Comparison of adherence, cytotoxicity, and Gal/GalNAc lectin gene structure in Entamoeba histolytica and Entamoeba dispar

The recent development of axenic culture for E. dispar allowed us to examine this ameba's ability to bind and lyse target cells and compare it to E. histolytica which has been axenically cultured for years. We found that under axenic conditions, E. dispar's adherence to target cells, ligand binding, and cytotoxicity were less than that of E. histolytica. These events were Gal/GalNAc-inhibitable supporting the idea that E. dispar expresses a lectin similar to E. histolytica. Genetic analysis showed that E. dispar had at least two members of the lectin heavy subunit family and four members of the lectin light subunit family that hybridized to ehhgl and ehlgl gene probes. A library screen produced clones which were isolated and sequenced. Derived amino acid sequences showed that the E. dispar heavy and light subunit clones were 86% and 79% identical, respectively, to their E. histolytica counterparts. In particular, the region which contains the epitopes for two adherence-enhancing monoclonal antibodies and a complement-sensitizing monoclonal antibody (amino acids 882–959 of the lectin heavy subunit) were conserved between the species.     

Use of monoclonal anti-light subunit antibodies to study the structure and function of theEntamoeba histolytica Gal/GalNAc adherence lectin

Adherence ofEntamoeba histolytiea trophozoites to host cells is medicated by a galactose (Gal) andN-acetylgalactosamine (GalNAc)-specific surface lectin. The lectin is a heterodimeric protein composed of heavy (170kDa) and light (35-31 kDa) subunits linked by disulfide bonds. Polyclonal and monoclonal antibodies (mAb) raised against a light subunit-glutathione-S-transferase fusion protein were used to probe its structure and function. Four light subunit-specific mAb were produced which recognized distinct epitopes on five different light subunit isoforms. Immunoblots with these mAb demonstrated co-migration of light and heavy subunits when nonreduced trophozoite proteins were analysed by SDS-PAGE, indicating that the subunits do not exist free of the heterodimer in significant quantities. While anti-heavy subunit antibodies had previously been shown to alter adherence, anti-light subunit antibodies did not, suggesting that the heavy subunit contains the carbohydrate recognition domain.     

Effect of alcohols on binding of camphor to cytochrome P450cam: spectroscopic and stopped flow transient kinetic studies

Addition of alcohols to cytochrome P450cam (CYP101) was shown to release the substrate camphor from the heme pocket of the enzyme. The release of the substrate was found to be caused both due to increased solubility of the substrate in solution in presence of alcohol and due to change in the tertiary structure of the active site of the enzyme. The far-UV CD and near-UV CD spectra reveal that addition of alcohols to cytochrome P450cam cause a small change in the secondary structural elements but a significant change in the tertiary structural organization of this enzyme. The CD spectra at the heme region at various concentrations of alcohols indicate a substantial change in the tertiary structural organization around the heme moiety too. The equilibrium constant associated with the binding of camphor to Cyt P450cam is strongly dependent on the concentration of alcohols and the corresponding free energy associated with the binding is found to scale linearly with the concentration of alcohols. Kinetic experiments on binding of camphor to Cyt P450cam show that both k(on) and k(off) rate constants are strongly affected by addition of alcohols suggesting that alcohol expel camphor out of the heme cavity of Cyt P450cam by affecting tertiary structure of Cyt P450cam as well as by modifying the solubility properties of camphor in aqueous medium.     

Structural requirements for the binding of oligosaccharides to immobilized lectin ofErythrina variegata (Linn) var.Orientalis

The structural requirements for the interaction of asparagine-linked oligosaccharide moieties of glycoproteins withErythrina variegata agglutinin (EVA) were investigated by means of affinity chromatography on an EVA-Sepharose column. Some of the branched poly-N-acetyllactosamine-type oligosaccharides obtained from human erythrocyte band 3 glycoprotein were found to show high affinity to EVA-Sepharose, whereas complex-type oligosaccharides were shown to have low affinity. Hybrid type, oligomannose-type and unbranched poly-N-acetyllactosamine-type oligosaccharides bound very little or not at all to EVA-Sepharose. To further study the carbohydrate-binding specificity of this lectin, we investigated the interaction of immobilized EVA and oligosaccharide fragments obtained through partial hydrolysis from branched poly-N-acetyllactosamine-type oligosaccharides. Branched poly-N-acetyllactosamine-type oligosaccharides were subjected to limited hydrolysis with 0.1% trifluoroacetic acid at 100°C for 40 min and then separated on an amino-bonded silica column. One of pentasaccharides thus prepared strongly bound to the EVA-Sepharose column. Structural analysis of this pentasaccharide showed that the Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal sugar sequence, which is an l-antigen determinant, was essential for the high affinity binding of the oligosaccharides to the EVA-Sepharose column.Abbreviations EVA Erythrina variegata agglutinin - WGA wheat germ agglutinin - STA potato lectin - LEA tomato lectin - DSA Datura stramonium agglutinin - PBS 0.01 M sodium phosphate buffer, pH 7.3, containing 0.15 M NaCl - Galol galactitol     

