A ganglioside-specific sialyltransferase localizes to axons and non-Golgi structures in neurons.

To investigate the tissue distribution and subcellular localization of ST3GalV (CMP-NeuAc:lactosylceramide alpha2,3 sialyltransferase/GM3 synthase) in the adult mouse, we generated two antisera against mouse ST3GalV that were designated CS2 (directed against amino acids K227-I272) and CS14 (directed against amino acids D308-H359). We previously reported that CS2 antiserum stains medial and trans-Golgi cisternae in all cell types investigated. In neural tissue, however, CS14 antiserum reveals a subpopulation of ST3GalV with a subcellular distribution complementary to CS2 antiserum. CS14 antiserum strongly stains axons in cortical, cerebellar, brainstem, and spinal cord tissue sections. The subcellular localization of neuronal ST3GalV is maintained in primary cultures of rat hippocampal neurons and in PC12 cells. In PC12 cells, ST3GalV localization evolves during NGF-induced differentiation such that a pool of enzyme leaves the Golgi for a distal compartment in conjunction with neurite outgrowth. In PC12 cells transfected with an epitope-tagged form of ST3GalV, staining for the epitope tag coincides with expression of endogenous enzyme. The non-Golgi pool of ST3GalV does not colocalize with markers for the trans-Golgi network, endosome, or synaptic vesicles, nor is it detected on the cell surface. Distinct subpopulations of ST3GalV imply that ganglioside synthesis can occur outside of the Golgi or, alternatively, that a portion of the total ST3GalV pool subserves a nonenzymatic function. Significantly fewer transfected cells were found in PC12 cultures treated with plasmid encoding ST3GalV than in cultures treated with control plasmid, indicating that the expression of ST3GalV in excess of endogenous levels results in either cell death or a decreased rate of cell division.

Synaptic neurochemical alterations associated with neuronal degeneration in an inherited cerebellar ataxia of Gordon Setters.

Canine Inherited Ataxia (CIA) is an autosomal recessive cerebellar disease of Gordon Setters associated with degeneration of Purkinje and granule cells. To define specific biochemical correlates of neuronal loss, synaptic neurochemical parameters were measured in three cerebellar regions (vermis, "pars intermedia," and hemisphere) at early and late stages of this disease. At one and a half years of age, affected dogs showed the most severe lesions in the "pars intermedia," with a 39% decrease in the number of Purkinje cells and a 29% decrease in granule cells. Neurochemical measurements demonstrated decreased [3H]muscimol binding and elevations in norepinephrine concentration (248% above control) and [3H]glutamate receptor binding (118% above control). At five years of age, reduction of Purkinje cells in the three cerebellar regions ranged from 65 to 91% while loss of granule cells was between 13 and 53%. [3H]Muscimol binding remained low throughout the cerebellum (38 to 59% of control) and norepinephrine concentration and [3H]glutamate binding were markedly reduced from the levels observed at one and a half years. Glutamate decarboxylase activity, [3H]QNB binding and GABA concentration were relatively unaffected. Our results indicate that neurochemical parameters associated with cerebellar neuronal systems demonstrate specific alterations in a chronic degenerative disorder. This study also indicates the importance of evaluating neurochemical measurements with regard to both spared and degenerating neuronal systems and emphasizes the role of compensatory neurochemical alterations in cerebellar degenerative disorders.

Morphologic and neurochemical studies of embryonic brain development in murine trisomy 16.

