Sabine Kuchler

Isolation and Immunochemical Study of a Soluble Cerebellar Lectin Delineating Its Structure and Function

Abstract: A procedure of sequential extractions of cerebellar tissue was set up, which allowed specific solubilization of endogenous lectins by mannose. Two cerebellar soluble lectins, CSL1 (Mr= 33,000) and CSL2 (Mr= 31,500), were isolated. They appeared to consist of structurally and immunologically related polypeptides chains. By immunoaffinity, another minor component (Mr= 45,000) was isolated. Immunological studies suggested that the minor component is the precursor of the two other, i.e., CSL1 and CSL2, subunits. CSL1 (mainly lysosomal) possesses an additional peptide compared with CSL2 (mainly cytoplasmic and extracellular), which seems to be implicated in the signal for secretion and release.

Cerebellar Soluble Lectin Is Responsible for Cell Adhesion and Participates in Myelin Compaction in Cultured Rat Oligodendrocytes

Cultures of rat oligodendrocytes were used to test the possible role of the cerebellar soluble lectin (CSL) in myelin formation. Immunocytochemistry at the ultrastructural level showed that the lectin is present in the cytoplasm of the perikaryon of cultured oligodendrocytes and also on the plasma membrane of the cell body and processes. It is present in compact myelin and in the zones of contacts between different myelin sheaths or oligodendrocyte membranes. Staining of blots of the cultures with iodinated CSL indicated that endogenous glycoprotein ligands for CSL are present in the culture, rendering probable the hypothesis that cell contacts between different oligodendrocytes or between adjacent lamellae in myelin are mediated by lectin-glycoprotein interactions. This hypothesis was demonstrated by two effects of anti-CSL Fab fragments (4 micrograms/ml) on oligodendrocyte cultures: (1) the almost complete detachment of the cell layer from the culture substratum, and (2) the loss of myelin compaction by a separation of lamellae at the intraperiod line. The present findings could explain the complexity of the contacts between cultured oligodendrocyte processes by the formation of CSL bridges between glycoproteins of the membranes of these cells. CSL seems to be a key molecule in adhesion both for intercellular contacts and fixation of cells to the substratum. The small number of glycoprotein subunits found in oligodendrocytes that interact with CSL suggests that CSL-mediated cell adhesion involves a special class of glycoprotein glycans.

Immunohistochemical Localization of a β-Galactoside-Binding Lectin in Rat Central Nervous System

An endogenous brain lectin exhibiting β-galactoside specificity (RBL-16) was localized during postnatal cerebellum development both at the light- and electron-microscopical level. The lectin was widely distributed in neurons, astroglial and perivascular cells. Its levels were nearly constant during development in the two latter cell types. The lectin was developmentally regulated with a transient accumulation in Purkinje dendritic spines between the 10th- and 13th day, then it decreased until adult age. From electron-microscopical observations, it could be concluded that, in Purkinje cells, the lectin remained in the intracellular compartment, in dendrites and cell bodies. It was never externalized in the region where synaptogenesis takes place. A role in the intracellular transport of molecules should be expected from such a localization. The lectin was also transiently found on the surface of postmitotic neuroblasts in the external germinative layer and on the parallel fibers of the upper part of the molecular layer. However, it was not expressed inside neuroblasts. This suggests that part of the lectin found on the surface of neuroblasts originates from heavily stained astrocytes which could secrete it. RBL-16 could be making bridges between neuroblasts in the premigratory zone and between growing axons. A role in transient neuroblast adhesion in the external germinative layer and in parallel fiber fasciculation is expected from such a localization.

Glycoproteins and lectins in cell adhesion and cell recognition processes

SummaryThe discovery of endogenous lectins having specific and high affinity for the carbohydrate portions of glycoproteins has opened up new directions in the field of cell adhesion and cell recognition. Two endogenous lectins, termed as CSL and R1, initially isolated from the rat cerebellum and having a wide distribution in mammalian tissues, have been shown to participate in essential mechanisms of cell adhesion. The membrane-bound lectin R1 seems to be involved in transient recognition between neuronal cells, followed by elimination of the glycoprotein ligands at the surface of the recognized cell. In contrast, CSL is a molecule involved in adhesion between various normal or transformed cells since it participates in the formation of tight junctions. The glycoprotein ligands recognized with higher affinity by these two lectins seem to possess a special structure which defines a sub-class of oncofetal HNK-1 glycans. The over-expression of the glycoprotein ligands of these lectins in most transformed cells provides new tools for understanding the underlying mechanism of malignant transformation as well as the generation of signals through cell adhesion.

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.

Mannose dependent tightening of the rat ependymal cell barrier. in vivo and in vitro study using neoglycoproteins.

