Structural studies of POL (Pleurotus ostreatus Lectin), a fungal lectin of medical interest

Lectins are proteins widely diffuse in nature that interact non-covalently with carbohydrates [1]. Of all the mushroom proteins, lectins are probably the most extensively investigated because it has been observed that they can exhibit antitumour activity on human cancer cells [2]. Among them, a lectin from the fruiting bodies of the edible oyster mushroom Pleurotus ostreatus was isolated since it appears to be able to inhibit the growth of human neoplastic cells [3]. It was named POL, Pleurotus ostreatus lectin and in our laboratory it is purified using two chromatographic steps: a hog gastric mucin column followed by a Sephacryl S-100 gel filtration column. Two alternative ways of elution from the affinity column (with lactose 0.2 M and with EDTA 5 mM) give the same yield (1-1.5 mg) of protein starting with 500 g of mushrooms. Crystals of 0,1-0,3 mm can be grown in two crystallization conditions: 1) 0.1 M Na Hepes pH 7.5 in the presence of 0.8 M potassium/sodium tartrate tetrahydrate and 2) 1.6 M Ammonium sulphate, 0.1 MES pH 6.5 and 10% v/v Dioxane. We have collected X-ray diffraction data at various beamlines of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France. The structure was solved by Single Isomorphous Replacement (SIR) with anomalous dispersion. The model was built with the program Coot and refinement was carried out with data collected from apo crystals at 2.05 \uc5 using RefMac 5. The unknown preliminary amino acid sequence of the polypeptide chain was obtained from the electron density maps. The asymmetric unit contains one monomer with two domains of 22 \u3b2-strand only: 10 forming the domain near the N-terminus and 12 the C-terminus nearer, with a conformation that resembles the \u3b2-barrel fold. \u3b2-sheets are radially arranged around a central tunnel packing face-to-face. Since there seems to be an enzymatic activity associated to POL, the purified lectin was routinely checked with DLS experiments to ensure that the eventual enzymatic activity was only due to the lectin and not to other contaminants [4]. The presence of a single peak confirmed the purity of the sample and so it was decided to perform enzymatic assays with four nitrophenol derivatives. The most reactive substrate for POL was 4-nitrophenyl-\u3b2-D-glucopyranoside with a Vmax=87.21 nmol sec-1 mg-1, kcat=43s-1 and Km=240 \ub5M. Since in the POL electron density maps there was a region too big for water fitting the presence of a metal cofactor was suspected. Experiments with the spectrofluorimeter, analyzing fluorescence protein quenching upon the addition of a metal, were carried out and confirmed the presence of Calcium bound to the lectin. As POL density maps did not reveal any density regions that could be ascribed to a carbohydrate, it will be necessary to crystallize the lectin with specific inhibitors bound at the active site (for example nojirimycin). In addition, POL was also tested on human pancreatic cancer cells (MiaPaCa-2) and its therapeutic effect was evident. The antitumoral activity of POL might be exploited to direct PLGA, poly(lactic-co-glycolic acid) nanoparticles, to different melanoma cell lines, and also to prepare POL-filled nanoparticles emulsions or patches applicable on melanomas. For this purpose, since the total yield of purified POL is very low, attempts of heterologous expression in Pichia pastoris and E. coli ,with the protein sequence optimized for the expression in this bacterial system, are still in progress

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS)

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS

Human plasma retinol-binding protein can physiologically be bound to palmitic acid; new information from old crystals

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in serum as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesised in the liver but is also produced by adipose tissue (about 20-40 % of the amounts released by the liver) and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass to about 80,000 and thus avoid its elimination by glomerular filtration. The RBP-TTR complex dissociates readily upon interaction with the RBP receptor, STRA6, that removes the vitamin from the transporter and facilitates its entrance into the cell. When retinol is not present in the complex, RBP dissociates from TTR and is eliminated in urine. We previously reported the X-ray structure of human holo RBP4 and what we expected to be the apo form, i.e. after the loss of retinol, to 2.5 \uc5 resolution. Our most important finding was the observation of a well-defined conformational transition involving a loop at the entrance of the ligand binding site. We also reported that the protein molecule without retinol contained residual electron density in the central cavity that we interpreted as ordered solvent molecules. This job reports the three-dimensional structure of human holo-RBP and of the protein naturally deprived from retinol purified from plasma, urine and amniotic fluid determined to resolutions of 1.5, 2.0, 1.5 and 1.7 \uc5 respectively. It is remarkable that the crystals used in this study of the plasma RBP4 holo and deprived from the retinol molecule are of the same batch as those used to determine the 2.5 \uc5 resolution structure more than 20 years ago. In all the crystal forms of the RBP4 naturally deprived from the ligand we found palmitic acid bound in the hydrophobic ligand-binding site, a result that we confirmed by mass spectrometry measurements. The interactions of all-trans retinol with the protein at this significantly improved resolution as well as the conformational changes induced by vitamin deprivation are discussed in detail as is the structure of the complex of human RBP4 with palmitic acid

