Lectins from Parkia biglobosa and Parkia platycephala: A comparative study of structure and biological effects.

The relation structure-activity of the Mimosoideae lectins of Parkia platycephala (PPL) and Parkia biglobosa (PBL) was analyzed in this study. PBL was solved by X-ray crystallography at a resolution of 2.1Å, and the crystal structure belonged to the C2221 space group. Structural organization and binding sites were also characterized. Specifically, PBL monomer consists of three ß-prism domains tandemly arranged with each one presenting a different carbohydrate recognition domain (CRD). PPL showed antinociceptive activity in the mouse model of acetic acid-induced writhes with maximal inhibitory effect by 74% at 1mg/mL. PPL also demonstrated anti-inflammatory effect causing inhibition of leukocyte migration induced by both direct and indirect chemoattractants. These PPL activities were compared to that of PBL described previously. Molecular docking of both PBL and PPL demonstrated some differences in carbohydrate-lectin interaction energy. Comparing structure and biological effects of the two lectins provided new data about their structure and the relation with its biological activities.

Purification and primary structure of a mannose/glucose-binding lectin from Parkia biglobosa Jacq. seeds with antinociceptive and anti-inflammatory properties.

Parkia biglobosa (subfamily Mimosoideae), a typical tree from African savannas, possess a seed lectin that was purified by combination of ammonium sulfate precipitation and affinity chromatography on a Sephadex G-100 column. The P. biglobosa lectin (PBL) strongly agglutinated rabbit erythrocytes, an effect that was inhibited by d-mannose and d-glucose-derived sugars, especially α-methyl-d-mannopyranoside and N-acetyl-d-glucosamine. The hemagglutinating activity of PBL was maintained after incubation at a wide range of temperature and pH and also was independent of divalent cations. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, PBL exhibited an electrophoretic profile consisting of a single band with apparent molecular mass of 45 kDa. An analysis using electrospray ionization-mass spectrometry indicated that purified lectin possesses a molecular average mass of 47 562 ± 4 Da, and the analysis by gel filtration showed that PBL is a dimer in solution. The complete amino acid sequence of PBL, as determined using tandem mass spectrometry, consists of 443 amino acid residues. PBL is composed of a single non-glycosylated polypeptide chain of three tandemly arranged jacalin-related domains. Sequence heterogeneity was found in six positions, indicating that the PBL preparations contain highly homologous isolectins. PBL showed important antinociceptive activity associated to the inhibition of inflammatory process.

Structural basis for both pro- and anti-inflammatory response induced by mannose-specific legume lectin from Cymbosema roseum.

Legume lectins, despite high sequence homology, express diverse biological activities that vary in potency and efficacy. In studies reported here, the mannose-specific lectin from Cymbosema roseum (CRLI), which binds N-glycoproteins, shows both pro-inflammatory effects when administered by local injection and anti-inflammatory effects when by systemic injection. Protein sequencing was obtained by Tandem Mass Spectrometry and the crystal structure was solved by X-ray crystallography using a Synchrotron radiation source. Molecular replacement and refinement were performed using CCP4 and the carbohydrate binding properties were described by affinity assays and computational docking. Biological assays were performed in order to evaluate the lectin edematogenic activity. The crystal structure of CRLI was established to a 1.8Å resolution in order to determine a structural basis for these differing activities. The structure of CRLI is closely homologous to those of other legume lectins at the monomer level and assembles into tetramers as do many of its homologues. The CRLI carbohydrate binding site was predicted by docking with a specific inhibitory trisaccharide. CRLI possesses a hydrophobic pocket for the binding of α-aminobutyric acid and that pocket is occupied in this structure as are the binding sites for calcium and manganese cations characteristic of legume lectins. CRLI route-dependent effects for acute inflammation are related to its carbohydrate binding domain (due to inhibition caused by the presence of α-methyl-mannoside), and are based on comparative analysis with ConA crystal structure. This may be due to carbohydrate binding site design, which differs at Tyr12 and Glu205 position.

A simple micro-method for determining precise oligosaccharidic specificity of mannose-binding lectins.

