Inhibition of conjugation in Tetrahymena pyriformis by ConA : Localization of ConA-binding sites

Two patterns of ConA binding to starved mating types of Tetrahymena pyriformis were observed depending on its time of addition. When ConA was added upon mixing of the mating types, at zero time of conjugation, it was first bound to the oral region and subsequently was taken into intracellular vacuoles. When it was added to conjugants, it was specifically bound as a ring around the conjugation area. The ability of the cells to form vacuoles, assayed by addition of carmine particles, declined prior to pair formation. The relationship between the above phenomena and the ability of ConA to inhibit conjugation is discussed.     

Killing of Trypanosoma brucei by concanavalin A: structural basis of resistance in glycosylation mutants

Concanavalin A (Con A) kills procyclic (insect) forms of Trypanosoma brucei by binding to N-glycans on EP-procyclin, a major surface glycosyl phosphatidylinositol (GPI)-anchored protein which is rich in Glu-Pro repeats. We have previously isolated and studied two procyclic mutants (ConA 1-1 and ConA 4-1) that are more resistant than wild-type (WT) to Con A killing. Although both mutants express the same altered oligosaccharides compared to WT cells, ConA 4-1 is considerably more resistant to lectin killing than is ConA 1-1. Thus, we looked for other alterations to account for the differences in sensitivity. Using mass spectrometry, together with chemical and enzymatic treatments, we found that both mutants express types of EP-procyclin that are either poorly expressed or not found at all in WT cells. ConA 1-1 expresses mainly EP1-3, a novel procyclin that contains 18 EP repeats, is partially N-glycosylated, and bears hybrid-type glycans. On the other hand, ConA 4-1 cells express almost exclusively EP2-3, a novel non-glycosylated procyclin isoform with 23 EP repeats and no site for glycosylation. The predominance of EP2-3 in ConA 4-1 cells explains their high resistance to ConA killing. Thus, switching the procyclin repertoire, a process that could be relevant to parasite development in the insect vector, modulates the sensitivity of trypanosomes to cytotoxic lectins.     

Nuclear glycoproteins of hamster liver and Kirkman-Robbins hepatoma recognized by concanavalin A.

1. Glycoproteins recognized by Concanavalin A (ConA) have been identified in nuclei and nuclear fractions differing in sensitivity to micrococcal nuclease digestion from hamster liver and Kirkman-Robbins hepatoma. 2. The major ConA binding proteins from hamster liver and Kirkman-Robbins hepatoma nuclei have molecular weights about 27,000 and 57,000, and 38,000 and 49,000, respectively. 3. A distinct distribution of glycoproteins between fractions differing in sensitivity to nuclease digestion has not been observed.     

Binding of concanavalin A to intact cells and spheroplasts ofAnacystis nidulans

Native cells of the cyanobacterium (blue-green alga)Anacystis nidulans did not bind fluorescein isothiocyanate-conjugated concanavalin A (FITC-ConA) as measured by fluorescent spectrophotometry. By contrast, spheroplasts ofA. nidulans underwent rapid and specific agglutination in the presence of ConA thus showing appreciable affinity towards the lectin. After treatment with 0.01–0.05% (wt/vol) cetyltrimethylammonium bromide (CTAB) intact cells also became liable to ConA binding, which was not accompanied by significant agglutination. Detergents, other than CTAB, were far less effective. Specific and nonspecific binding was discriminated, as usual, with the aid of methyl -d-mannoside. Conditions are described that allow specific binding of up to 7×10⁴ molecules of FITC-ConA per cell. The binding of ConA to pretreated cells ofA. nidulans was verified by freeze-etching electron microscopy using ferritin-ConA conjugate. Our results appear to be first to demonstrate lectin binding to a cyanobacterium.     

Change of concanavalin A induced agglutinability during preimplantation mouse development

Mouse embryos during early cleavage (zygote to eight-cell stage) were agglutinable with a low concentration (10 μg/ml) of concanavalin A (ConA). This agglutinability was reduced during the first mitotic division. Morulae were agglutinable with a slightly higher concentration (100 μg/ml), whereas blastocysts were not agglutinable even with ConA at a concentration of 5000 μg/ml; however, isolated inner cell masses agglutinated readily at 10 μg/ml of ConA. Embryos grown in vitro behaved as did those isolated directly from the genital tract. Treatment with proteolytic enzymes did not induce agglutinability of mouse blastocyst. The change in agglutinability of trophoblastic cells reflects dramatic changes in the cell surface.     

