Concanavalin A Receptors Associated with Rat Brain Synaptic Junctions Are High Mannose-Type Oligosaccharides

Abstract: Glycoproteins were isolated from a rat brain synaptic junction fraction by affinity chromatography on Concanavalin A-agarose. The isolated glycoproteins were digested with pronase and radiolabeled with ¹²⁵I-Bolton Hunter reagent, and ¹²⁵I-Concanavalin A-binding glycopeptides were isolated by chromatography on Concanavalin A-agarose. Treatment of the ¹²⁵I-Concanavalin A-binding glycopeptides with either α-mannosidase or endo-β- N -acetylglucosaminidase-C₁₁ abolished their interaction with Concanavalin A. The pronase digest was reacted with endo-β-N-acetylglucosaminidase-C₁₁ and released oligosaccharides were reduced with NaB³H₄. Following affinity chromatography on Concanavalin A-agarose, Concanavalin A-binding [³H]oligosaccharides were chromatographed on Biogel P₄. Two major oligosaccharides corresponding to standard carbohydrates containing eight and five mannose residues were identified. Treatment of these oligosaccharides with α-mannosidase converted them to smaller saccharides having a mobility on Biogel P₄ columns equal to the standard disaccharide mannose-β-1-4- N '-acetylglucosamine. These results demonstrate that the Concanavalin A receptor activity associated with CNS synaptic junctions resides in asparaginelinked oligosaccharides of the high-mannose type.     

Conjugation in the Ciliate Euplotes octocarinatus: Comparison of Ciliary and Cell Body-Associated Glycoconjugates of Non-mating-Competent, Mating-Competent, and Conjugating Cells

Pair formation in the hypotrichous ciliate Euplotes octocarinatus is a poorly understood phenomenon. In order to obtain information about the molecules involved in this process, we compared ciliary and cell body-associated glycoconjugates of non-mating-competent, mating-competent, and conjugating cells. Detection of glycoconjugates was carried out on Western blots by immunostaining of oxidized, digoxigenin-labeled carbohydrate moieties. Using this method, in both of two complementary mating types a 130-kDa glycoprotein was identified, which appeared on cilia during acquisition of mating competence and was reduced during cell pairing. This suggests an active role of this glycoprotein in ciliary adhesion during pair formation, Additionally, in both of the two mating types a cell body-associated 135-kDa glycoprotein was detected, which is present in non-mating-competent cells as well as in mating-competent cells, but is strongly reduced in conjugating cells. In contrast to the ciliary 130-kDa glycoprotein, the cell body-associated 135-kDa glycoprotein is not surface-exposed [8]. We therefore propose that the cell body-associated glycoprotein is either involved in the preparation for cell fusion or meiosis or that it serves as a cytoplasmic pool for the ciliary 130-kDa glycoprotein.     

The histochemistry of glycoconjugates in the colonic epithelium of the chicken

Summary In the colonic epithelium of the chicken, glycoconjugates have been studied by means of selected histochemical methods of light and electron microscopy. According to the results obtained, most of the colonic goblet cells contained acidic and neutral glycoconjugates with sulphate and vicinal diol groupings, -D-mannose and -D-glucose residues and sialic acid-galactose dimers. These goblet cells were found to undergo changes in histochemical reactivity during upward migration along the crypts; -D-mannose and -D-glucose residues and terminal sialic acidgalactose dimers increased in amount. The striated border of the colonic columnar cells has, likewise, been found to contain such glycoconjugates as were similar in reactivity to those of the goblet cells. The histophysiological significances of glycoconjugates involved in the chicken colonic epithelium have been discussed with special reference to the functional activities of the carbohydrates.     

