Kyria S. Nascimento

Binding Studies of α-GalNAc-specific Lectins to the α-GalNAc (Tn-antigen) Form of Porcine Submaxillary Mucin and Its Smaller Fragments

Isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements demonstrate that a chemically and enzymatically prepared form of porcine submaxillary mucin that possesses a molecular mass of ∼106 daltons and ∼2300 α-GalNAc residues (Tn-PSM) binds to the soybean agglutinin (SBA) with a Kd of 0.2 nm, which is ∼106-fold enhanced affinity relative to GalNAcα1-O-Ser (Tn), the pancarcinoma carbohydrate antigen. The enzymatically derived 81 amino acid tandem repeat domain of Tn-PSM containing ∼23 α-GalNAc residues binds with ∼103-fold enhanced affinity, while the enzymatically derived 38/40 amino acid cleavage product(s) of Tn-PSM containing ∼11-12 α-GalNAc residues shows ∼102-fold enhanced affinity. A natural carbohydrate decorated form of PSM (Fd-PSM) containing 40% of the core 1 blood group type A tetrasaccharide, and 58% peptide-linked GalNAcα1-O-Ser/Thr residues, with 45% of the peptide-linked α-GalNAc residues linked α-(2,6) to N-glycolylneuraminic acid, shows ∼104 enhanced affinity for SBA. Vatairea macrocarpa lectin (VML), which is also a GalNAc binding lectin, displays a similar pattern of binding to the four forms of PSM, although there are quantitative differences in its affinities as compared with SBA. The higher affinities of SBA and VML for Tn-PSM relative to Fd-PSM indicate the importance of carbohydrate composition and epitope density of mucins on their affinities for lectins. The higher affinities of SBA and VML for Tn-PSM relative to its two shorter chain analogs demonstrate that the length of a mucin polypeptide and hence total carbohydrate valence determines the affinities of the three Tn-PSM analogs. The results suggest a binding model in which lectin molecules “bind and jump” from α-GalNAc residue to α-GalNAc residue along the polypeptide chain of Tn-PSM before dissociating. The complete thermodynamic binding parameters for these mucins including their binding stoichiometries are presented. The results have important implications for the biological activities of mucins including those expressing the Tn cancer antigen.

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(α1–6)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.

Antidepressant‐like effect of lectin from Canavalia brasiliensis (ConBr) administered centrally in mice

This study investigates the action of the central administration of the lectins isolated from Canavalia brasiliensis seeds (ConBr) and from Canavalia ensiformes seeds, (Concanavalin A, ConA) in the forced swimming test (FST) in mice. ConBr (1-10 micro g/site, i.c.v.), but not ConA, produced a decrease in the immobility time in the FST (observed at the time points 15, 30, 60 and 120 min after the injection), without changing the locomotor activity in the open-field test. The effect of ConBr in the FST was dependent on its protein structure integrity. ConBr (0.1 micro g/site, i.c.v.) caused a potentiation of the action of fluoxetine, a selective 5-HT reuptake inhibitor. The anti-immobility effect elicited by ConBr (10 micro g/site, i.c.v.) in the FST was prevented by the pretreatment of mice with pindolol (32 mg/kg, a 5-HT(1A/1B) receptor/beta-adrenoceptor antagonist), NAN-190 (0.5 mg/kg, a 5-HT(1A) receptor antagonist), ketanserin (5 mg/kg, a 5-HT(2A/2C) receptor antagonist), sulpiride (50 mg/kg, a D(2) receptor antagonist) or yohimbine (1 mg/kg, an alpha(2)-adrenoceptor antagonist), but not with SCH 23390 (0.05 mg/kg, a D(1) receptor antagonist) or prazosin (1 mg/kg, an alpha(1)-adrenoceptor antagonist). These results indicate that the antidepressant-like effect of ConBr in the FST is dependent on its interaction with the serotoninergic (via 5-HT(1A) and 5-HT(2)), noradrenergic (via alpha(2)-adrenoceptors) and dopaminergic (via D(2) receptors) systems. Considering the presence of lectins in the brain and based on the results, it will be important to determine a possible role of endogenous lectins in the modulation of the central nervous system function.

In vitro inhibition of oral streptococci binding to the acquired pellicle by algal lectins

Aims:  The initial colonization of the tooth by streptococci involves their attachment to adsorbed components of the acquired pellicle. Avoiding this adhesion may be successful in preventing caries at early stages. Salivary mucins are glycoproteins that when absorbed onto hydroxyapatite may provide binding sites for certain bacteria. Algal lectins may be especially interesting for oral antiadhesion trials because of their great stability and high specificity for mucins. This work aimed to evaluate the potential of two algal lectins to inhibit the adherence of five streptococci species to the acquired pellicle in vitro.

Toxicity of some glucose/mannose-binding lectins to Biomphalaria glabrata and Artemia salina.

Schistosomiasis or bilharzia, which affects millions of people living in Africa, Asia and Latin America, is closely associated with certain species of aquatic snails. One way of attacking the disease is to eradicate the host snails. Molluscicidal activities of natural compounds are especially important in the widespread control of this tropical disease. As part of our search for natural compounds with molluscicidal properties for the vector control of schistosomiasis, we are now evaluating for the first time the toxicity of the plant lectins from Canavalia brasiliensis (ConBr), Cratylia floribunda (CFL), Dioclea guianensis (Dgui), Dioclea grandiflora (DGL) and Dioclea virgata (Dvir) to Biomphalaria glabrata Say and Artemia salina Leach. Results indicate that all the samples were toxic to A. salina Leach, some of them with values of lethal concentration that kills 90% of the population (LC(90))<10 microg mL(-1). They are also active against B. glabrata Say, killing 100% of adult snails, at a concentration of 50 microg mL(-1). The lectins CFL and Dgui possess properties lethal to mollusks, with values of LC(90)=50.3 microg mL(-1) and LC(90)=41.0 microg mL(-1), respectively.