The possible role of carbohydrate binding proteins (lectins) and glycoconjugates in the formation of junctions ensuring tightening between ependymal cells was studied using synthetic glycoconjugates, the neoglycoproteins. These compounds are prepared by substituting bovine serum albumin with sugar residues and additional labelling (or not) with fluorescein or biotin. Injections of these components into the cerebral ventricles of adult rats resulted in a binding pattern which could be related to their carbohydrate composition. Mannose-containing neoglycoproteins were bound to ependymal cell cilia and penetrated rapidly the brain tissue. Such phenomenon was not seen with glucose- or galactose-containing neoglycoprotein molecules. In contrast, mannose-, galactose- and glucose-containing neoglycoproteins bound strongly to some endothelial cells around blood vessels. Fluorescent unglycosylated serum albumin did not bind to any brain structures. In contrast, co-injection of mannose-containing non-fluorescent neoglycoproteins with the other fluorescent compounds (including fluorescent sugar-free BSA) resulted in the penetration of the fluorescent compounds into the brain tissue. This internalization into brain was attributed to disaggregation of junctions between ependymal cells. Cultured ependymal cells behaved likewise. In short term experiments (5 min-1 h), only the mannose-containing neoglycoproteins bound strongly to the ependymal cells, particularly to the cilia. In long term experiments (1-9 days), mannose-containing neoglycoproteins specifically induced the disappearance of junctions between the cultured cells. These results emphasize the importance of mannose-dependent recognition system in the maintenance of junctions between ependymal cells, where a mannose-binding lectin has been previously detected.

Endogenous lectin CSL is present on the membrane of cilia of rat brain ependymal cells

SummaryAn endogenous brain lectin, with a great affinity for oligomannosidic glycans, called CSL (for ‘cerebellar soluble lectin’), was detected on the surface of the cilia of ependymal cells both in cultures andin vivo. The lectin is not synthesized by the ependymal cells themselves.In vivo it is neither found in cerebrospinal fluid nor in cells of the choroid plexus. Probably, lectin CSL is produced by subependymal astrocytic cells. The membranes of ependymal cells seem to possess glycoprotein ligands for the lectin which explain the specific adhesion of CSL on the surface of these cells, particularly on the cilia. The localization of this adhesive molecule on cilia of ependymal cells suggests that it may play a role in trapping foreign cells, micro-organisms or debris.

Lesion-induced re-expression of neonatal recognition molecules in adult rat cerebellum

It has been previously shown that sectioning of parallel fibers in the cerebellar molecular layer of adult rats gave rise to rapid reinnervation of the target cells, i.e., Purkinje cells. This paper reports that such a reinnervation is accompanied by reexpression (partial and total) of two developmentally regulated complementary molecules. These are an endogenous mannose-binding lectin, called R1, which reappears at the surface of the dendrites of Purkinje cells, and an endogenous glycoprotein ligand of R1, the 31 kDa glycoprotein, which seems to be neosynthetized and transported to the surface of parallel fibers. In this system, embryonic N-CAM is not reexpressed in neurons but reappears in reactive astrocytes in the vicinity of the lesion. The reexpression of recognition molecules (lectin and glycoprotein ligand) involved in normal synaptogenesis, may constitute the molecular basis for repair of nervous circuits in the adult as well.

Carbohydrate moieties of myelin-associated glycoprotein, major glycoprotein of the peripheral nervous system myelin and other myelin glycoproteins potentially involved in cell adhesion.

The myelin-associated glycoprotein (MAG) and the major glycoprotein of the peripheral nervous system myelin (P0) are two members of the family of cell adhesion molecules (CAMs). A role in cell adhesion of the carbohydrate moiety of these molecules has been attributed to the presence of N-glycans bearing the HNK-1 carbohydrate epitope. On the other hand, it has been suggested that these glycoproteins could be ligands of an endogenous mannose-binding lectin present in myelin, the cerebellar soluble lectin (CSL). In order to further document the heterogeneity of the glycans of these two CAMs, we have used several probes: an anti-carbohydrate antibody of the HNK-1 type, called Elec-39, the plant lectin concanavalin A (ConA), and the endogenous lectin CSL involved in myelin compaction. This study shows that CSL binds to a small proportion of the polypeptide chains of MAG found in adult CNS of rats and man and the polypeptide chains of P0 molecules from adult human and rat sciatic nerve. For MAG from adult rat brain, the binding of CSL is restricted to glycans of polypeptide chains which could be separated from the others according to their solubility properties. These MAG molecular entities react also with the Elec-39 antibody and with ConA. These results confirm that P0 and MAG are heterogeneous in their carbohydrate moieties.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and Immunohistochemical Localization of a Chondroitin Sulfate Proteoglycan from Adult Rat Brain

Abstract: A chondroitin sulfate proteoglycan called PGM 1 has been isolated from the particulate fraction of adult rat forebrain. Delipidation of the material, solubilization of proteoglycans in guanidinium chloride, precipitation at low ionic strength, and final extraction at pH 5.0 were used for its isolation. Proteoglycans were subjected to further purification by diethylaminoethyl‐cellulose chromatography. Individual components were separated by gel filtration. PGM 1 appeared to be a high‐molecular‐weight chondroitin sulfate proteoglycan, capable of strong interaction with hy‐aluronic acid. It was finally isolated by gel filtration on Ultrogel AcA 22 in the presence of 4 M guanidinium chloride. Monospecific antibodies obtained in rabbits against the purified molecule did not cross‐react with other brain proteoglycans. Immunocytochemical techniques revealed an almost unique association of this compound with axons, particularly those known to contain neurofilaments. However, not all these axons and all parts of these axons contained PGM 1. This component was not detectable in liver, intestine, spleen, kidney, lung, heart, skin, hair, lens, and muscle, a finding suggesting a specificity for the nervous tissue. This component is expressed in neural cell cultures. Despite the preservation of the neuronal specificity, it seems to lose its specific axonal localization in vitro.