Telencephalic and diencephalic/brainstem regions from embryonic trisomy-16 mice (Ts16) between gestational days 15-18 were analyzed for alterations of morphologic and neurochemical parameters and compared to phenotypically normal littermates. Mean trisomic wet weights from both regions were significantly diminished (greater than 20%) and total protein content was reduced. Ratios of the thickness of the ventricular (germinal) zone to the thickness of the whole cortex were increased, suggesting a delay in neuronal differentiation. Pre- and postsynaptic markers for GABAergic, cholinergic, catecholaminergic and serotonergic transmitter systems were compared. A significant impairment of the trisomic brain catecholaminergic and serotonergic system development was observed, based upon regional reductions in norepinephrine, dopamine and serotonin content. Choline acetyltransferase activity in the diencephalon/brainstem was reduced by 21-26% in contrast to normal levels within the cerebral hemispheres. Presynaptic GABAergic markers were not affected in the Ts16 embryos. It is concluded that although a genetic imbalance involving chromosome 16 in the mouse embryo produces a delay in neurogenesis, it has a more selective effect on the catecholaminergic, serotonergic and cholinergic systems than on GABAergic neurons.

Mouse chimeras composed of trisomy 16 and normal (2N) cells: preliminary studies.

Many humans with trisomy 21 (Down Syndrome (DS)) have psychomotor and cognitive retardation, congenital heart disease, and hematologic abnormalities. Partial genetic homology exists between mouse chromosome 16 and human chromosome 21; thus, studies of the development of mice with trisomy 16 (Ts16) may provide important insights into the pathogenesis of these defects and into the mechanisms by which they arise in humans. Since Ts16 mice do not survive the late fetal period, chimeras have been formed between Ts16 and normal (2N) mouse embryos to rescue the Ts16 cells for postnatal studies. In this preliminary study of the postnatal development of such chimeras, we examined the proportion of Ts16 cells in a variety of tissues, including the coat, blood, placenta, heart, and brain. The Ts16 cells made significant contributions to almost all tissues examined. Aspects of the behavior and the neurochemistry of adult Ts16 less than-greater than 2N chimeras were found to differ significantly from control (2N less than-greater than 2N) chimeras and from animals of the two donor embryo strains. Ts16 cells comprised a substantial, but not predominant, proportion of cells in each brain region examined. Suggestions for definitive analyses are reviewed.

Function and structure of Drosophila glycans.

Through the application of classic organismal genetic strategies, such as mutagenesis and interaction screens, Drosophila melanogaster provides opportunities to understand glycan function. For instance, screens for Drosophila genes that establish dorsal-ventral polarity in the embryo or that influence cellular differentiation through signal modulation have identified putative glycan modifying enzymes. Other genetic and molecular approaches have demonstrated the existence of phylogenetically conserved and novel oligosaccharide processing activities and carbohydrate binding proteins. While the structural characterization of Drosophila oligosaccharide diversity has lagged behind the elucidation of glycan function, landmarks are becoming apparent in the carbohydrate terrain. For instance, O-linked GlcNAc and mucins, spatially and temporally regulated N-linked oligosaccharide expression, glycosphingolipids, heparan sulfate, chondroitin sulfate and polysialic acid have all been described. A major challenge for Drosophila glycobiology is to expand the oligosaccharide structural database while endeavoring to link glycan characterization to functional analysis. The completion of the Drosophila genome sequencing project will yield a broad portfolio of glycosyltransferases, glycan modifying enzymes and lectins requiring characterization. To this end, the great range of genetic tools that allow the controlled spatial and temporal expression of transgenes in Drosophila will permit unprecedented manipulation of glycosylation in a whole organism.

Gliolectin-mediated carbohydrate binding at the Drosophila midline ensures the fidelity of axon pathfinding.

Gliolectin is a carbohydrate-binding protein (lectin) that mediates cell adhesion in vitro and is expressed by midline glial cells in the Drosophila melanogaster embryo. Gliolectin expression is maximal during early pathfinding of commissural axons across the midline (stages 12-13), a process that requires extensive signaling and cell-cell interactions between the midline glia and extending axons. Deletion of the gliolectin locus disrupts the formation of commissural pathways and also delays the completion of longitudinal pathfinding. The disruption in commissure formation is accompanied by reduced axon-glial contact, such that extending axons grow on other axons and form a tightly fasciculated bundle that arches over the midline. By contrast, pioneering commissural axons normally cross the midline as a distributed array of fibers that interdigitate among the midline glia, maximizing contact and, therefor, communication between axon and glia. Restoration of Gliolectin protein expression in the midline glia rescues the observed pathfinding defects of null mutants in a dose-dependent manner. Hypomorphic alleles generated by ethylmethanesulfonate mutagenesis exhibit a similar phenotype in combination with a deletion and these defects are also rescued by transgenic expression of Gliolectin protein. The observed phenotypes indicate that carbohydrate-lectin interactions at the Drosophila midline provide the necessary surface contact to capture extending axons, thereby ensuring that combinatorial codes of positive and negative growth signals are interpreted appropriately.