Influence of temperature on the interaction of Concanavalin A with chick fibroblasts during embryo development

The interaction between Concanavalin A and chick embryo fibroblasts was studied. Cells from younger and older embryos had the same number of lectin receptor sites per cell at 4°, 21° and 37°C but the affinity constants increased with increasing temperature. Analysis of the binding data according to Scatchard showed that the apparent changes in binding as a function of temperature might be related to thermodynamic properties. The lectin binding sites on the cell surface proved homogeneous with regard to binding properties.The age-related differences noted in the affinities of the cells to bind Concanavalin A could be related to differences in the degree of rearrangement of the cell surface components and/or to a change in the structure of cell surface glycoconjugates, and may serve to explain the differences in the effect of Concanavalin A on cell growth.     

Local and Temporal Pattern of Con A-Binding Site Aggregation During Conjugation in Euplotes vannus (Ciliophora,Hypotrichida)1

ABSTRACT. Conjugating cells of Euplotes vannus (syngens Naples and Barcarès) were investigated for Con A-binding sites by means of fluorescence microscopy. Cells fixed with 0.4% paraformaldehyde and incubated with 15 μg/ml FITC-Con A showed a distinct region of strong fluorescence, the size, shape and localization of which characteristically changed during the course of conjugation, first appearing at courtship stage (cells contact without forming pairs) and vanishing soon after the migration pronuclei have been exchanged, but before cells have separated. Con A binding and conjugation were blocked by cyclohexamide and α-galactosidase. Con A-binding was also inhibited by α-mannosidase, α-glucosidase and β-galactosidase without affecting conjugation except a delay of pair formation. The results suggest an involvement of newly formed or translocated glycoconjugates in cell pairing during conjugation.     

Detection of a Concanavalin A binding protein in the mollicute Spiroplasma citri and purification from the plasma membrane

A protein that binds Concanavalin A (Con A) was detected on Western blots of Spiroplasma citri proteins. Its apparent molecular weight was 84000. It was localized in the plasma membrane. Affinity chromatography on Con A-agarose was used to isolate this protein. The glycosylation inhibitor, tunicamycin, inhibits S. citri growth and seems to block the glycosylation of the Con A-binding protein.     

Affinity-Purification of Concanavalin A-Binding Ciliary Glycoconjugates of Starved and Feeding Tetrahymena thermophila

Development of mating competency in Tetrahymena thermophila requires starvation for at least 70 min in low ionic strength buffer. Pair formation between conjugating cells is blocked at early stages by the lectin Concanavalin A (Con A). To investigate the role of Con A-binding proteins in this induced cellular change and pairing, and to confirm and extend an earlier study from our laboratory, a method was developed for preparation of Con A-binding proteins from ciliary membrane-rich fractions of T. thermophila. Con A-binding ciliary proteins were prepared from non-starved and starved cells from two wild type strains and a mating mutant, RH179E1. Comparison of these proteins by SDS-PAGE revealed on overall reduction in number of wild-type bands after starvation. In particular, a major band at 28 kDa was present in non-starved cells and absent in starved cells. However, in the mating mutant, no change in banding profile was seen after starvation: the 28 kDa band was present in both non-starved and starved cells. This, Con A-binding ciliary membrane proteins undergo a major change during starvation-induced development of mating competency in wild-type T. thermophila. In contrast, the mutant differed from wild-type in overall composition of its ciliary Con A-binding glycoproteins and in the response of these proteins to starvation, suggesting that it may be deficient in its ability to be initiated by starvation. Our results are consistent with the hypothesis that a change affecting ciliary membrane Con A-binding proteins is essential for the cellular response to mating signals.     

Transient Concanavalin A-Binding Glycoproteins of the Parallel Fibres of the Developing Rat Cerebellum: Evidence for the Destruction of Their Glycans

Abstract: The lifetime of the glycoprotein glycans of the rat cerebellum was followed in the 2nd and 3rd weeks of postnatal age, after injection of labelled glucosamine. It appears that a particular class of glycans binding to Concanavalin A synthesized at an early age has a short lifetime. These results indicate that the glycans of the Concanavalin A-binding glycoproteins abundant on the newly formed parallel fibres are rapidly degraded between the 14th and the 18th postnatal day. Reeber A. et al. Transient Concanavalin A-binding glycoproteins of the parallel fibres of the developing rat cerebellum: Evidence for the destruction of their glycans. J. Neurochem. 35 , 1273–1277 (1980).     