A simple and inexpensive method was developed to rapidly define the specificity of mannose-specific lectins toward oligomannoside-type structures. The method involved the interaction of a mixture of N-[(14)C]-acetylated glycoasparagines, prepared by exhaustive pronase digestion of bovine pancreatic ribonuclease B and N-[(14)C]-acetylation with [(14)C]-acetic anhydride and containing all the possible oligomannoside-type N-glycans, with the lectin immobilized on Sepharose-4B. After exhaustive desalting, the obtained fractions were separated by high-performance thin-layer chromatography on silica gel plates and visualized by autoradiography with intensifying screen. As an example of the usefulness of this method, the fine specificity of artocarpin, the mannose-specificity lectin isolated from seeds of jackfruit (Artocarpus integrifolia) toward oligomannoside-type structures is presented. On the basis of such a determination, the best oligomannosidic ligand recognized by a mannose-specific lectin can be selected for studies of crystal structures of the lectin in complex with the defined ligand. Furthermore, some of these immobilized lectins, after definition of their precise specificities with the method, could represent valuable tools for the fractionation and characterization of oligomannose-type structures, present in complex mixtures.

Purification, characterization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds.

The unique carbohydrate-binding property of lectins makes them invaluable tools in biomedical research. Here, we report the purification, partial primary structure, carbohydrate affinity characterization, crystallization, and preliminary X-ray diffraction analysis of a lactose-specific lectin from Cymbosema roseum seeds (CRLII). Isolation and purification of CRLII was performed by a single step using a Sepharose-4B-lactose affinity chromatography column. The carbohydrate affinity characterization was carried using assays for hemagglutination activity and inhibition. CRLII showed hemagglutinating activity toward rabbit erythrocytes. O-glycoproteins from mucine mucopolysaccharides showed the most potent inhibition capacity at a minimum concentration of 1.2 microg mL(-1). Protein sequencing by mass spectrometry was obtained by the digestion of CRLII with trypsin, Glu-C, and AspN. CRLII partial protein sequence exhibits 46% similarity with the ConA-like alpha chain precursor. Suitable protein crystals were obtained with the hanging-drop vapor-diffusion method with 8% ethylene glycol, 0.1 M Tris-HCl pH 8.5, and 11% PEG 8,000. The monoclinic crystals belong to space group P2(1) with unit cell parameters a = 49.4, b = 89.6, and c = 100.8 A.

Lectins from the Red Marine Algal Species Bryothamnion seaforthii and Bryothamnion triquetrum as Tools to Differentiate Human Colon Carcinoma Cells.

The carbohydrate-binding activity of the algal lectins from the closely related red marine algal species Bryothamnion triquetrum (BTL) and Bryothamnion seaforthii (BSL) was used to differentiate human colon carcinoma cell variants with respect to their cell membrane glyco-receptors. These lectins interacted with the cells tested in a dose-dependent manner. Moreover, the fluorescence spectra of both lectins clearly differentiated the cells used as shown by FACS profiles. Furthermore, as observed by confocal microscopy, BTL and BSL bound to cell surface glycoproteins underwent intense internalization, which makes them possible tools in targeting strategies.

Purification and molecular cloning of a new galactose-specific lectin from Bauhinia variegata seeds.

A new galactose-specific lectin was purified from seeds of a Caesalpinoideae plant, Bauhinia variegata, by affinity chromatography on lactose-agarose. Protein extracts haemagglutinated rabbit and human erythrocytes (native and treated with proteolytic enzymes), showing preference for rabbit blood treated with papain and trypsin. Among various carbohydrates tested, the lectin was best inhibited by D-galactose and its derivatives, especially lactose. SDS-PAGE showed that the lectin, named BVL, has a pattern similar to other lectins isolated from the same genus, Bauhinia purpurea agglutinin (BPA). The molecular mass of BVL subunit is 32 871 Da, determined by MALDI-TOF spectrometry. DNA extracted from B.variegata young leaves and primers designed according to the B. purpurea lectin were used to generate specific fragments which were cloned and sequenced, revealing two distinct isoforms. The bvl gene sequence comprised an open reading frame of 876 base pairs which encodes a protein of 291 amino acids. The protein carried a putative signal peptide. The mature protein was predicted to have 263 amino acid residues and 28 963 Da in size.

cDNA cloning and 1.75 A crystal structure determination of PPL2, an endochitinase and N-acetylglucosamine-binding hemagglutinin from Parkia platycephala seeds.

Parkia platycephala lectin 2 was purified from Parkia platycephala (Leguminosae, Mimosoideae) seeds by affinity chromatography and RP-HPLC. Equilibrium sedimentation and MS showed that Parkia platycephala lectin 2 is a nonglycosylated monomeric protein of molecular mass 29 407+/-15 Da, which contains six cysteine residues engaged in the formation of three intramolecular disulfide bonds. Parkia platycephala lectin 2 agglutinated rabbit erythrocytes, and this activity was specifically inhibited by N-acetylglucosamine. In addition, Parkia platycephala lectin 2 hydrolyzed beta(1-4) glycosidic bonds linking 2-acetoamido-2-deoxy-beta-D-glucopyranose units in chitin. The full-length amino acid sequence of Parkia platycephala lectin 2, determined by N-terminal sequencing and cDNA cloning, and its three-dimensional structure, established by X-ray crystallography at 1.75 A resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (betaalpha)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.