Gliolectin is a novel carbohydrate-binding protein expressed by a subset of glia in the embryonic Drosophila nervous system.

Interactions between embryonic neural cells generate the specific patterns of connectivity observed in nervous systems. Cell surface carbohydrates have been proposed to function in cellular recognition events guiding such interactions. Carbohydrate-binding proteins (lectins) that recognize specific oligosaccharide ligands in embryonic neural tissue provide a molecular mechanism for carbohydrate-mediated cell-cell interactions in neural development. Therefore, we have screened an embryonic Drosophila melanogaster cDNA library, expressed in COS1 cells, for carbohydrate-binding activity. COS1 cells expressing putative Drosophila lectins were identified and recovered based on their adhesion to immobilized preparations of neutral and zwitterionic glycolipids extracted from Drosophila embryos. We have identified an endogenous lectin expressed during Drosophila embryogenesis. The cloned lectin, designated 'gliolectin', possesses a novel protein sequence with a calculated molecular mass of 24,993. When expressed in Drosophila S2 cells, the lectin mediates heterophilic cellular aggregation. In embryos, gliolectin is expressed by a subset of glial cells found at the midline of the developing nervous system. Expression is highest during the formation of the Drosophila embryonic axonal commissures, a process requiring midline glial cell funcion. Immunoprecipitation with a monoclonal antibody against gliolectin yields a protein of Mr=46,600 from Drosophila embryonic membranes, suggesting that post-translational modification of gliolectin is extensive. Epitope- tagged chimericproteins composed of the amino terminal one-half of gliolectin and the Fc region of human IgG bind a small subset of the total glycolipids extracted from Drosophila embryos, demonstrating that the lectin activity of gliolectin can discriminate between oligosaccharide structures. The presence of gliolectin in the developing Drosophila embryonic nervous system further supports a role for cell surface carbohydrates in cell-cell recognition and indicates that the molecular diversity of animal lectins is not yet completely defined.

A membrane receptor for gangliosides is associated with central nervous system myelin.

A binding protein specific for major neuronal gangliosides was detected on rat brain membranes using a synthetic ganglioside-protein conjugate, 125I-(GT1b)4BSA (bovine serum albumin derivatized with 4 mol of ganglioside GT1b/mol of protein), as a radioligand. Specific binding of the ligand displayed marked regional variation within the brain, with white matter-enriched regions demonstrating the highest binding activity. Autoradiographic localization of 125I-(GT1b)4BSA binding to tissue sections revealed selective association with myelinated pathways throughout the brain. The ligand also bound preferentially to brain subcellular fractions enriched in myelin, even after removal of axolemma. In contrast, peripheral nerve myelin had little binding activity. The myelin-associated ganglioside receptor detected by 125I-(GT1b)4BSA binding appears to be a novel oligodendroglial membrane protein which preferentially recognizes neuronal gangliosides.

Molecular identification, tissue distribution and subcellular localization of mST3GalV/GM3 synthase.