The cytochemistry of glycoconjugates in the planum nasolabial gland of the goat as studied by electron microscopic methods

Summary In the planum nasolabial glands of the goat, glycoconjugates of glandular and duct cells have been studied by means of a series of electron microscopic cytochemical methods. In the glandular cells glycoconjugates with vicinal diol groupings were present in secretory granules, certain elements of the Golgi complex, lysosome-like dense bodies, the surface coat of the plasma membrane, the majority of intracellular cytomembranes, glycogen particles and the basal lamina. In duct cells, glycoconjugates with the same properties were localized in similar ultrastructures, except for secretory granules, which were not detected in these cells. By lectin cytochemistry, glycoconjugates in glandular cell secretory granules contained a variety of saccharide residues such as -d-mannose, -d-glucose,N-acetyl-d-glucosamine and -l-fucose. The cytochemical properties of the secretory glycoconjugates are discussed in relation to the physiological functions performed by the planum nasolabial glands in the goat.     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: Starved versus nonstarved

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent (“initiated”) cells were compared with those from non-starved, mating-incompetent (“non-initiated”) cells, by gel electro-phoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glyco-conjugate as having a potential role in control of pair formation during mating. © 1992 Wiley-Liss, Inc.     

Asymmetric distribution of Concanavalin A binding sites on yeast plasmalemma and vacuolar membrane

Isolated vacuoles of Saccharomyces cerevisiae did not bind Concanavalin A (labelled with tritium or with a fluorescent dye) unless the vacuoles were rendered permeable and their inner membrane surface made accessible. Yeast protoplasts, on the other hand, bound large amounts of Concanavalin A on their surface, and the number of binding sites was not increased after a gentle lysis expected to expose also the inner surface of the plasmalemma. It is concluded that both the plasmalemma and the vacuolar membrane carry Concanavalin A binding sites exclusively on the surface opposite to the cytoplasmic matrix.Non-Standard Abbreviations ConA concanavalin A - MDPF 2-methoxy-2,4-diphenyl-3(2H)-furanone - -MM -methyl-D-mannopyranoside - Pipes piperazine-N,N-bis-2-ethanesulfonic acid - DNP potassium dinitrophenolate     

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts.

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.     

Glycoconjugates in the secretory epithelium of the chicken mandibular gland

Summary In the secretory epithelium of the chicken mandibular gland, glycoconjugates have been studied by means of histochemical methods of light and electron microscopy. In light microscopy, a series of histochemical procedures have been employed which included lectin—peroxidase—diaminobenzidine methods and a digestion technique with neuraminidase or-amylase. In electron microscopy, a battery of methods were used that corresponded to those employed in light microscopy. In the secretory cells of the chicken mandibular gland, vicinal diol- and sulphate-containing glycoconjugates with sialic acid,-d-mannose,-d-glucose and-d-galactose residues were visualized and the possible histophysiological significances of such glycoconjugates were discussed with special reference to the functions of the salivary gland.     

Inhibition of Herpes Virus-Induced Cell Fusion by Concanavalin A, Antisera, and 2-Deoxy-d-Glucose

Pseudorabies virus-induced cell fusion in rabbit kidney cells can be prevented by Concanavalin A added early after infection. The infected cells are not agglutinated and the infectivity of cell-free virus is not reduced. Sera from productively infected animals also inhibit polykaryocytosis, whereas a hyperimmune serum directed against virus structural components has no effect. 2-Deoxy-d-glucose reversibly disturbs virus-induced fusion and reduces significantly the virus infectivity.     

Identification of Tetrahymena ciliary surface proteins labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate and Concanavalin A and fractionated with Triton X-114.

Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from greater than 350 kDa to less than 20 kDa, were resolved. The major surface 50-60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg(2+)-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

Concanavalin A binding to Chlamydomonas eugametos flagellar proteins and its effect on sexual reproduction

The relative amounts of Concanavalin A (Con A) bound by gamete and vegetative flagella of both mating types (mt ⁺ and mt ^-) of Chlamydomonas eugametos were determined using ¹²⁵I-Con A. Con A agglutinated all cell types by cross-linking their flagella in a random manner. No correlation was found between the extent of Con A-binding and Con A-mediated isoagglutination. Con A inhibited the sexual interaction between gametes at various levels. In mt ⁺ gametes it blocked sexual agglutination, whereas in mt ^- gametes it prevented papillar fusion. By SDS-gel electrophoresis nine Con A-binding components were found to be present in flagella. However, it was not possible to allocate a role in sexual agglutination to any of these components since they were present in all cell types, including vegetative cells which are not able to sexually agglutinate.Abbreviations Con A concanavalin A - SDS sodium dodecyl sulphate - TB Tris buffer - PBS phosphate buffered saline - HRP horse radish peroxidase - SEM scanning electron microscope - PAS periodic acid Schiff     

Ciliary polypeptides and glycoconjugates of wild-type and mutant Tetrahymena thermophila: starved versus nonstarved.

To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating.     

C-2 Epimer Formation of Tetrose, Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg -1 min -1 from d -glucose, 0.56 mg mg -1 min -1 from d -arabinose and 3.0 mg mg -1 min -1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.     

Identification of Tetrahymena Ciliary Surface Proteins Labeled with Sulfosuccinimidyl 6-(Biotinamido) Hexanoate and Concanavalin A and Fractionated with Triton X-114

ABSTRACT. Tetrahymena thermophila cells were labeled with sulfosuccinimidyl 6-(biotinamido) hexanoate, a sensitive nonradioactive probe for cell surface proteins, and Western blots of axonemes and ciliary membrane vesicles were compared to cilia fractionated with Triton X-114 (TX-114) in order to study the orientation of ciliary membrane proteins. Greater than 40 ciliary surface polypeptides, from >350 kDa to <20 kDa, were resolved. The major surface 50–60 kDa proteins are hydrophobic and partition into the TX-114 detergent phase. Two high molecular weight proteins, one of which is biotinylated, comigrate with the heavy chains of ciliary dynein, sediment at 14S in a sucrose gradient, and partition into the TX-114 aqueous phase. Fractions containing these high molecular weight proteins as well as fractions enriched in 88-kDa and 66-kDa polypeptides contain Mg²⁺-ATPase activities. Detergent-solubilized tubulins partition into the TX-114 aqueous phase, are not biotinylated, and must not be exposed to the ciliary surface. The detergent-insoluble axoneme and membrane fraction contains a 36-kDa polypeptide and a portion of the 50-kDa polypeptides that otherwise partition into the detergent phase. These polypeptides could not be solubilized by ATP or by NaCl extraction and appear to be associated with pieces of ciliary membrane tightly linked to the axoneme. The ciliary membrane polypeptides were also tested for Concanavalin A binding and at least sixteen Con A-binding polypeptides were resolved. Of the major Con A-binding polypeptides, three are hydrophobic and partition into the TX-114 detergent phase, three partition into the TX-114 aqueous phase, and four partition exclusively in the detergent-insoluble fraction, which contains axonemes and detergent-resistant membrane vesicles.     

C-2 Epimer Formation of Tetrose,Pentose and Hexose Sugars by Xylose Isomerase

We describe novel tetrose isomerizations and C-2 epimerizations by the industrially important d -xylose ketol-isomerase (E.C.5.3.1.5) with both the d - and l -forms of the sugars. We further show that in addition to isomerization to d -fructose, d -glucose is slowly C-2 epimerized to d -mannose. The formation rate of the C-2 epimer was 0.03 mg mg &#109 1 min &#109 1 from d -glucose, 0.56 mg mg &#109 1 min &#109 1 from d -arabinose and 3.0 mg mg &#109 1 min &#109 1 from d -erythrose. The equilibria of the reaction products as a function of temperature were measured for threose/erythrulose/erythrose, arabinose/ribulose/ ribose and glucose/fructose/mannose.