Purification and structural studies of a Tremella fuciformis mushroom lectin

Lectins are carbohydrate-binding proteins or glycoproteins of non-immune origine widely distributed in living organisms including animals, plants and fungi. They play a role in different biological processes mediating cellular signaling, differentiation, tissue metastasis and host-pathogen interactions. Moreover they serve as storage proteins, are fundamental during fungi and plant morphogenesis and development and take part into their defense processes [1].Thanks to their carbohydrate specific binding, some lectins are able to recognize, in a reversible way, the sugar moieties on the erythrocytes cell surface (N-acetylgalactosamine, D-galactosamine), causing a phenomena called hemagglutination. Furthermore some lectins have been found to possess antitumoral properties [2]. Specifically they recognize the Tn-antigenic determinant (Gal\u3b21-3GalNAc\u3b1) on the malignant cells surface causing apoptosis, cytotoxicity, inhibition of tumor growth and preventing the proliferation of tumor cells. Considering the fact that this kind of residues are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target only malignant cells, also because the Tn-antigen is the most specific human cancer-associated structure, expressed in about 90% of the human carcinomas.For the reasons described above, during the last decades lectins have been extensively investigated for their potential therapeautical effects and biotechonological applications, especially fungal lectins which have unique carbohydrate specificities. However, altough the function and the biological properties of many lectins have been determined, their structural characterization lags behind.As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties and have shown to possess anticancer, anti-inflammatory, antioxidant and neuroprotective activities. In the light of above the crude extract proteins have been checked to assess the presence of lectins [3]. To this purpose, the mushrooms dried fruiting bodies of Tremella fuciformis were homogenized and extracted in a phospate buffer at 4\ub0C and neutral pH. The crude extract was then precipitated using a high concentration of (NH4)2SO4 and dyalised against TRIS buffer in order to remove the precipitant. A lectin was eluted from a hog gastric mucin affinity column and purified first with a DEAE-cellulose column and then with a size exclusion SEPHACRYL G-100 column. An electrophoresis gel was required to precisely define the lectin molecular weight, which is 22 kDa. The purified lectin has been used for testing several crystal screening conditions

Purification and structural studies of a Tremella fuciformis mushroom lectin

Lectins are carbohydrate-binding proteins of non-immune origine widely distributed in living organisms. They play a role in different biological processes, serve as storage proteins, are fundamental during fungi and plant morphogenesis and development and take part in their defense processes [1]. Due to their carbohydrate specific binding, some lectins are able to recognize, in a reversible way, the sugar moieties present on the surface of erythrocytes (N-acetylgalactosamine, D-galactosamine), causing a phenomena called hemagglutination. Furthermore some lectins have been found to possess antitumoral properties [2]. Specifically they recognize the Tn-antigenic determinant (Gal\u3b21-3GalNAc\u3b1) on the malignant cells surface causing apoptosis, cytotoxicity, inhibition of tumor growth and preventing the proliferation of tumor cells. Considering the fact that this kind of residues are masked on healthy cells, the highly specific carbohydrate-lectin interaction can be exploited to target malignant cells. The Tn-antigen is the most specific human cancer-associated structure, expressed in about 90% of human carcinomas. Although the function and biological properties of several lectins have been determined, there are still many lectins that remain to be structurally and functionally characterized. As reported in the literature, some Tremella fuciformis proteins have been investigated for their potential therapeutical properties [3] and in the light of this, we have examined the crude extract proteins of this fungus to assess the presence of lectins. A lectin of 22 KDa was isolated and purified from the dried fruiting bodies and used for testing several crystal screening conditions. Crystals were grown in 0,1 M TRIS pH 8.5, 1,5 potassium phosphate dibasic and preliminary data sets were collected at the ESRF of Grenoble. The space group is P21 and the cell parameters are a= 61,6 \uc5, b= 61,8 \uc5, c= 67,8 \uc5 with \u3b2= 106,87 \ub0. The highest resolution of these crystals is 1,5 \uc5 and the total number of reflections collected were 740651. Dynamic Light Scattering (DLS) analysis reveals that TFL is a monomer under normal conditions. The distribution plot shows a size distribution of 2,9 nm \ub1 0,2 nm, with a polydispersity index (PDI) of 0,4 \ub1 0,1. Thermal protein stability was examined by means of differential scanning calorimetry, while chemical and pH-induced unfolding was investigated using fluorescence spectroscopy. Isothermal titration calorimetry yielded preliminary data on sugar binding, justifying a more detailed study to be undertaken in the future. It has also been observed that Tremella fuciformis lectin shows no cytotoxicity on malignant and healthy cells and its antitumoral properties are currently being investigated. [1] A. Varrot, S.M. Basheer, A. Imberty, Current opinion in structural biology , 2013, 23, 678-685. [2] Ju T., Otto VI, Cummings R.D., Angew. Chem. Int. Ed Engl., 2011, 50(8), 1770-91. [3] Hung C.L., Chang A-J.,Kuo, X-K and Sheu F., J.Agric.Food Chem., 2014, 62(7), 1526-35

