Glycoproteins from adult rat brain synaptic vesicles. Fractionation on four immobilized lectins.

Glycoproteins obtained from large amounts of highly purified synaptic vesicles isolated from adult rat brain were fractionated by sequential affinity chromatography in the presence of SDS on four different immobilized lectins: concanavalin A, Ulex europeus lectin, Ricinus sanguinis lectin and wheat germ agglutinin. 83% of the total protein-bound sugar of synaptic vesicles can be adsorbed on the lectins and separated from the bulk of carbohydrate free proteins. Nine fractions containing only glycoproteins and differing by their terminal sugars were separated by analysed for their carbohydrate composition and electrophoretic profiles. A considerable heterogeneity of the glycoprotein population was observed which cannot be explained solely by the microheterogeneity of the glycans of the synaptic vesicle glycoproteins.

Purification and properties of the membrane-bound acetylcholinesterase from adult rat brain.

The membrane-bound acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) from adult rat brain has been purified to homogeneity using sequential affinity chromatography on Con A-Sepharose and on dimethyl-aminoethylbenzoic acid-Sepharose 4B followed by DEAE-cellulose chromatography. The yield of the purified enzyme (specific activity: 3068 U/mg protein) is higher than 50%. Polyacrylamide gel electrophoresis in the presence of Triton X-100 gives only one band with acetylcholinesterase activity. With the exception of electrofocusing and pore gradient electrophoresis, where a multiple band pattern was detected (which seems to be artefactual), the enzyme appears to be homogeneous. Gel filtration and sucrose density gradient centrifugation in the presence of Triton X-100 give only one symmetrical peak, with a calculated molecular weight of 328 000. Since polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) and mercaptoethanol gives only one band with a molecular weight of 74 500, a tetrameric structure can be postulated for the membrane-bound acetylcholinesterase from rat brain.

Structure and immunological localization of spleen sulfolipid.

By using chemical and immunological techniques, the structure of the spleen sulfolipid was confirmed as being a sulfogalactosylceramide. This lipid was localized in the spleen granulocytes (polymorphonuclear leukocytes).

Effect of okadaic acid on O-linked N-acetylglucosamine levels in a neuroblastoma cell line.

O-Linked N-Acetylglucosamine (O-GlcNAc) is a major form of post-translational modification found in nuclear and cytoplasmic proteins. Several authors have advanced the hypothesis according to which phosphorylation and O-GlcNAc glycosylation are reciprocally related to one another [1,2]. In order to test this hypothesis we have investigated the effect of a broad spectrum phosphatase inhibitor, okadaic acid (OA), generally used to induce protein hyperphosphorylation, on the GlcNAc content of cellular glycoproteins. We demonstrate that in neuronal cells lines OA decreases the level of O-GlcNAc in both nuclear and cytoplasmic proteins with a greater effect in the nuclear fraction. This phenomenon was demonstrated by the use of three different procedures for the detection of O-GlcNAc in conjunction with a systematic treatment with PNGase F. O-Linked GlcNAc was characterized using respectively lectin staining with WGA, galactosyltransferase labeling and metabolic labeling of cultured cells with [3H]glucosamine. Although the effects on individual proteins varied, a less pronounced effect was observed on HeLa or COS cell total homogenates. When Kelly cells were treated with OA, the major observation was a decrease in O-GlcNAc content of nuclear proteins. The measurement of the UDP-GlcNAc level clearly demonstrates that the decrease on the O-GlcNAc level in the neuroblastoma cell line after treatment with okadaic acid is not a consequence of the modification of the UDP-GlcNAc pool.

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat: an ultrastructural study.

In a previous paper (Kuchler et al., Eur. J. Cell Biol. 52, 87-97 (1990)), endogenous carbohydrate-binding sites were studied at the optical level during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidin-peroxidase staining. It was shown that these tools were capable of revealing carbohydrate-binding sites during development of the rat cerebellum. The staining pattern with the individual probes disclosed variable developmental regulation and consequently suggested that recognition processes during cerebellar development may include several types of carbohydrate determinants. However, studies at the light microscope could not give information on potential membrane-bound localization of carbohydrate-binding sites and therefore discern the possible involvement of these molecules in cell adhesion processes. Furthermore, nuclear staining was suggested at the optical level. In order to elucidate these points, we examined the localization of mannose-, fucose- and heparin-binding sites at the electron microscopic level. Ultrastructural studies demonstrate that these tools are very efficient in detecting intracellular carbohydrate-binding sites, but failed to detect most of them expressed at the cell surface when using immunocytochemical techniques for known receptors, probably because of the interaction of these carbohydrate-binding sites with endogenous membrane-bound ligands. The significance of the nuclear staining and of part of the nucleolus found with fucose-containing neoglycoprotein and of the nuclear staining found with heparin are discussed.