Purification, partial characterization and preliminary X-ray diffraction analysis of a mannose-specific lectin from Cymbosema roseum seeds.

A lectin from Cymbosema roseum seeds (CRL) was purified, characterized and crystallized. The best crystals grew in a month and were obtained by the vapour-diffusion method using a precipitant solution consisting of 0.1 M Tris-HCl pH 7.8, 8%(w/v) PEG 3350 and 0.2 M proline at a constant temperature of 293 K. A data set was collected to 1.77 A resolution at a synchrotron-radiation source. CRL crystals are orthorhombic, belonging to space group P2(1)2(1)2(1). Crystallographic refinement and full amino-acid sequence determination are in progress.

Beta-propeller crystal structure of Psathyrella velutina lectin: an integrin-like fungal protein interacting with monosaccharides and calcium.

The lectin from the mushroom Psathyrella velutina recognises specifically N-acetylglucosamine and N-acetylneuraminic acid containing glycans. The crystal structure of the 401 amino acid residue lectin shows that it adopts a very regular seven-bladed beta-propeller fold with the N-terminal region tucked into the central cavity around the pseudo 7-fold axis. In the complex with N-acetylglucosamine, six monosaccharides are bound in pockets located between two consecutive propeller blades. Due to the repeats shown by the sequence the binding sites are very similar. Five hydrogen bonds between the protein and the sugar hydroxyl and N-acetyl groups stabilize the complex, together with the hydrophobic interactions with a conserved tyrosine and histidine. The complex with N-acetylneuraminic acid shows molecular mimicry with the same hydrogen bond network, but with different orientations of the carbohydrate ring in the binding site. The beta-hairpin loops connecting the two inner beta-strands of each blade are metal binding sites and two to three calcium ions were located in the structure. The multispecificity and high multivalency of this mushroom lectin, combined with its similarity to the extracellular domain of an important class of cell adhesion molecules, integrins, are another example of the outstanding success of beta-propeller structures as molecular binding machines in nature.

Helianthus tuberosus agglutinin directly induces neutrophil migration, which can be modulated/inhibited by resident mast cells.

We investigated the effect of Helianthus tuberosus agglutinin (HTA) on neutrophil migration in vivo and in vitro. The role of resident cells in this effect was analyzed. Peritonitis was induced by injecting stimuli into rat (150-200 g) peritoneal cavities, and in vitro neutrophil chemotaxis was performed using a Boyden microchamber. HTA (80, 200, or 500 microg/mL per cavity) induced significant in vivo neutrophil migration (p < 0.05); in vitro assays showed that this lectin also induced neutrophil chemotaxis, an effect inhibited by the incubation of lectin associated with alpha-D(+)-mannose, its specific binding sugar. Depletion of the resident-cell population by peritoneal lavage did not alter HTA-induced neutrophil migration (200 microg/mL per cavity). The opposite strategy, increasing peritoneal macrophages by intraperitoneally injecting rats with thioglycollate, did not enhance the neutrophil migration produced by HTA (200 microg/mL per cavity). In addition, injection of supernatant from HTA-stimulated macrophage culture (300 microg/mL) into rat peritoneal cavities did not induce neutrophil migration. However, reduction of the peritoneal mast-cell population potentiated the neutrophil migration (p < 0.05) induced by HTA (200 microg/mL per cavity). Lectin from H. tuberosus has a direct neutrophil chemotatic effect that is modulated by mast cells.

HCA and HML isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis define a novel lectin family.

HCA and HML represent lectins isolated from the red marine algae Hypnea cervicornis and Hypnea musciformis, respectively. Hemagglutination inhibition assays suggest that HML binds GalNAc/Gal substituted with a neutral sugar through 1-3, 1-4, or 1-2 linkages in O-linked mucin-type glycans, and Fuc(alpha1-6)GlcNAc of N-linked glycoproteins. The specificity of HCA includes the epitopes recognized by HML, although the glycoproteins inhibited distinctly HML and HCA. The agglutinating activity of HCA was inhibited by GalNAc, highlighting the different fine sugar epitope-recognizing specificity of each algal lectin. The primary structures of HCA (9193+/-3 Da) and HML (9357+/-1 Da) were determined by Edman degradation and tandem mass spectrometry of the N-terminally blocked fragments. Both lectins consist of a mixture of a 90-residue polypeptide containing seven intrachain disulfide bonds and two disulfide-bonded subunits generated by cleavage at the bond T50-E51 (HCA) and R50-E51 (HML). The amino acid sequences of HCA and HML display 55% sequence identity (80% similarity) between themselves, but do not show discernible sequence and cysteine spacing pattern similarities with any other known protein structure, indicating that HCA and HML belong to a novel lectin family. Alignment of the amino acid sequence of the two lectins revealed the existence of internal domain duplication, with residues 1-47 and 48-90 corresponding to the N- and C-terminal domains, respectively. The six conserved cysteines in each domain may form three intrachain cysteine linkages, and the unique cysteine residues of the N-terminal (Cys46) and the C-terminal (Cys71) domains may form an intersubunit disulfide bond.