DURATIONS OF GAMETE MOTILITY AND CONJUGATION ABILITY OF ULVA COMPRESSA (ULVOPHYCEAE)1

The present study was designed to develop a technique for crossing and to gain insight into how sexual reproduction contributes to the maintenance of local populations of Ulva compressa L. To examine the durations of gamete motility and conjugation ability, freshly released gametes were incubated for various periods of time prior to mixing both mating types. The conjugation ability of the gametes gradually declined after being released from the thalli when the gametes were incubated without mixing with the opposite mating type. The ability to conjugate decreased by half after 6 h, although most of the gametes remained motile. The gametes released 4 h later had the same level of conjugation ability when mixed immediately after releasing. When the mature thalli were wrapped in a moist paper towel to prevent gametes from being released, the gametes were preservable for 7 h without a significant decrease in their conjugation ability. Conjugation occurred soon after mixing gametes of both mating types and reached a plateau after 30 s. However, conjugation rates did not exceed a rate of ～70%, even though freshly released gametes were used. Interestingly, a portion of the gametes newly conjugated 30 min after mixing both mating types, and conjugation rates reached a second plateau at ～90%. Gametes with delayed conjugation are provided some period of time that allows them to be transported away and increases their chances of mating with more distant populations, thus contributing to the maintenance of genetic variation.     

Human gastric glycoproteins corresponding to paradoxical concanavalin a staining

Summary Glycoproteins were isolated from human gastric mucosa, and their reactivities with concanavalin A, periodate oxidation and subsequent reduction, are described. Gastric glycoproteins corresponding to the paradoxical concanavalin A staining-class II and III mucins were proved biochemically. The analytical results suggest that N-acetylglucosamine residues in the glycoproteins mediate the interaction with concanavalin A.     

Properties of cell surface carbohydrates in sexual reproduction of the Closterium peracerosum–strigosum–littorale complex (Zygnematophyceae,Charophyta)

The Closterium peracerosum–strigosum–littorale complex is a best characterized zygnematophycean green alga with respect to the process of sexual reproduction. Intercellular communication mediated by two sex pheromones has been well documented in this organism, but information concerning direct cell–cell recognition and fusion of cells involved in conjugation processes has not yet been clarified. In this study, we examined the properties of cell surface carbohydrates in vegetative and reproductive cells using a variety of fluorescein isothiocyanate labeled lectins as probes. Among 20 lectins tested, 10 bound to the Closterium cell surface, and eight of these were specific for the cells involved in sexual reproduction. In addition, some of the lectins inhibited the progress of zygote formation. In particular, Lycopersicon esculentum lectin (LEL) and ConcanavalinA (ConA) considerably inhibited zygote formation (23.6% and 0% of zygotes formed, respectively, compared with the control). LEL mainly accumulated on conjugation papillae and on the surface and lumens of empty cell walls remaining after zygote formation. ConA bound to both vegetative and sexually reproductive cells and strongly accumulated on the conjugation papillae of the latter, indicating ConA binding material(s) are non‐specifically present in Closterium cells but some of the material(s) would be essential for zygote formation. These results suggest that different carbohydrates specifically recognized by these lectins are involved in cell recognition and/or fusion during conjugation processes in the C. psl. complex.     

The role of metal ions in substrate recognition and stability of concanavalin A: a molecular dynamics study

The binding of carbohydrate substrates to concanavalin A (Canavalia ensiformis agglutinin (ConA)) is essential for its interaction with various glycoproteins. Even though metal ions are known to control the sugar binding ability of legume lectins, the interplay between sugar and metal ion binding to ConA has not been elucidated in a detailed manner at the atomic level. We have carried out long, explicit solvent molecular dynamics simulations for tetrameric, dimeric, and monomeric forms of ConA in both the presence and absence of trimannoside and metal ions. Detailed analyses of these trajectories for various oligomeric forms under different environmental conditions have revealed dynamic conformational changes associated with the demetalization of ConA. We found that demetalization of ConA leads to large conformational changes in the ion binding loop, with some of the loop residues moving as far as 17 Å with respect to their positions in the native trimannoside and metal ion-bound crystal structure. However, the β-sheet core of the protein remains relatively unperturbed. In addition, the high mobility of the ion binding loop results in drifting of the substrates in the absence of bound metal ions. These simulations provide a theoretical rationale for previous experimental observations regarding the abolition of the sugar binding ability upon demetalization. We also found that the amino acid stretches of ConA, having high B-factor values in the crystal structure, show relatively greater mobility in the simulations. The overall agreement of the results of our simulations with various experimental studies suggests that the force field parameters and length of simulations used in our study are adequate to mimic the dynamic structural changes in the ConA protein.     