Detection of binding sites for biotinylated neoglycoproteins and heparin (endogenous lectins) during cerebellar ontogenesis in the rat.

Endogenous carbohydrate-binding sites were studied during rat cerebellar development on sections of fixed tissue using synthetic tools, biotinylated neoglycoproteins, in conjunction with subsequent avidinperoxidase staining. Neoglycoproteins were constructed by chemically coupling the histochemically pivotal carbohydrate moieties to an inert carrier protein. The sugar part of the neoglycoproteins included common constituents of the carbohydrate part of cellular glycoconjugates, namely mannose, galactose, fucose, N-acetyl-glucosamine, N-acetylgalactosamine and N-acetyl-neuraminic acid to probe for the presence of respective endogenous receptors. Heparin was biotinylated after mild cyanogen bromide activation and aminoalkylation. Specific positive reactions were obtained for all neoglycoproteins and heparin. The staining pattern with the individual probes disclosed variable developmental regulation. Consequently, these results suggest that recognition processes during cerebellar development may include several types of carbohydrate determinants. In two instances, the binding of neoglycoproteins could be compared to endogenous lectin-specific antibodies. Despite a significant extent of accordance the comparison revealed notable differences. These differences were attributed primarily to fixation and the presence of physiological ligands that can mask the active endogenous carbohydrate-binding proteins. In any case, histochemical application of labeled neoglycoproteins is valuable to discern the presence, localization and developmental pattern of binding sites for the carbohydrate part of glycoconjugates, on which further biochemical and cell biological studies can consequently be based.

Involvement of the endogenous lectin CSL in adhesion of Chinese hamster ovary cells.

Immunochemical localization of an endogenous mannose-binding protein, the cerebellar soluble lectin (CSL; Zanetta et al., J. Neurochem. 49, 1250-1257 (1987)), in Chinese hamster ovary cells indicated its high concentration in areas of contact between cells. This suggested its role in cell adhesion. The pattern of staining differed significantly in the cells cultured in suspension from that grown as monolayer. In cells maintained for a short time as suspension, the extracellular CSL immunoreactivity was found mainly in close apposition to the plasma membrane including contact areas. In cells cultured as monolayer, extracellularly, the lectin was found both at the cell surface and in a 75-nm thick layer between two cells, apparently adhering to the cell surface through bridges. Endogenous glycoprotein ligands of CSL were present in the cultures of CHO cells, both as membrane-bound glycoproteins and as glycoprotein ligands soluble in the presence of mannose in the absence of detergent. The lectin CSL induced adhesion between these cells as evident by low concentration of anti-CSL Fab fragments inhibiting such adhesion. These data suggested that adhesion between CHO cells occurs, in part, through a glycobiological recognition system involving CSL. This mechanism should be taken into account for the interpretation of experiments of transfection in CHO cells of the genes of glycoproteins involved in cell adhesion.

Malignant cells have increased levels of common glycoprotein ligands of the endogenous cerebellar soluble lectin CSL.

The glycoprotein composition of various transformed cells or malignant tumors was analyzed and compared to their respective non-malignant control cells or tissues of several species, including man, using an endogenous carbohydrate-binding protein, the cerebellar soluble lectin CSL (Zanetta et al., J. Neurochem. 49, 1250-1257 (1987). A large variety of transformed cells contain a much higher number and larger quantity of glycoprotein ligands of CSL than the control cells or normal tissues. The glycoprotein profiles were, in most cases, independent of the nature of the cell transformation of the degree of differentiation, of the tissue and species. Thus, it is suggested that many transformed cells have, as a common anomaly, the increased synthesis of the special type of glycan recognized by CSL, expressed on the same polypeptide chains.

Occurrence of ceramides and neutral glycolipids with unusual long-chain base composition in purified rat liver mitochondria.

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three-fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long-chain bases found in free ceramides were d18:1 sphingosine, d18:0 3-ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3-ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long-chain base of glucosylceramide of outer membranes.