Structural and biophysical studies on the lectin domain of GalNAc-T6 for therapeutic applications

The expression of glycoproteins containing immature truncated O-glycans such as the Thomsen-Friedenreich antigen (Ser/Thr-O-Gal\u3b21\u20133GalNAc; T-antigen) and the Lewis antigen (sialyl-T-antigen) is a characteristic feature observed on almost all malignant epithelial cells. Therefore, there is a particular interest in their application not only as prognostic markers but also as therapeutic targets [1]. These antigens can be recognized by lectins, a group of highly specific carbohydrate-binding proteins that have been proposed as useful tools for antitumor drug-targeting [2].The three-dimensional structure of several lectins with antitumor properties has been determined in our laboratory by X-ray crystallography. N-\u3b1-acetylgalactosaminyltransferase-6 (GalNAc-T6) is an enzyme present also in humans which contains a catalytic domain and a lectin domain with a binding site for N-acetylgalactosamine (GalNAc), one of the saccharides exposed by cancer cells (Tn-antigen). Unlike other lectins with these properties, the lectin domain of GalNAc-T6 presents a structural fold found also in other human proteins, unlocking the opportunity to use protein engineering tools to design new anticancer therapeutics [3]. The three-dimensional structure of GalNAc-T6 has not been determined so far, neither has been its substrate specificity. Therefore, the production of a recombinant form containing only the lectin domain can contribute to these two critical points that need to be considered to evaluate its possible use in cancer therapies. The lectin domain of this enzyme was expressed by cloning the C-terminal portion of the DNA coding sequence and introducing it into Pichia pastoris for its recombinant production. Biophysical methods such as spectrofluorimetry and isothermal titration calorimetry were used to analyze the ability of the engineered protein to bind the T-antigen monosaccharides. The binding dissociation constant (Kd) of the protein-carbohydrate interaction was determined. The stability of the protein was also studied through its thermodynamic parameters of unfolding using differential scanning calorimetry. Crystallization screenings were set up using a broad variety of precipitants in order to produce crystals to be used to study the three-dimensional structure of the engineered protein using X-ray diffraction. The crystals that were grown were taken to the European Synchrotron Radiation Facility (ESRF) in Grenoble (France) to carry out the diffraction experiments. Although we were able to collect data up to a resolution of 2.8 \uc5 (854,648 reflections) all the crystals we have examined so far were found to be twinned making the assignment of a definitive space group uncertain. We are currently working on correcting this problem using both the appropriate software and attempting to grow better crystals. Our goal is to produce an engineered human protein that specifically recognizes cancer specific carbohydrates and is thus suitable for protein therapeutics applied in drug-delivery methods for cancer treatment. The present structural and biophysical data are the prerequisite for future studies regarding the biological and clinical properties of the lectin. [1] Stowell, S. R. Tongzhong J. and Cummings R. D. Protein Glycosylation in Cancer. Annu Rev Pathol 2015. 10: 473\u2013510. [2] Sharon, N., and Lis, H. Lectins: from hemagglutinins to biological recognition molecules. A historical overview. Glycobiology. 2004. 14: 53\u201362. [3] Berois, N., Mazal, D. et al. UDP-N-Acetyl-D-Galactosamine: N-acetylgalactosaminyltransferase-6 as a New Immunohistochemical Breast Cancer Marker. Journal of Histochemistry & Cytochemistry. 2006. 54(3): 317\u2013328

Structural studies of human acidic fibroblast growth factor mutants to be used in anticancer therapy