The endocytosis of concanavalin A by Dictyostelium discoideum cells

Differentiating cells of D. discoideum in suspension bind ConA. The proportion of the bound lectin which is competitively removed by methyl-α- mannopyranoside decreases with the time of exposure. Ferritin conjugated ConA is seen to bind both to the cell surface and to be taken into the cells, the proportion of the ConA inside the cells increasing with time. The surface bound ConA is removed by washing with methyl-α- mannopyranoside while the endocytosed ConA appears unaffected. It is suggested that much of the [¹²⁵I]ConA, uncompetable by methyl-α- mannopyranoside in our and other binding studies, may be this intracellular ConA.     

The inactivation by concanavalin A of the ecto-ATPase and ectoacetylcholinesterase of intact skeletal muscles

The (Ca2+ or Mg2+)-activated ectophosphohydrolase of intact frog muscle liberates, in situ, about 37 mumol inorganic phosphate/g muscle in 20 min at 20 degrees C with 10 mM ATP. Pretreatment with concanavalin A (ConA) at 4 degrees C for 18 h caused ectoenzyme inactivation which plateaued at 35-40% of the control rate. The inhibition was concentration dependent, being maximal at about 500 micrograms ConA/mL Ringer's solution. The lectin mediated its effect via the membrane glycoproteins since the inhibition was specifically prevented by alpha-methyl D-mannopyranoside. As the temperature increased from 10 to 40 degrees C, the ectoenzyme activity of untreated muscles increased linearly between 10 and 35 degrees C, with a "break point" and a clear change in slope at 35 degrees C. When treated with ConA the activity increased linearly from 10 to 40 degrees C, eliminating the transition temperature. The findings suggested that a phase transition toward fluidity in the lipid bilayer may have occurred at 35 degrees C and that this was abolished by the lectin binding. Hence we perturbed the surface membrane phospholipids of muscle pretreated with the lectin. Phospholipase C increased the activation by the lectin; phospholipase D had no effect, but phospholipase A2 completely prevented it. The lectin may require the more fluid fatty acyl chains of membrane lipids to achieve inhibition of this ecto-ATPase. Ectoacetylcholinesterase, in situ, and its inactivation by ConA were measured directly on whole, intact skeletal muscles.     

Reversible inhibition of cAMP-induced axon formation in neuroblastoma cells by concanavalin a and vinblastine : Relationship to microtubules and cytotoxicity

The formation of axons induced by dibutyryl-adenosine 3′,5′-cyclic monophosphate (db-cAMP) in neuroblastoma cells was inhibited by concanavalin A (ConA) and vinblastine. These compounds also caused the retraction of existing axons. After removal of ConA or vinblastine, addition of db-cAMP again resulted in axon formation. The cytotoxicity of ConA and vinblastine for neuroblastoma cells was reduced when cell multiplication was inhibited by db-cAMP. Linearly growing normal fibroblasts were also more sensitive to the cytotoxic effect of ConA than confluent non-multiplying fibroblasts. The effects of ConA and vinblastine were additive both in their effects on axon formation and cytotoxicity. Wheat germ agglutinin (WGA) and lumicolchicine did not affect axon formation or reduce cell viability. It is suggested that ConA bound to the cell surface can interfere with the assembly of cytoplasmic microtubules involved in axon formation and cell division.     

Human gastric glycoproteins corresponding to paradoxical concanavalin A staining

Glycoproteins were isolated from human gastric mucosa, and their reactivities with concanavalin A, periodate oxidation and subsequent reduction, are described. Gastric glycoproteins corresponding to the paradoxical concanavalin A staining-class II and III mucins were proved biochemically. The analytical results suggest that N-acetylglucosamine residues in the glycoproteins mediate the interaction with concanavalin A.     