Role of oligomannosidic N-glycans in the proliferation, adhesion and signalling of C6 glioblastoma cells.

The potential role of glycoprotein N-glycans in the proliferation and adhesion of C6 glioblastoma cells was investigated using a set of N-glycosylation inhibitors (tunicamycin, deoxynojirimycin, castanospermine, deoxymannojirimycin, swainsonine), and traffic (monensin). It was observed that both the proliferative and adhesive properties of C6 cells were dependent upon the expression at the cell surface of glycoproteins with oligomannosidic and hybrid type N-glycans, whereas the absence of N-glycans (tunicamycin) or the presence of glucosyl-oligomannosides (deoxynojirimycin and castanospermine) and the absence of glycoproteins at the cell surface (monensin) reduced both the proliferative and adhesive properties of C6 cells. Studies of the classical elements of signalling pathways indicated that the different inhibitors have a low impact on tyrosine phosphorylations and oncogene product expression (except the ras oncogene product), except on phosphorylations on other residues. An endogenous soluble lectin (CSL; J. Neurochem. 49 (1987) 1250), specific for oligomannosidic and hybrid type N-glycans, was present and externalised by the cells through a pinching-off of large intracellular vesicles, a mechanism that was not blocked by monensine; in contrast with the externalisation of its glycoprotein ligands. The inhibitory effect of anti-CSL Fab fragments on adhesion indicates that the polyvalent CSL acts as a bridging molecule for a family of surface glycoproteins expressed at the surface of C6 cells. The inhibitory effect of the same Fab fragments on the proliferation indicates that CSL is a mitogen for these cells, possibly involved in clustering its surface glycoprotein ligands. A mechanism for the loss of contact inhibition is discussed based on the over-expression of CSL ligands in C6 glioblastoma cells relative to normal cells.

Carbohydrate and glycoprotein specificity of two endogenous cerebellar lectins.

Two endogenous cerebellar mannose binding lectins have been isolated in an active form by immunoaffinity chromatography employing their respective immobilized antibodies. One of them, termed cerebellar soluble lectin (CSL), was extracted in the absence of detergents, whereas the other, called Receptor 1 (R1), was soluble only in the presence of detergents. Tests of inhibition of agglutination of erythrocytes were performed with mono-, oligo and polysaccharides, as well as glycoconjugates of known structures. On the basis of agglutinating activities these 2 lectins are different from the previously reported lectins in brain, since they were not inhibited by galactosides and lactosides and were only marginally inhibited by glycosaminoglycans. CSL and R1 were better inhibited by mannose-rich glycopeptides as compared to the corresponding oligosaccharides. The different inhibition patterns obtained with glycans of known structures indicated that these lectins are very discriminative. Although CSL and R1 have similar specificities, they differed in their binding properties towards glycopeptides of ovalbumin. Both lectins showed considerable affinity for endogenous cerebellar glycopeptides, also rich in mannose. These glycopeptides belong to a few endogenous Con A-binding cerebellar glycoprotein subunits and are not present on other endogenous Con A-binding glycoproteins. In the forebrain, where CSL and R1 were also present, at least some of the glycoproteins interacting with the lectins were different from that observed in the cerebellum. Our data overall suggest that specific cell recognition in the nervous system could be invoked via the interactions between widely distributed lectins and cell-specific glycoproteins.

The SPASIBA force field as an essential tool for studying the structure and dynamics of saccharides.

The SPASIBA force field has been applied to the determination of the structure and dynamical properties of various disaccharides. It has been shown that the experimental properties (structure, dipole moment, conformational relative energies) are satisfactorily predicted. The anomeric and exo-anomeric effects are confidently reproduced without specific terms for the alpha and beta anomers and the type of glycosidic linkages.

Involvement of glycosylation in the intracellular trafficking of glycoproteins in polarized epithelial cells.

The surface of epithelial cells is composed of apical and basolateral domains with distinct structure and function. This polarity is maintained by specific sorting mechanisms occurring in the Trans-Golgi Network. Peptidic signals are responsible for the trafficking via clathrin-coated vesicles by means of an interaction with an adaptor complex (AP). The basolateral targeting is mediated by AP-1B, which is specifically expressed in epithelial cells. In contrast, the apical targeting is proposed to occur via apical raft carriers. It is thought that apically targeted glycoproteins contain glycan signals that would be responsible for their association with rafts and for apical targeting. However, the difficulty in terms of acting specifically on a single step of glycosylation did not allow one to identify such a specific signal. The complete inhibition of the processing of N-glycans by tunicamycin often results in an intracellular accumulation of unfolded proteins in the Golgi. Similarly, inhibition of O-glycosylation can be obtained by competitive substrates which gave a complex pattern of inhibition. Therefore, it is still unknown if glycosylation acts in an indirect manner, i.e. by modifying the folding of the protein, or in a specific manner, such as an association with specific lectins.