Binding and precipitating activities ofErythrina lectins with complex type carbohydrates and synthetic cluster glycosides. A comparative study of the lectins fromE. corallodendron,E. cristagalli,E. flabelliformis,andE. indica

Erythrina lectins possess similar structural and carbohydrate binding properties. Recently, tri- and tetra-antennary complex type carbohydrates with non-reducing terminal galactose residues have been shown to be precipitated as tri- and tetravalent ligands, respectively, with certainErythrina lectins [Bhattacharyya L, Haraldsson M, Brewer CF (1988) Biochemistry 271034-41]. The present work describes a comparative study of the binding and precipitating activities of fourErythrina lectins,viz. E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica, with multi-antennary complex type carbohydrates and synthetic cluster glycosides. The results show that though their binding affinities are very similar, theErythrina lectins show large differences in their precipitating activities with the carbohydrates. The results also indicate significant dependence of the precipitating activities of the lectins on the core structure of the carbohydrates. These findings provide a new dimension to the structure-activity relationship of the lectins and their interactions with asparagine-linked carbohydrates.Abbreviations EAL, ECorL, ECL, EFL, and EIL represent the lectins from the seeds ofErythrina arborescens, - E. corallodendron, E. cristagalli, E. flabelliformis, andE. indica respectively - AFOS thetri-antennary complex type oligosaccharide from asialofetuin - AFGP the tri-antennary glycopeptide from asialofetuin - MeGal methyl -d-galactopyranoside Unless stated otherwise all sugars are in thed-configuration.     

Preparation of cluster glycosides ofN-acetylgalactosamine that have subnanomolar binding constants towards the mammalian hepatic Gal/GalNAc-specific receptor

Preparation of di-and tri-valent cluster glycosides containingN-acetyl-d-galactosamine (GalNAc) is described. Oligopeptides that contain a protected amino group and two or three free carboxyl groups are activated by methyl chloroformate and then coupled to 6-aminohexyl 2-acetamido-2-deoxy--d-galactopyranoside. The concentrations of the divalent GalNAc glycosides needed to produce 50% inhibition of the binding of asialoorosomucoid to the isolated, purified rat liver receptor specific for galactose and GalNAc and to the receptor on the hepatocyte surface were of the order of 10^–8 M and 10^–9 M, respectively. The binding affinity of the trivalent glycoside was 10-to 20-fold stronger than the divalent glycosides towards both the soluble receptor and intact hepatocyte.Abbreviations Z benzyloxycarbonyl - EDAC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride - AH 6-aminohexyl - ASOR aslaloorosomucoid - DMF N-dimethylformamide - DMSO dimethylsulfoxide - Lac lactosyl     

Differential binding of lectins to haemocytes of the mussel Mytilus edulis

Summary Pre- and post-embedding techniques were used to investigate the ultrastructural binding of a range of lectins to the haemocytes of the mussel Mytilus edulis. Direct and indirect labelling procedures were employed using colloidal gold and ferritin-labelled lectins, or biotinylated lectins followed by gold-labelled streptavidin. Cell surface receptors were present for lectins from Helix pomatia (HPA), Helix aspersa (HAA), Triticum vulgaris (WGA) and Tetragonolobus purpureas (TPA). Double labelling of haemocytes with HPA and WGA demonstrated binding sites for both lectins on the plasma membrane of the majority of haemocytes. Endocytosis of colloidal gold-labelled HPA was observed for unfixed haemocytes. Three classes of haemocyte were identified by use of morphological criteria: hyalinocytes; granulocytes containing small granules; and granulocytes containing large granules. Lectin binding showed the small granules of the granulocytes to be HPA-positive and the large granules of the granulocytes to be WGA-positive. The WGA-positive granules demonstrated a differential pattern of binding according to granule size. Binding sites for the lectin from Arachis hypogaea (PNA) were not demonstrated on the cell surface, but did show an affinity for the heterochromatin region of the nucleus in post-embedding protocols.     