An overview of lectins purification strategies.

Lectins hold great promise not only as reagents for diagnostics and drug discovery but also as a novel class of biopharmaceutical products. In fact, new research directions in the last years have led to major developments in the uses of plant lectins as therapeutic agents against numerous diseases in an ageing society. It is even expected that lectins may occupy an important place in the biopharmaceutical industry next to monoclonal antibodies. All these new trends are placing a tremendous emphasis on the development of new approaches for faster lectins development, selection, and optimization, including alternatives methods of purification. This article reviews the isolation and purification methods used for lectins purification. Origins and applications of lectins are described, highlighting the special features of this class of proteins, such as the carbohydrated‐binding domains and their importance in the development of affinity methodologies to increase and facilitate lectins purification. Published strategies for the purification of lectins from different sources are analyzed in relation to the purification methods used, their sequence, and the number of times they are used in a purification procedure. The purity of lectins is analyzed in relation to the average overall yield and purification factors obtained for each purification scheme for these proteins and the purification steps necessary. New directions are described for improving lectins separation and purification. Copyright

Antinociceptive and Anti-inflammatory Effects of a Lectin-Like Substance from Clitoria fairchildiana R. Howard Seeds

Lectins are proteins that have the ability to bind specifically and reversibly to carbohydrates and glycoconjugates, without altering the structure of the glycosyl ligand. They are found in organisms such as viruses, plants and humans, and they have been shown to possess important biological activities. The objective of this study was to purify and characterize lectins in the seeds of Clitoria fairchildiana, as well as to verify their biological activities. The results indicated the presence of a lectin (CFAL) in the glutelin acid protein fraction, which agglutinated native rabbit erythrocytes. CFAL was purified by column chromatography ion-exchange, DEAE-Sephacel, which was obtained from a peak of protein retained in the matrix by applying 0.5 M NaCl using the step-wise method. Electrophoretic analysis of this lectin in SDS-PAGE indicated a two band pattern protein molecular mass of approximately 100 and 116 kDa. CFAL proved to be unspecific to all carbohydrates/glycoconjugates in common use for the sugar inhibition test. This lectin showed no significant cytotoxicity to human red blood cells. It was observed that CFAL has anti-inflammatory activity in the paw edema induced by carrageenan model, in which a 64% diminution in edema was observed. Antinociceptive effects were observed for CFAL in the abdominal writhing test (induced by acetic acid), in which increasing doses of the lectin caused reduction in the number of contortions by up to 72%. It was concluded that the purified and characterized lectin from the seeds of Clitoria fairchildiana has anti-inflammatory and antinociceptive activity, and is not cytotoxic to human erythrocytes.

Pharmacological analysis of the neutrophil migration induced by D. rostrata lectin: involvement of cytokines and nitric oxide.

In the present study, we investigated the involvement of resident cell and inflammatory mediators in the neutrophil migration induced by chemotactic activity of a glucose/mannose-specific lectin isolated from Dioclea rostrata seeds (DrosL). Rats were injected i.p. with DrosL (125-1000 microg/cavity), and at 2-96 h thereafter the leukocyte counts in peritoneal fluid were determined. DrosL-induced a dose-dependent neutrophil migration accumulation, which reached maximal response at 24 h after injection and declines thereafter. The carbohydrate ligand nearly abolished the neutrophil influx. Pre-treatment of peritoneal cavities with thioglycolate which increases peritoneal macrophage numbers, enhanced neutrophil migration induced by DrosL by 303%. However, the reduction of peritoneal mast cell numbers by treatment of the cavities with compound 48/80 did not modify DrosL-induced neutrophil migration. The injection into peritoneal cavities of supernatants from macrophage cultures stimulated with DrosL (125, 250 and 500 microg/ml) induced neutrophil migration. In addition, DrosL treatment induced cytokines (TNF-alpha, IL-1beta and CINC-1) and NO release into the peritoneal cavity of rats. Finally, neutrophil chemotaxis assay in vitro showed that the lectin (15 and 31 microg/ml) induced neutrophil chemotaxis by even 180%. In conclusion, neutrophil migration induced by D. rostrata lectin occurs by way of the release of NO and cytokines such as IL-1beta, TNF-alpha and CINC-1.

PURIFICATION AND CHARACTERIZATION OF A NEW LECTIN FROM THE RED MARINE ALGA HYPNEA MUSCIFORMIS

A lectin from the red marine alga Hypnea musciformis (HML) was purified by extraction with 20 mM PBS, precipitation with 70% saturated ammonium sulphate, ion-exchange DEAE-Cellulose chromatography and RP-HPLC. The 9.3 kDa polypeptide agglutinates erythrocytes from various sources and shows oligomerization tendencies under certain MALDI-TOF/MS conditions. Preliminary N-terminal sequencing and biological assays strongly suggest that the HML may belong to a new class of algae lectins.

cDNA cloning and 1.75 Å 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 β(1–4) glycosidic bonds linking 2‐acetoamido‐2‐deoxy‐β‐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 Å resolution, showed that Parkia platycephala lectin 2 is homologous to endochitinases of the glycosyl hydrolase family 18, which share the (βα)8 barrel topology harboring the catalytic residues Asp125, Glu127, and Tyr182.