Lectins are carbohydrate-binding proteins present ubiquitously in nature. They play a role in biological recognition phenomena involving cells and proteins. The interaction lectin-carbohydrate is highly specific, and can be exploited for the development of nanoparticles containing on their surface specific lectins that are directed to carbohydrate residues present only on malignant cells and absent on healthy ones [1].Lectins have been found to possess several anticancer properties and they are proposed as therapeutic agents, binding to cancer cell membrane receptors, causing cytotoxicity, apoptosis and inhibition of tumor growth. Some lectins are able to prevent the proliferation of malignant tumor cells because they recognize the T-antigen (Gal \u3b2 1\u20133GalNAc) found specifically on the surface of tumor cells [2]. The main problem is that their use as a detection agent for the T-antigen in clinical studies is not possible because the immune system can recognize them as foreign molecules and develop an immune response.Previous studies in our laboratory have characterized a lectin found in Boletus edulis mushrooms called BEL \u3b2-trefoil which has antiproliferative activity on tumor cell lines, because it contains three binding sites for the T-antigen. Unlike other lectins with this property, BEL \u3b2-trefoil shows structural homology with a human protein, acidic Fibroblast Growth Factor (FGF1) [3]. Superposition of the two structures suggests that the human protein could be mutated to contain at least one of the binding sites for the T-antigen. Such mutations should create in FGF1 the potential capacity of recognizing tumor cells with less immunogenicity than the fungal protein.FGF1 is a mitogenic and chemotactic protein that mediates cellular functions by binding to transmembrane receptors, which are activated by ligand-induced dimerization requiring heparin as co-receptor.To reach our purpose FGF1 cDNA was cloned into a bacterial plasmid and then mutated in two positions to prevent its binding to the natural receptor, thus suppressing its physiological activity. Loss of function was tested in fibroblast growth tests and then site-directed mutagenesis was performed in three specific positions to produce an FGF1 capable to bind T-antigen. Ligand-protein binding affinity was measured using fluorimetric and isothermal titration calorimetric techniques. Attempts to crystalize the mutants of FGF1 were made using the hanging drop technique with the final aim to carry out their structural characterization by X-ray diffraction analysis of the crystals

Crystallographic studies on carp Fishelectin (FEL)

A few years ago we isolated and sequenced a novel glycoprotein present in the eggs of the carp (Cyprinus carpio). The protein, that binds to a Sepharose 4B matrix column and can be eluted with 0.4 M N-acetyl glucosamine, behaves like a lectin of molecular mass 26686 Da. On the basis of DLS experiments the lectin is present in solution as a stable dimer. We have determined its 238 amino acid long sequence, the position of its 4 disulfide bridges and the structure of its single N-linked carbohydrate chain. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram positive and negative bacteria, these last interactions are inhibited by N-acetyl glucosamine. A data base search shows that its amino acid sequence is significantly similar to that of the members of an invertebrate lectin family that includes tachylectin-1, present in the amebocytes of the horseshoe crab Tachypleus tridentatus, and known to participate in the innate defense system of this species and two other lectins, characterized in the plasmodium Physarum polycephalum, that are called Tectonins I and II and are located in the external surface of the plasma membrane. We have proposed the name fishelectins (by analogy with tachylectins) for this new vertebrate protein family. Homologous genes are present in other bony fish. The carp protein has 85 % identity with a gene expressed in the crucian carp (Carassius auratus gibelio) and 78 % identity with a gene in the cDNA library of the zebrafish (Danio rerio). We have prepared three different crystal forms of the apo protein and two of co-crystals with N-acetyl glucosamine. The orthorhombic form of the apoprotein belongs to space group P212121 and was solved first using the MIR method. Our poster will present the data collection statistics of the best apo and holo forms of the lectin and the refinement statistics of the holo form and will discuss the structure and similarity of this newly identified family of vertebrate proteins to a well known invertebrate protein family

Crystallographic studies on carp Fishelectin (FEL)

A few years ago we isolated and sequenced a novel glycoprotein present in the eggs of the carp (Cyprinus carpio) (1). The protein, that binds to a Sepharose 4B matrix column and can be eluted with 0.4 M N-acetyl glucosamine, behaves like a lectin of molecular mass 26686 Da. On the basis of DLS experiments the lectin is present in solution as a stable dimer. We have determined its 238 amino acid long sequence, the position of its 4 disulfide bridges and the structure of its single N-linked carbohydrate chain. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram positive and negative bacteria, these last interactions are inhibited by N-acetyl glucosamine. A data base search shows that its amino acid sequence is significantly similar to that of the members of an invertebrate lectin family that includes tachylectin-1, present in the amebocytes of the horseshoe crab Tachypleus tridentatus, and known to participate in the innate defense system of this species (2.3) and two other lectins, characterized in the plasmodium Physarum polycephalum, that are called Tectonins I and II and are located in the external surface of the plasma membrane (4). We have proposed the name fishelectins (by analogy with tachylectins) for this new vertebrate protein family. Homologous genes are present in other bony fish. The carp protein has 85 % identity with a gene expressed in the crucian carp (Carassius auratus gibelio) (5) and 78 % identity with a gene in the cDNA library of the zebrafish (Danio rerio). We have prepared three different crystal forms of the apo protein and two of co-crystals with N-acetyl glucosamine. The orthorhombic form of the apoprotein belongs to space group P212121 and was solved first using the MIR method. Our poster will present the data collection statistics of the best apo and holo forms of the lectin and the refinement statistics of the holo form and will discuss the structure and similarity of this newly identified family of vertebrate proteins to a well known invertebrate protein family