A molecular screen for a mouse homologue of a Drosophila carbohydrate binding protein, called Gliolectin, yielded a cDNA encoding mST3GalV/GM3 synthase (CMP-NeuAc: lactosylceramide alpha2, 3-sialyltransferase). By in situ hybridization and immunohistochemistry, mST3GalV exhibits differential expression in neural and non-neural tissues. Although expressed by all neurons in the central nervous system, neuronal populations that contribute their axons to myelinated efferent projections, such as cerebellar Purkinje cells and spinal motorneurons, demonstrate the highest ST3GalV expression. When stained with anti-mST3GalV antiserum (designated CS2), subpopulations of neurons display an elaborate Golgi apparatus, frequently extending into one or more dendritic processes. The extended spatial distribution of the neuronal Golgi apparatus, particularly in spinal motorneurons, allowed the confocal immunohistochemical colocalization of mST3GalV with markers for medial/trans-Golgi but not the cis-Golgi or trans-Golgi network, consistent with previous observations suggesting that ganglioside glycosyltransferases are enriched in late Golgi compartments. Among non-neural tissues, liver and testes demonstrate cell-type specific CS2 staining. In liver, endothelial cells lining a ring of sinusoids, concentric with the central vein, express mST3GalV. Kupffer cells are also stained with CS2 antiserum but hepatocyte expression is undetectable. In the seminiferous tubules of the testes, ST3GalV is found in somatic (Leydig, Sertoli) and early germline cells (spermatogonia and primary spermatocytes); the epididymal epithelium exhibits intense ST3GalV expression. Since GM3 is a precursor for the synthesis of a- and b-series gangliosides, the range of mST3GalV/GM3 synthase expression among various cell populations indicates that certain cell types possess greater reliance on ganglioside function than others.

Zwitterionic and acidic glycosphingolipids of the Drosophila melanogaster embryo.

Defining glycosphingolipid structures in species amenable to genetic manipulation, such as Drosophila melanogaster, provides a foundation for investigating mechanisms that regulate glycolipid expression. Therefore, eight of the 12 major glycosphingolipids, accounting for 64% of lipid-linked carbohydrate in Drosophila embryos, were purified after separation into acidic and zwitterionic pools. The zwitterionic lipids possess phosphoethanolamine (PEtn) linked to one or more GlcNAc residues and comprise a family of serially related structures. The longest characterized glycolipid, an octaosylceramide, designated Nz28, has the structure: GalNAcbeta, 4(PEtn-6)GlcNAcbeta,3Galbeta,3GalNAcalpha,4Ga lNAcbeta, 4(PEtn-6)GlcNAcbeta,3Manbeta,4GlcbetaCer. Heptaosyl (Nz7), hexaosyl (Nz6), pentaosyl (Nz5) and tetraosyl (Nz4) forms of Nz28, sequentially truncated from the nonreducing terminus, possess only one PEtn moiety. The major acidic lipid, designated Az29, possesses two PEtn moieties and a glucuronic acid linked to a Gal-extended Nz28. Two other acidic glycolipids, Az9 and Az6, exhibit one PEtn moiety and the same hexose and N-acetylhexosamine composition as Az29 and Nz6, respectively. The fully extended Drosophila core oligosaccharide differs from that of other dipterans in the linkage at a single glycosidic bond, a distinction with significant structural and biosynthetic consequences. Furthermore, acidic species account for a larger proportion of total glycosphingolipid, and PEtn substitution of GlcNAc is more complete in the Drosophila embryo. Divergent characteristics may reflect interspecies variation or stage-specific glycosphingolipid expression in dipterans.

Biochemical and biological characterization of the protective Leishmania pifanoi amastigote antigen P-8.