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS)

Structural characterization and interaction studies of human lipocalin-type prostaglandin D synthase (L-PGDS

Structural characterization and interaction studies of humanlipocalin-type prostaglandin D synthase (L-PGDS)

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups in the presence of sulphydryl compounds. PGH2 is a common precursor of all prostanoids, which include thromboxanes, prostacyclins and prostaglandins. PGD2 is synthesized in both the central and peripheral nervous system and it is involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as \u3b2-trace protein, the second most abundant protein in human cerebro-spinal fluid. L-PGDS is also detected in brain, testis and prostate, endothelial cells, placenta and heart tissue and even in macrophages infiltrated in atherosclerotic plaques. In these tissues it participates in many physiological activities as well as in the response to diseases. Currently the main structural and biochemical studies, present in the literature, concern recombinant rat and mouse L-PGDS. In this work we use recombinant human L-PGDS in order to solve its three-dimensional structure by X-ray diffraction and test its affinity for several ligands using Surface Plasmon Resonance (SPR). Wild type human L-PGDS and three mutants (C65A; C65A-K59A; C89/186A) were expressed using E. coli cell strains and subsequently purified by a chitin affinity column, size exclusion and hydrophobic interaction chromatography. Large and highly ordered crystals were used to collect X-ray diffraction data using either a rotating-anode generator or a synchrotron source. The multiple isomorphous replacement method was used to solve the phase problem. In the electron density maps an unidentified density was observed apparently interacting with lysine 59 inside the L-PGDS-C65A cavity; the foreign molecule is probably PEG, an additive present in the crystallization liquors. This hypothesis is supported by the fact that the L-PGDS-C65A/K59A crystals, which grow without PEG, show a completely free protein cavity. A seeding experiment of L-PGDS-C65A/K59A crystal, grown in L-PGDS-C65A crystallization conditions, partially confirmed this hypothesis since the foreign molecule was present in the L-PGDS-C65A/K59A cavity. Another crystal form was obtained by mixing L-PGDS-C65A/K59A with the amyloid \u3b2 peptide (1-40). Although the amyloid \u3b2 peptide is not visible in the maps, the packing of the protein molecules has changed in the presence of the peptide suggesting interaction of the two molecules. Wild type L-PGDS small crystals were recently obtained and will be tested as soon beam time at a synchrotron source becomes available. SPR experiments are also in progress and will be used to verify interaction of L-PGDS with PEG, the amyloid \u3b2 peptide and other ligands and to determine their binding constants

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

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

High resolution structures of mutants of residues that affect access to the ligand-binding cavity of human lipocalin-type prostaglandin D Synthase

Lipocalin-type prostaglandin D synthase (L-PGDS) catalyzes the isomerisation of the 9,11-endoperoxide group of PGH2 (Prostaglandin H2) to produce PGD2 (Prostaglandin D2) with 9-hydroxy and 11-keto groups. The product of the reaction, PGD2, is the precursor of several metabolites involved in many regulatory events. L-PGDS, the first member of the important lipocalin family to be recognized as an enzyme, is also able to bind and transport small hydrophobic molecules and was formerly known as \u3b2-trace protein, the second most abundant protein in human cerebro-spinal fluid. Previous structural work on the mouse and human proteins has focused on the identification of the amino acids responsible and the proposal of a mechanism for catalysis. In this paper we present the X-ray structures of the apo and holo forms (bound to PEG) of the C65A mutant of human L-PGDS to 1.40 \uc5 resolution and of the double mutant C65A K59A to 1.60 \uc5 resolution. We have also studied the apo forms of the double mutants C65A W54F and C65A W112F and the triple mutant C65A W54F W112F. Mutation of the lysine residue does not seem to affect the binding of PEG to the ligand-binding cavity and mutation of a single or both tryptophanes appears to have the same effect on the position of these two aromatic residues at the entrance of the cavity. We have also identified a solvent molecule in an invariant position in the cavity of virtually all the molecules present in the 9 asymmetric units of the crystals that we have examined. Taken together our observations indicate that the residues we have mutated appear to indeed play a role in the entrance-exit process of the substrate and/or other ligands to the binding cavity of the lipocalin