Field and laboratory studies on the fungus Aspergillus parasiticus, a pathogen of the pink sugar cane mealybug Saccharicoccus sacchari

Infestation of sugar cane nodes by the mealybug Saccharicoccus sacchari (Cockerell) was studied in two commercial fields over a 7-month period in 1987. Natural enemies associated with S. sacchari were fungi Aspergillus parasiticus Speare, Metarhizium anisopliae (Metschnikoff) Sorokin, and Penicillium spp.; the dipteran Cacoxenus perspicax Knab; and the hymenopteran parasitoid Anagyrus saccharicola Timberlake. A. parasiticus was the predominent natural enemy of S. sacchari whereas all other natural enemies showed a low level of activity. The highest prevalence of A. parasiticus was in March when it occurred on 84% of S. sacchari-infested nodes. The prevalence of A. parasiticus declined rapidly during April and May and was absent in the winter months during which nodal infestation of S. sacchari increased. In laboratory bioassays all fungal isolates originating from S. sacchari were more virulent at 28°C than at 24°C. Laboratory studies supported the hypothesis based on field observations that temperature highly influenced the efficacy of A. parasiticus against S. sacchari.     

Purification and characterization of two major lectins fromVigna mungo (blackgram)

Blackgram (Vigna mungo L. Hepper)seeds contain two galactose-specific lectins, BGL-I and BGL-II. BGL-I was partially purified into two monomeric lectins which were designated as BGL-I-1 (94 kDa) and BGL-I-2 (89 kDa). BGL-II is a monomeric lectin of 83 kDA. The purified lectins were associated with galactosidase activities. BGL-I-1 and BGL-II were copurified with α-galactosidase activity while BGL-I-2 was largely associated with β-galactosidase activity. These lectins agglutinate trypsin treated rabbit erythrocytes, but not the human erythrocytes of A, B or O groups. They were stable between pH 3·5 and 7·5 for their agglutination. The lectins did not show any metalion requirement. They were inactivated at 50°C. The lectin activity was inhibited by D-galactose (0·1 mM). The Scatchard plots of galactose binding to these lectins are nonlinear and biphasic curves indicative of multiple binding sites. The data show that the monomeric lectins have both lectin and galactosidase activities suggestive of a bifunctional protein.     

Detection of gangliosides by direct binding ofLimax flavus agglutinin to thin layer chromotograms

A simple and sensitive method for detecting gangliosides on TLC plates is described. Gangliosides are extracted by phase partition in chloroform/methanol, developed on TLC plates in chloroform/methanol/0.25% aqueous KCl (5/4/1 by vol) and identified by binding of¹²⁵I-labelled, sialic acid-specificLimax flavus agglutinin (LFA) autoradiography and scanning densitometry. The detection limit of the method is below 1 ng (0.5 pmol) for GM3, GM1 and GT1b, and below 0.3 ng (0.2 pmol) for GM2 and GD1a. Binding of¹²⁵I-LFA is not inhibited by 10⁶-fold molar excess concentrations ofN-acetylneuraminic acid or lactose but is decreased in a dose-dependent manner by eitherN-acetylneuraminyllactose or unlabelled lectin. Gangliosides were not detected after their treatment byClostridium perfringens sialidase in the presence of taurocholic acid. Ten gangliosides were detected in human brain and seven in normal human serum. Extracts from 0.2 mg of brain and 20 l of serum were sufficient for the detection of major gangliosides.Abbreviations LFA Limax flavus agglutinin - ELLA Enzyme Linked Lectin Assay - PIM Poly(isobutyl methacrylate) - PVP Polyvinylpyrrolidone mol.wt. 40,000 - PBS Phosphate buffered saline - BSA Bovine serum albumin     

Equilibrium and kinetic studies on the binding of des-N-tetramethyltriostin A to DNA

The interaction between TANDEM (a des-methyl analogue of triostin A) and poly(dA-dT) results in extension of the helix by 6.8 Å for each ligand molecule bound, exactly as predicted for a bis-intercalation reaction. Cooperativity is evident in Scatchard plots for the interaction at ionic strengths of 0.2 and 1.0, where the binding constant is diminished compared to that which pertains at low salt concentration. Binding to a natural DNA (calf thymus), already considerably weaker than binding to poly(dA-dT), is also sensitive to increased ionic strength. With a self-complementary octanucleotide d(G-G-T-A-T-A-C-C) the binding curve indicates the presence of a single des-N-tetramethyltriostin A binding site per helical fragment with a non-cooperative association constant about 6·10⁶ M^?1. Detergent-induced dissociation of des-N-tetramethyltriostin A-poly(dA-dT) complexes results in a simple exponential decay at all levels of binding, but the time constant of decay is dependent upon the initial binding ratio. This behaviour cannot directly explain the cooperativity of equilibrium binding isotherms but suggests the occurrence of relatively long-lived perturbations of the helical structure by binding of the ligand. [Ala³, Ala⁷]des-N-tetramethyltriostin A, which has a more flexible octapeptide ring lacking the disulphide cross-bridge, dissociates from poly(dA-dT) much faster than des-N-tetramethyltriostin A. Dissociation of des-N-tetramethyltriostin A from calf thymus DNA is more rapid than dissociation of triostin A or other quinoxaline antibiotics, which may account for its low antimicrobial activity.     