The Leishmania pifanoi amastigote antigen P-8 has been previously shown to induce protective immunity in a murine model of cutaneous leishmaniasis (L. Soong, S. M. Duboise, P. Kima, and D. McMahon-Pratt, Infect. Immun. 63:3559-3566, 1995). As this antigen is of interest for further vaccine studies, the biochemical characterization of P-8 was undertaken. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western-blot analysis, and gel filtration chromatography revealed that P-8 antigen consisted of two proteoglycolipid complexes. The P-8 epitope is associated with the L. pifanoi amastigote-specific glycolipid components found in the two complexes. The P-8 complex 1 (P-8c1) consists of a 56-kDa serine metalloproteinase, apolipoprotein E (derived from fetal bovine serum), and amastigote-specific glycolipids. The P-8 complex 2 (P-8c2) consists of a 31-kDa cysteine proteinase associated with amastigote glycolipids. Biochemical analyses suggest that the P-8 antigenic glycolipids may be distinct from previously described Leishmania glycolipids (glycosylinositol-phospholipids and sphingoglycolipids). Protective immunity studies revealed that P-8c1 (serine metalloproteinase-glycolipid complex) confers comparable protection against infection as immunopurified P-8. The isolated P-8c2 (cysteine proteinase-glycolipid complex) does not provide significant protection, nor does stimulation with P-8c2 result in significant T-cell activation in P-8- or P-8c2-vaccinated mice. Consequently, the P-8c1 complex appears to be the immunodominant component of P-8.

Ganglioside-specific binding protein on rat brain membranes.

A derivative of ganglioside GT1b (IV3NeuAc,II3(NeuAc)2-GgOse4) with an active ester in its lipid portion was synthesized and covalently attached to bovine serum albumin (BSA). The conjugate, having four GT1b molecules per albumin molecule [GT1b)4BSA) was radioiodinated and used to probe rat brain membranes for ganglioside binding proteins. A ganglioside-specific, high affinity (KD = 2-4 nM), saturable (Bmax = 13-20 pmol/mg membrane protein) binding site for 125I-(GT1b)4BSA was demonstrated on detergent-solubilized rat brain membranes adsorbed to filters. 125I-(GT1b)4BSA binding was tissue-specific (more than 35-fold greater to brain than to liver membranes) and was nearly eliminated by pretreatment of brain membrane-adsorbed filters with trypsin (1 microgram/ml). Underivatized gangliosides added as mixed detergent-lipid micelles blocked 125I-(GT1b)4BSA binding to brain membranes; structurally related GQ1b, GT1b, and GD1b were the most potent (half-maximal inhibition at 70-110 nM), while half-maximal inhibition by other gangliosides (GD3, GD1a, GM3, GM2, and GM1) required 5-20-fold higher concentrations. Other sphingolipids, neutral glycosphingolipids, and glycoproteins were poor inhibitors, and treatment of (GT1b)4BSA with neuraminidase attenuated its binding. Although most phospholipids were noninhibitory, phosphatidylinositol and phosphatidylglycerol inhibited half-maximally at 400-600 nM. However, inhibition of 125I-(GT1b)4BSA binding by gangliosides was competitive and reversible while that by phosphatidylinositol and phosphatidylglycerol was not. Ganglioside-protein conjugate binding reveals ganglioside-specific brain membrane receptors.

Inactivation of GM1-ganglioside beta-galactosidase by a specific inhibitor: a model for ganglioside storage disease.

This study was designed to establish an in vitro model with biochemical and morphological similarities to the human neurodegenerative disease GM1 gangliosidosis. Utilizing a specific inactivator of the lysosomal enzyme GM1-ganglioside beta-galactosidase (beta-D-galactopyranosylmethyl-p-nitrophenyltriazene [beta-GalMNT]) and neuroblastoma X glioma hybrid cells (NG108-15), we suppressed beta-galactosidase activity for up to 72 hours. Coincidental with suppression of this enzyme to levels less than 1% of control, we found up to a nine-fold accumulation of its substrate, the GM1-ganglioside, and the ultrastructural appearance of membranous cytoplasmic bodies. beta-GalMNT treatment suppressed growth but had little effect on the specific activity of choline acetyltransferase, lactate dehydrogenase, or other lysosomal enzymes including galactosylceramidase. This model should permit studies of the neurophysiological effects of increased ganglioside accumulation and their reversibility.

The binding specificity of normal and variant rat Kupffer cell (lectin) receptors expressed in COS cells.

