Structural and biochemical characterization of a new type of lectin isolated from carp eggs.

A previously unidentified glycoprotein present in the eggs of the carp (Cyprinus carpio) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N-acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N-acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp (Carassius auratus gibelio) and 78% identity with a gene in the cDNA library of the zebrafish (Danio rerio)

BEL \u3b2\u2010Trefoil Reduces the Migration Ability of RUNX2 Expressing Melanoma Cells in Xenotransplanted Zebrafish

RUNX2, a master osteogenic transcript ion factor, is overexpressed in several cancer cells; in melanoma it promotes cells migration and invasion as well as neoangiogenesis. The annual mortality rates related to metastatic melanoma are high and novel agents are needed to improve melanoma patients\u2019 survival. It has been shown that lectins specifically target malignant cells since they present the Thomsen\u2013Friedenreich antigen. This disaccharide is hidden in normal cells, while it allows selective lectins binding in transformed cells. Recently, an edible lectin named BEL \u3b2-trefoil has been obtained from the wild mushroom Boletus edulis. Our previous study showed BEL \u3b2-trefoil effects on transcription factor RUNX2 downregulation as well as on the migration ability in melanoma cells treated in vitro. Therefore, to better understand the role of this lectin, we investigated the BEL \u3b2-trefoil effects in a zebrafish in vivo model, transplanted with human melanoma cells expressing RUNX2. Our data showed that BEL \u3b2-trefoil is able to spread in the tissues and to reduce the formation of metastases in melanoma xenotransplanted zebrafish. In conclusion, BEL \u3b2-trefoil can be considered an effective biomolecule to counteract melanoma disease

Structure of a lectin with antitumoral properties in king bolete (Boletus edulis) mushrooms.

A novel lectin has been isolated from the fruiting bodies of the common edible mushroom Boletus edulis (king bolete, penny bun, porcino or cep) by affinity chromatography on a chitin column. We propose for the lectin the name BEL (B. edulis lectin). BEL inhibits selectively the proliferation of several malignant cell lines and binds the neoplastic cell-specific T-antigen disaccharide, Galβ1-3GalNAc. The lectin was structurally characterized: the molecule is a homotetramer and the 142-amino acid sequence of the chains was determined. The protein belongs to the saline-soluble family of mushroom fruiting body-specific lectins. BEL was also crystallized and its three-dimensional structure was determined by X-ray diffraction to 1.15 Å resolution. The structure is similar to that of Agaricus bisporus lectin. Using the appropriate co-crystals, the interactions of BEL with specific mono- and disaccharides were also studied by X-ray diffraction. The six structures of carbohydrate complexes reported here provide details of the interactions of the ligands with the lectin and shed light on the selectivity of the two distinct binding sites present in each protomer

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 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

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

Can we obtain new information from old protein crystals? The plasma retinol-binding protein case.

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in plasma 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 and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass and thus avoid its elimination by glomerular filtration [1]. Many years ago we published the structure of RBP4 holo and believed to be apo at 2.5 \uc5 resolution [2] and wrote at the end of the abstract \u201cIn the case of the unliganded form, the central cavity that is occupied by the vitamin in the two human crystalline holo RBPs, is filled by electron density that, at the present resolution we interpret as solvent.\u201d The crystals used for that study had been prepared by microdialysis using protein purified from human plasma and, since they were still kept in our laboratory, we decided to test them at the ESRF a couple of years ago. We collected a full data set of both the liganded and unliganded crystals at a resolution of 1.5 \uc5 and 2.0 \uc5 respectively and were able to refine the unliganded form using about 20,000 reflections instead of the 10,000 that we had used in the original paper. The result was that we identified a fatty acid in the ligand-binding site of the protein believed to be apo. It is remarkable that the data for the holo and \u201capo\u201d protein purified from plasma could be collected using crystals from the same batch as those of the structures published more than 20 years ago. The significantly increased resolution can be attributed to the amazing developments in the X-ray data collection methods that occurred during the intervening years and was only possible thanks to the stability of protein crystals prepared by equilibrium dialysis. We also prepared crystals of RBP4 purified from human urine and amniotic fluid, two sources of protein that contain non fluorescent RBP4, i.e. not bound to retinol, and for that reason believed to be the apo form of the protein. In every case we found a fatty acid in the central cavity of the RBP4 molecule, a result that we confirmed by GC-MS analysis of the samples used in the crystallization experiments. This result changes substantially our perception of this protein that has so far been considered to be specific for retinol and is a good example of how simply increasing the quality of the diffraction data can change the perception of the function of a protein [3

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