Pro-inflammatory effect of Arum maculatum lectin and role of resident cells.

Arum maculatum agglutinin (AMA) is a monocot lectin isolated from tubers of Arum maculatum L. (Araceae) which exhibits different specificity towards oligo-mannosidic-type and N-acetyllactosaminic-type glycans. We have investigated the effect of this lectin on the cells of the immune system. Models of neutrophil migration in vivo, neutrophil chemotaxis in vitro and macrophage cultures were used to study the lectin inflammatory activity. When administered into rat peritoneal cavities, AMA (80, 200 and 500 microg/mL/cavity) induced significant and dose-dependent neutrophil migration. This effect was inhibited by incubation with alpha-methyl-d-mannoside. A 83% depletion in the number of resident cells following peritoneal lavage did not reduce the AMA-induced neutrophil migration, as compared to sham animals (not washed). However, pre-treatment with 3% thioglycolate which increases the peritoneal macrophage population by 236%, enhanced the neutrophil migration induced by AMA (200 microg/mL/cavity) (119%, p < 0.05). Reduction of peritoneal mast cell population by chronic treatment of cavities with compound 48/80 did not modify AMA-induced neutrophil migration. The neutrophil chemotaxy assay in vitro shows that the lectin (300 microg/mL) induces neutrophil chemotaxy (368% p < 0.05) compared to RPMI. Finally, injection into peritoneal cavities of supernatants from macrophage cultures obtained after stimulation with AMA (300 microg/mL) enhanced neutrophil migration (110% p < 0.05). Summarizing, our data suggest that A. maculatum agglutinin presents pro-inflammatory activity, inducing neutrophil migration by two ways, one which is independent on resident cells and another one dependent on the presence of these cells.

Crystallization and preliminary X-ray diffraction analysis of a new chitin-binding protein from Parkia platycephala seeds.

A chitin-binding protein named PPL-2 was purified from Parkia platycephala seeds and crystallized. Crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 55.19, b = 59.95, c = 76.60 A, and grew over several days at 293 K using the hanging-drop method. Using synchrotron radiation, a complete structural data set was collected to 1.73 A resolution. The preliminary crystal structure of PPL-2, determined by molecular replacement, presents a correlation coefficient of 0.558 and an R factor of 0.439. Crystallographic refinement is in progress.

Specificity of Amaranthus leucocarpus syn. hypocondriacus lectin for O-glycopeptides.

Amaranthus leucocarpus syn. hypochondriacus lectin (ALL) has been shown to be specific for N-acetyl-D-galactosamine (GalNAc). In this work, we determined a value of 1.0 x 10(-2) M for the association constant of ALL for GalNAc, calculated using fluorescence spectroscopy assays. Using neoglycopeptides obtained by in vitro O-glycosylation, we determined the main features of O-glycopeptides recognized by ALL using molecular dynamics simulations, capillary electrophoresis, and ELISA. Neo-glycopeptides were obtained by in vitro O-glycosylation reaction using microsomal preparations of murine thymocytes, human gastric fundus and colonic mucosa. ELISA assays were performed with peroxidase-labeled murine monoclonal IgG2, kappa light chain (5D4) antibodies against ALL. Among the in vitro neoglycopeptides, only those of TTSAPTTS containing GalNAc at Thr in #2 and #6 reacted with ALL. Neither the TTSAPTTS glycopeptide, containing a unique GalNAc residue at Thr in #2, nor others (with more than two GalNAc residues) interacted with the lectin. Computational docking assays of the lower energy conformers for interactions between glycopeptides and lectins confirmed that ALL recognized GalNAc residues when they are spaced out in glycan structures, whereas GalNAc residues arranged in clusters prevented interaction with the lectin, indicating that ALL is specific for a special GalNAc-containing motif found in different O-glycoproteins.