Inhibition of the glycosylation and alteration in the intracellular trafficking of mucins and other glycoproteins by GalNAcalpha-O-bn in mucosal cell lines: an effect mediated through the intracellular synthesis of complex GalNAcalpha-O-bn oligosaccharides.

To address the function of carbohydrates in mucins, GalNAcalpha-O-bn has been used in in vivo experiments on several human mucosal cultured cells as a potential competitor of the glycosylation of N-acetylgalactosamine residues. GalNAcalpha-O-bn is metabolized by glycosyltransferases expressed in the cell, and give rise to different internal derivatives starting in particular from the formation of the disaccharide Galalpha1-3GalNAcalpha-O-bn. In this line, GalNAcalpha-O-bn exposure inhibits peripheral glycosylation according a cell-type specific manner. The metabolic alterations are very important in HT-29 cell line, leading to a massive accumulation of GalNAcalpha-O-bn oligosaccharide derivatives and to a strong inhibition of the terminal elongation of O-glycans by alpha2,3 sialyltransferase ST3Gal I. GalNAcalpha-O-bn treatment also induced alterations at the cellular level, exhibiting a large scale in HT-29 cells, i.e. 1) an inhibition of mucin secretion, 2) a blockade in the targeting of some membrane glycoproteins (brush border glycoproteins such as dipeptidylpeptidase IV, carcinoembryonic antigen and the mucin-like glycoprotein MUC1, and the basolateral cell adhesion molecule CD44), 3) an inhibition in the processing of lysosomal enzymes. Morphological abnormalities have been evidenced in GalNAcalpha-O-bn treated cells, in particular the accumulation of numerous intracellular vesicles in HT-29 cells. Taken together, these data suggest that O-glycosylation might be involved in the regulation of the targeting of O-glycosylproteins through carrier vesicles.

Cerebrospinal fluid anti-cerebellar soluble lectin antibodies in human immunodeficiency virus type 1 infection.

Cerebrospinal fluid samples from 14 human immunodeficiency type 1 (HIV-1) seropositive patients in various stages of HIV infection were tested for the presence of autoantibodies to an endogenous manose-binding protein, the cerebellar soluble lectin (CSL), which has recently been found to be detected in a high proportion of patients with multiple sclerosis. An immunoblotting test was used with rat CSL as antigen. Seven patients were positive for anti-CSL and seven were negative. The seven anti-CSL-positive patients had signs of intrathecal immunoglobulin G production measured as an elevated IgG index, while the seven anti-CSL-negative patients had a normal IgG index. There was no apparent relation between infectious stage and the presence of anti-CSL. Immunological reactions such as anti-CSL autoantibodies may be a similar pathogenic mechanism in HIV and multiple sclerosis brain disease.

Presence of anti-CSL antibodies in the cerebrospinal fluid of patients: a sensitive and specific test in the diagnosis of multiple sclerosis.

The carbohydrate-binding protein (lectin) CSL is an antigen involved in the stabilization of the myelin structure by interacting with the carbohydrate moiety of myelin glycoproteins. Since anti-CSL Fab fragments were able to produce destruction of CNS myelin in vitro, CSL was considered as a potential immunological target in multiple sclerosis. The presence of anti-CSL antibodies has been examined in the cerebrospinal fluid of 1388 different patients with various neurological diseases. It is concluded that the presence of anti-CSL antibodies in the cerebrospinal fluid of patients less than 50 years old constitutes a very sensitive and specific test for multiple sclerosis.

Location of a transiently expressed glycoprotein in developing cerebellum delineating its possible ontogenetic roles.