Linker histone binding to superhelical DNA: Electrophoretic and filter binding studies

It has been known for years that linker histones bind preferentially to supercoiled DNA. This preference has been demonstrated by a number of different techniques including deoxyfibonucleoprotein electrophoresis, sedimentation, and filter binding under non-competitive conditions. In an attempt to further study this issue, we used one- and two-dimensional electrophoretic gels and filter binding under competitive conditions, with all DNA forms of interest being simultaneously present in the incubation mixture. Comparison between results obtained by the two methods showed that whereas the preference for superhelical molecules was clearly seen in the electrophoretic gels, the filter binding assay failed to reveal this preference. These results reveal limitations to the filter binding technique, which must be borne in mind in studies involving superhelical DNA molecules.     

Specific binding of a Verticillium dahliae phytotoxin to protoplasts of cotton, Gossypium hirsutum

Summary The occurrence of specific, high-affinity binding sites for a protein-lipopolysaccharide (PLP) phytotoxin purified from culture filtrates of a virulent Vertidllium dahliae isolate has been demonstrated in cotton protoplasts. Binding of the ¹²⁵I-radiolabelled PLP-complex to protoplasts from cotyledon tissue was saturable and with an affinity (K_d = 17.3 nM) comparable with the concentration required for biological activity. A single class of binding site, accessible at the surface of the intact protoplasts, was found and the maximal number of binding sites were estimated as 2.41 × 10^–16 moles per protoplast. The binding affinity to protoplasts proved near identical to that found with purified plasma membrane fractions from roots. When cultivars exhibiting resistance or susceptibility towards the pathogen were compared, no significant differences were found in the affinity of binding, but five times as many binding sites per protoplast and sixteen times as many binding sites per mg membrane protein were found in the resistant cultivar.Abbreviations PLP protein-lipopolysaccharide - k_d dissociation constant - B_max maximal number of binding sites - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The binding of lectins to carbohydrate moieties on haemocytes of the insects,Blaberus craniifer (Dictyoptera) and Extatosoma tiaratum (Phasmida)

Summary The specificity and distribution of carbohydrate moieties, which act as receptors for lectins on the haemocytes of two insect species, Blaberus craniifer and Extatosoma tiaratum, were investigated. Four peroxidase-labelled lectins were utilised as probes: wheat-germ agglutinin, Ricinus communis (120) agglutinin, concanavalin A and Lens culinaris agglutinin, and binding visualised by reaction with DAB/H₂O₂. The lectin-binding studies indicated that Blaberus and Extatosoma plasmatocytes, and Extatosoma spreading granular cells possess surface N-acetyl-D-glucosamine, D-galactose and mannose moieties; Extatosoma cystocytes have D-galactose and mannose; whilst Blaberus granular cells/cystocytes and Extatosoma fine granular cells have mannose determinants.     

Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines.

Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.     

Larvicidal activity of lectins onLucilia cuprina: mechanism of action

Foraging behaviour and host-instar preference of young and old females of the solitary aphid parasitoid,Lysiphlebus cardui Marshall (Hymenoptera: Aphidiidae), were studied in the laboratory. The analysis of interactions between parasitoids and different stages ofAphis fabae cirsiiacanthoidis Scop. (Homoptera: Aphididae) revealed that encounter rates between aphids and parasitoid females and defence reactions of the aphids influenced the degree to which a particular aphid age class is parasitized. Encounter rates between hosts and parasitoid females depended on the foraging pattern of the parasitoid, which varied with age. In mixed aphid colonies patch residence time increased with parasitoid age. Furthermore, younger parasitoids (≦1 day old) laid more eggs into second and third instars, while older parasitoids (≧4 days old) did not show distinct host instar preferences. It is suggested that the oviposition behaviour ofL. cardui is influenced by the physiological state, i.e. the age of the wasp.     

Kinetic studies of killer toxin K1 binding to yeast cells indicate two receptor populations

A recently described new method for determination of killer toxin activity was used for kinetic measurenments of K1 toxin binding. The cells of the killer sensitive strain Saccharomyces cerevisiae S6 were shown to carry two classes of toxin binding sites differing widely in their half-saturation constants and maximum binding rates. The low-affinity and high-velocity binding component (K _T1=2.6x10⁹ L.U./ml, V _max1=0.19 s^-1) probably reflects diffusion-limited binding to cell wall receptors; the high-affinity and low-velocity component (K _T2=3.2x10⁷ L.U./ml, V _max2=0.03 s^-1) presumably indicates the binding of the toxin to plasma membrane receptors. Adsorption of most of the killer toxin K1 to the surface of sensitive cells occured within 1 min and was virtually complete within 5 min. The amount of toxin that saturated practically all cell receptors was about 600 lethal units (L.U.) per cell of S. cerevisiae S6.