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

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

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

An unusual aspartic acid cluster in the reovirus attachment fiber s1 mediates stability at low pH and preserves trimeric organization

The reovirus attachment protein σ1 mediates cell attachment and receptor binding and is thought to undergo conformational changes during viral disassembly. σ1 is a trimeric filamentous protein with an α-helical coiled-coil tail, a triple-β-spiral body, and a globular head. At the trimer interface, the head domain features an unusual and conserved aspartic acid cluster, which forms the only significant intratrimer interactions in the head and must be protonated to allow trimer formation. To define the role of pH on σ1 stability and conformation, we tested its domains over a wide range of pH values. We show that all domains of σ1 are remarkably thermostable, even at the low pH of the stomach. We determined the optimal pH for stability to be between pHs 5 and 6, a value close to the pH of the endosome and of the jejunum. The σ1 head is stable at acidic and neutral pH but detrimerizes at basic pH. When Asp(345) in the aspartic acid cluster is mutated to asparagine (D345N), the σ1 head loses stability at low pH and is more prone to detrimerize. Although the D345N mutation does not affect σ1 binding affinity for the JAM-A receptor, the overall binding stoichiometry is reduced by one-third. The additional replacement of the neighboring His(349) with alanine disrupts inner trimer surface interactions, leading to a less thermostable and monomeric σ1 D345N head that fails to bind the JAM-A receptor. When the body is expressed together with the head domain, the thermostability is restored and the stoichiometry of the binding to JAM-A receptor is preserved. Our results confirm a fundamental role of the aspartic acid cluster as a pH-dependent molecular switch controlling trimerization and enhancing thermostability of σ1, which represent essential requirements to accomplish reovirus infection and entry and might be common mechanisms among other enteric viruses. IMPORTANCE: Enteric viruses withstand the highly acidic environment of the stomach during transmission, and many of them use low pH as a trigger for conformational changes associated with entry. For many nonenveloped viruses, the structural basis of these effects is not clear. We have investigated the stability of the reovirus attachment protein σ1 over a range of pHs and find it to be remarkably thermostable, especially at low pH. We identify a role for the aspartic acid cluster in maintaining σ1 thermostability, trimeric organization, and binding to JAM-A receptor especially at the gastric pH reovirus has to withstand while passing the stomach. The understanding of monomer-trimer dynamics within σ1 enhances our knowledge of reovirus entry and has implications for stability and transmission of other enteric viruses

Pegylated silica nanoparticles: cytotoxicity and macrophage uptake

Here, we present a thorough study of pegylated silica nanoparticle (SNP) interaction with different biological environments. The SNPs have a mean diameter of about 40 nm and are coated with polyethylene glycol (PEG) of different molecular weights. The physicochemical characterization of SNPs allowed the confirmation of the binding of PEG chains to the silica surface, the reproducibility of the synthesis and the narrow size-dispersion. In view of clarifying the SNP interaction with biological environments, we first assessed the SNP reactivity after the incubation with two cell lines (macrophages RAW 264.7 and primary human fibroblasts), observing a reduced toxicity of pegylated SNPs compared to the bare ones. Then, we investigated the effect of the protein adsorption on the SNP surface using the model serum protein, bovine serum albumin (BSA). We found that the protein adsorption takes place more heavily on poorly pegylated SNPs, promoting the uptake of the latter by macrophages and leading to an increased mortality of these cells. To better understand this mechanism by means of flow cytometry, the dye Ru(bpy)3Cl2 was incorporated in the SNPs. The overall results highlight the SNP potentialities as a drug delivery system, thanks to the low interactions with the macrophages