Production of monoclonal antibodies against a cell surface concanavalin a binding glycoprotein

Concanavalin A-binding (Con A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate-polyacrylamide gels than did the Tritonsoluble surface components, which were not retarded by the Con A-Sepharose column. [¹²⁵I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate-polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten α-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [¹²⁵I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [¹²⁵I]-labeled CHO surface component of ～265,000 daltons.     

The Relation of Concanavalin A Receptor Distribution to the Conjugation Process in Tetrahymena thermophila

By using fluorescent isothiocyanate-conjugated concanavalin A (FITC-Con A), cell surface events were examined under a light microscope during the early period of the conjugation process in Tetrahymena thermophila. Until the two complementary mating types (D-III and IV) were mixed, Con A-binding activities were hardly detected on the cell surface of ciliates. After mixing, however, the FITC-Con A (25 μg/ml) bound especially to the anterior cell surface at the early stage of conjugation, followed by characteristic changes of the Con A-binding pattern and, subsequently, by formation of a bright fluorescent ring around the area of contact between conjugants. Such alterations of FITC-Con A-binding pattern were found to be interrupted or eliminated by cycloheximide (2 μg/ml). These findings are related to the onset and subsequent conjugation in T. thermophila.     

The binding of haptoglobin to apolipoprotein AI: influence of hemoglobin and concanavalin A

Haptoglobin (Hp) can be purified by affinity chromatography using hemoglobin (Hb)-linked Sepharose. Elution with 8 M urea is generally performed, resulting in heavy contamination of the Hp preparation by apolipoprotein AI (ApoAI), and partial loss of Hb binding activity. Hp, separated from ApoAI, was recovered by elution with glycine-HCl at pH 3. Complexes of the isolated protein with Hb or ApoAI were detected by enzyme-linked immunosorbent assay (ELISA). Competition between the two ligands in their interaction with Hp was observed. Concanavalin A (ConA), which binds the Hp carbohydrate chains, did not influence Hp binding to ApoAI. These results suggest that changes in the plasma levels of ApoAI or Hb affect the Hp role in regulating the reverse transport of cholesterol or preventing Hb-dependent oxidative damage.     

Mutations Affecting Sexual Conjugation and Related Processes in SACCHAROMYCES CEREVISIAE. I. Isolation and Phenotypic Characterization of Nonmating Mutants

Nonmating mutants were also isolated from haploid strains of yeast of both mating types. The mutants were characterized with respect to their ability to produce and respond to specific yeast sex factors, their ability to mate at low frequencies, and the ability of the low-frequency diploids to sporulate. Loss of the ability to mate by either mating type was invariably accompanied by the loss of one or more, and in some cases, all, of the above capabilities. The results strongly indicate that the sex factors are functionally involved in the conjugation process.     

Conjugal transfer of streptococcal plasmids to strains of Bacillus sphaericus

Abstract The streptococcal plasmids pIP501, pDC10535 and pSM15346 coding for MLS resistance have been successfully transferred to Bacillus sphaericus strains 1593, 2297, 2362 and local strains after mating on filter. The transfer occurred at high frequency and was demonstrated electrophoretically. Conjugation in liquid media also took place but at lower frequency. The conjugation process was studied by electron microscopy. A kind of a bridge of electron-dense material between the mating cells has been observed. The addition of trypsin did not change significantly the transfer frequency in our experimental conditions.     

Genetic diversity in populations of a freshwater ciliate

RAPD fingerprinting with nine different primers revealed that all of 18 E. aediculatus isolates from nine ponds and streams in western Germany, France and the U.S.A. were genetically different. The extent of genetic similarity between genotypes from different waters did not show a significant relationship with the geographical distance among habitats, although genotypes isolated from the same habitat showed a higher genetic similarity than genotypes isolated from different habitats. Phylogenetic analyses of RAPD patterns indicate a separation of E. aediculatus strains into subgroups within one species, but all strains were genetically more similar to one another than to strains from two other Euplotes species. Crossings of the different E. aediculatus strains revealed they belonged to seven mating types of one gene pool. The high genetic diversity observed is explained by a frequent occurrence of conjugation in the studied populations.