A receptor uniquely found on the surface of rat Kupffer cells was shown previously to bind oligosaccharides terminating in galactose, N-acetylgalactosamine, and fucose. To analyze further the binding specificity of the receptor, receptor-mediated adhesion of transfected COS cells to immobilized glycolipids of known structure was measured. The glycolipid Gb4Cer (GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) was the best ligand. Gb5Cer (GalNAc alpha 1-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc beta 1Cer) and LacCer (Gal beta 1-4Glc beta 1Cer) bound more weakly (five times less than Gb4Cer) and Gb3Cer (Gal alpha 1-4Gal beta 1-4Glc beta 1Cer), and g3Cer(GalNAc beta 1-4Gal beta 1-4Glc beta 1Cer) bound even more weakly (60 times less than Gb4Cer). Gangliosides did not support adhesion of transfected cells. The adhesion of COS cells transfected with plasmids encoding variants of the receptor was also examined. In each variant, either tryptophan 498 or 523, which are conserved in most C-type lectins, was replaced by one of several amino acids. Variants that retained binding activity had the same specificity as the normal receptor. Differences between variants were noted, however, in maximal levels of adhesion and these differences correlated with altered expression of the receptor variants in COS cells.

Carbohydrate ligands for endothelial-leukocyte adhesion molecule 1.

The acute inflammatory response requires that circulating leukocytes bind to and penetrate the vascular wall to access the site of injury. Several receptors have been implicated in this interaction, including a family of putative carbohydrate-binding proteins. We report here the identification of an endogenous carbohydrate ligand for one of these receptors, endothelial-leukocyte adhesion molecule 1 (ELAM-1). Radiolabeled COS cells transfected with a plasmid containing the cDNA for ELAM-1 were used as probes to screen glycolipids extracted from human leukocytes. COS cells transfected with this plasmid adhered to a subset of sialylated glycolipids resolved on TLC plates or adsorbed on polyvinyl chloride microtiter wells. Adhesion to these glycolipids required calcium but was not inhibited by heparin, chondroitin sulfate, keratan sulfate, or yeast phosphomannan. Monosaccharide composition, linkage analysis, and fast atom bombardment mass spectrometry of the glycolipids indicate that the ligands for ELAM-1 are terminally sialylated lactosylceramides with a variable number of N-acetyllactosamine repeats and at least one fucosylated N-acetylglucosamine residue.

Cell surface ligands for rotavirus: mouse intestinal glycolipids and synthetic carbohydrate analogs.

Rotaviral binding to receptors on epithelial cells in the small intestine is thought to be a key event in the infection process and may be carbohydrate-mediated. Strain SA11 of rotavirus bound in vitro both to glycolipids isolated from mouse small intestine and to authentic glycolipids using thin layer chromatography overlay and microtiter well adsorption assays. Neutral mouse intestinal glycolipids which bound rotavirus were GA1 (Gal beta 1----3GalNAc beta 1---4Glc beta 1----4Glc beta 1----1-ceramide) and pentaosylceramides with terminal N-acetylgalactosamine, while acidic lipids which bound rotavirus included cholesterol 3-sulfate and two compounds termed bands 80 and 81. Digestion with ceramide glycanase suggested that bands 80 and 81 have lactosyl ceramide cores and an unidentified acidic moiety(s). No sialic-acid-containing glycolipids tested were active in viral binding. Band 81, which may have a ganglio core, bound rotavirus with greatest avidity, followed by GA1. Of authentic glycolipids assayed, only GA1 and GA2 (GalNAc beta 1----4Gal beta 1----4Glc beta 1----1-ceramide) displayed rotaviral binding. A phosphatidylethanolamide dipalmitoyl-containing neoglycolipid analog of GA2 bound rotavirus with avidity similar to native GA2. Substitution of beta 1----4-linked GlcNAc or beta 1----3-linked GalNAc for terminal GalNAc of GA2 neoglycolipid supported rotaviral binding, while other substitutions abrogated it. These findings suggest that a carbohydrate epitope similar to that of GA2 is sufficient for in vitro rotaviral binding, although binding may be enhanced by galactose and/or an acidic moiety in a secondary epitope.