The development pattern of a 31,000 mol. wt phosphatidyl inositol-anchored membrane glycoprotein was followed during development in mouse and rat cerebellum using monoclonal antibody 194-653. The epitope was developmentally regulated and particularly abundant in post mitotic precursors of granule cells, newly formed parallel fibres and unmyelinated axons of the white matter between the 5th and the 15th postnatal days. It decreased considerably thereafter. In the adult, a significant although relatively low staining was observed only in white matter. Observation at the ultrastructural level showed that most of the 31,000 mol. wt glycoprotein was very concentrated on neuronal plasma membranes. A little immunoreactivity was also found intracellularly at the perinuclear membrane of neuroblasts of the external germinal layer. The antigen was present in the coated pits and intracellularly in coated vesicles. Immunochemical studies indicated that 31,000 mol. wt antigen was very likely to be a previously identified transient concanavalin A-binding glycoprotein insoluble in neutral detergents (Reeber et al., 1981; Brain Res. 229, 53-65). It appeared to be one of the glycoprotein ligands for two endogenous mannosyl-lectins isolated from rat cerebellum (Zanetta et al., 1985, Devl. Brain Res. 17, 233-243, Zanetta et al., 1987, J. Neurochem. 49, 1250-1257). The affinity of the 31,000 mol. wt glycoprotein for the two endogenous lectins, together with its developmental pattern and localization indicate that it could be an important molecule for contact guidance during migration of neurons and for myelination and could take part in other ontogenetic steps.

Expression and localization in the developing cerebellum of the carbohydrate epitopes revealed by Elec-39, an IgM monoclonal antibody related to HNK-1.

The immunochemical and immunocytochemical reactivity of an anti-carbohydrate monoclonal antibody (Elec-39), obtained against acetylcholinesterase from Electrophorus electricus electric organ, was followed during the postnatal development of the rat cerebellum. The specificity of this antibody resembles that of a family of anti-carbohydrate antibodies that includes HNK-1, L2, NC-1 and NSP-4, as well as IgMs that occur in some human neuropathies. As revealed by immunoblotting techniques, the reactivity of Elec-39 is maximum around postnatal days 10-12. At this age, the antibody reveals eight major proteins of mol. wt ranging between 14 and 150 kDa. Some of them (with mol. wts of 14, 18, 28 and 31 kDa) are transiently expressed. They correspond to previously identified glycoproteins binding to the plant lectin concanavalin A and binding also to the endogenous mannose-binding lectin CSL and endogenous membrane-bound mannose-binding lectin. In young animals, an important staining with the Elec-39 antibody can be observed on postmitotic precursors of granule cells, on astrocyte processes in the external granular layer, on newly formed parallel fibres and on unmyelinated axons of the white matter. In adult animals, the labelling is localized essentially in myelin and also in the cytoplasm of astrocytes. These results are discussed in relation to ontogenetic phenomena occurring during cerebellar development and the potential role of the carbohydrate epitope revealed with Elec-39 as a determinant in cell adhesion processes.

Carbohydrates and soluble lectins in the regulation of cell adhesion and proliferation.

There is a large body of suggestions that complex carbohydrates play a role in the regulation of cell adhesion and cell proliferation. Many reports have emphasized that proteoglycans, glycoproteins or glycolipids are participating to cell adhesion mechanisms. The use of polyvalent anti-carbohydrate antibodies and plant lectins as well as the use of glycosylation inhibitors suggested that cell proliferation can be modulated by surface carbohydrates. The dating experiment of Burger and Noonan (1970) showing restoration of contact inhibition of malignant cells by monovalent concanavalin A was a determining experiment. However, in the latter as in the others, no precise mechanism was demonstrated how carbohydrates can be involved in adhesion and proliferation. New insights were opened with the discovery of vertebrate membrane-bound and soluble lectins. The latter generally display agglutinating activities in in vitro systems, suggesting that they were potential cell adhesion molecules, by forming bridges between cell surface carbohydrates. These polyvalent molecules may be also considered as clustering agents for their cell surface ligands, consequently generating signals for cell proliferation and/or differentiation.

Lectin activities of cytokines and growth factors: function and implications for pathology.

The discovery that certain cytokines have carbohydrate-binding (lectin) properties opens new concepts in the understanding of their mechanism of action. The carbohydrate-recognition domain, which is localized opposite to the receptor-binding domain, makes these molecules bi-functional. The expression of the biological activity of the cytokine relies on its carbohydrate-binding activity which allows the association of the cytokine receptor with molecular complexes comprising the specific kinase involved in receptor phosphorylation and in specific signal transduction. It is expected that blood accumulation of free or membrane-bound glucan ligands of cytokines may dramatically perturb their endogenous function inducing specific immunodeficiencies.