Search for strong gravity in multijet final states produced in pp collisions at √s=13 TeV using the ATLAS detector at the LHC

A search is conducted for new physics in multijet final states using 3.6 inverse femtobarns of data from proton-proton collisions at √s = 13TeV taken at the CERN Large Hadron Collider with the ATLAS detector. Events are selected containing at least three jets with scalar sum of jet transverse momenta (HT) greater than 1TeV. No excess is seen at large HT and limits are presented on new physics: models which produce final states containing at least three jets and having cross sections larger than 1.6 fb with HT > 5.8 TeV are excluded. Limits are also given in terms of new physics models of strong gravity that hypothesize additional space-time dimensions

Measurement of jet charge in dijet events from √s = 8 TeV pp collisions with the ATLAS detector

The momentum-weighted sum of the charges of tracks associated to a jet is sensitive to the charge of the initiating quark or gluon. This paper presents a measurement of the distribution of momentum-weighted sums, called jet charge, in dijet events using 20.3 fb−¹ of data recorded with the ATLAS detector at √s = 8 TeV in pp collisions at the LHC. The jet charge distribution is unfolded to remove distortions from detector effects and the resulting particle-level distribution is compared with several models. The pT dependence of the jet charge distribution average and standard deviation are compared to predictions obtained with several leading-order and next-to-leading-order parton distribution functions. The data are also compared to different Monte Carlo simulations of QCD dijet production using various settings of the free parameters within these models. The chosen value of the strong coupling constant used to calculate gluon radiation is found to have a significant impact on the predicted jet charge. There is evidence for a pT dependence of the jet charge distribution for a given jet flavor. In agreement with perturbative QCD predictions, the data show that the average jet charge of quark-initiated jets decreases in magnitude as the energy of the jet increases

Measurement of the correlation between flow harmonics of different order in lead-lead collisions at √sNN = 2.76 TeV with the ATLAS detector

Correlations between the elliptic or triangular flow coefficients vm (m=2 or 3) and other flow harmonics vn (n=2 to 5) are measured using √sNN=2.76 TeV Pb+Pb collision data collected in 2010 by the ATLAS experiment at the LHC, corresponding to an integrated luminosity of 7 μb−1. The vm−vn correlations are measured in midrapidity as a function of centrality, and, for events within the same centrality interval, as a function of event ellipticity or triangularity defined in a forward rapidity region. For events within the same centrality interval, v3 is found to be anticorrelated with v2 and this anticorrelation is consistent with similar anticorrelations between the corresponding eccentricities, ε2 and ε3. However, it is observed that v4 increases strongly with v2, and v5 increases strongly with both v2 and v3. The trend and strength of the vm−vn correlations for n=4 and 5 are found to disagree with εm−εn correlations predicted by initial-geometry models. Instead, these correlations are found to be consistent with the combined effects of a linear contribution to vn and a nonlinear term that is a function of v22 or of v2v3, as predicted by hydrodynamic models. A simple two-component fit is used to separate these two contributions. The extracted linear and nonlinear contributions to v4 and v5 are found to be consistent with previously measured event-plane correlations

Search for vectorlike B quarks in events with one isolated lepton, missing transverse momentum, and jets at √s = 8 TeV with the ATLAS detector

A search has been performed for pair production of heavy vectorlike down-type (B) quarks. The analysis explores the lepton-plus-jets final state, characterized by events with one isolated charged lepton (electron or muon), significant missing transverse momentum, and multiple jets. One or more jets are required to be tagged as arising from b quarks, and at least one pair of jets must be tagged as arising from the hadronic decay of an electroweak boson. The analysis uses the full data sample of pp collisions recorded in 2012 by the ATLAS detector at the LHC, operating at a center-of-mass energy of 8 TeV, corresponding to an integrated luminosity of 20.3 fb −1 . No significant excess of events is observed above the expected background. Limits are set on vectorlike B production, as a function of the B branching ratios, assuming the allowable decay modes are B → Wt/Zb/Hb. In the chiral limit with a branching ratio of 100% for the decay B → Wt, the observed (expected) 95% C.L. lower limit on the vectorlike B mass is 810 GeV (760 GeV). In the case where the vectorlike B quark has branching ratio values corresponding to those of an SU(2) singlet state, the observed (expected) 95% C.L. lower limit on the vectorlike B mass is 640 GeV (505 GeV). The same analysis, when used to investigate pair production of a colored, charge 5/3 exotic fermion T 5/3 , with subsequent decay T 5/3 → Wt, sets an observed (expected) 95% C.L. lower limit on the T 5/3 mass of 840 GeV (780 GeV)