility of appropriate binding sites or a differential response to binding.

Isolation and immunochemical characterization of the Euonymus europaeus lectin receptor from the major sialoglycoprotein of human O erythrocytes.

Purified and desialized blood‐group‐M glycoprotein from human O erythrocytes precipitated with a lectin isolated from Euonymus europaeus seeds. The precipitation was inhibited completely by lacto‐N‐fucopentaose I and lactose. The H2 N‐terminal tryptic fragment of glycoprotein M (MT), containing most of carbohydrate and all fucose of glycoprotein M [Lisowska and Jeanloz (1973) Carbohydr. Res. 29, 181 ‐ 191], did not precipitate with the lectin but did inhibit the precipitation. Reduced alkali‐labile oligosaccharides and glycopeptide containing alkali‐stable oligosaccha‐ride were isolated from the desialized MT glycopeptide by alkaline borohydride degradation and gel nitration. Only glycopeptide with the alkali‐stable oligosaccharide inhibited the precipitation and on a molar basis it was as active as glycopeptide MT‐ Both gave 50% inhibition at about 0.76 mM. Two desialized oligosaccharides obtained as hydrazinolysis products of glycoprotein M, were also as active as glycopeptide MT in the inhibition test. This shows that both oligosaccharides as well as alkali‐stable oligosaccharide linked to glycopeptide MT have determinants of the same structure. The chemical composition of alkali‐stable oligosaccharides and their pattern of inhibition of precipitation strongly suggest that l‐fucose α1‐linked at the nonreducing end is an immuno‐dominant group and that grouping l‐Fucα1 → DGal →(GlcNAc, Gal, Man) is present in their molecules. The inhibition of the precipitation by glycopeptide MT is in agreement with the presence of only one alkali‐stable oligosaccharide chain on the glycoprotein M molecule [Tomita, Furthmayr and Marchesi (1978) Biochemistry, 17, 4756 ‐ 4770]. These results as well as the restriction of lectin receptor site to alkali‐stable oligosaccharide, show that glycopeptide MT is monovalent in the reaction with the lectin.