Guido Tettamanti

A new procedure for the extraction, purification and fractionation of brain gangliosides

Abstract 1. 1. A procedure for the extraction, separation and purification of brain gangliosides is described, which involves: exhaustive extraction of brain with buffered tetrahydrofuran; partition of the extract first with ethyl ether, then with distilled water; dialysis of the obtained aqueous phase, and chromatography of the dialyzed solution on silica gel column. The residue after exhaustive extraction contains all brain glycoproteins. 2. 2. This procedure, compared with the conventional chloroform-methanol procedure, was proved to achieve a more complete extraction of gangliosides with no concurrent solubilization of glycoproteins and to guarantee the absence of any significant degradation. Thus, the present procedure is able to provide a final preparation of gangliosides free of glycoproteins, and a preparation of glycoproteins free of gangliosides.

Purification and characterization of bovine and ovine submaxillary mucins

Abstract The procedure of Tsuiki et al. for the isolation of bovine submaxillary mucin was modified but gave a product (BSM-T) very similar to that reported earlier. When applied to ovine submaxillary glands, it gave a very similar product (OSM-T) with a somewhat simpler composition. When treated with hydroxyapatite gel, a minor component (5–15%) was separated from each of both the ovine and bovine products. The major mucins had compositions similar to those of the untreated products. The minor mucins had larger amounts of protein, l -fucose, and d -galactose, and of the “minor” amino acids. The molecular weights of the major mucins were about 4 × 10 5 . A possible physiological role is suggested.

Activation of (Na+, K+)‐ATPase by Nanomolar Concentrations of GM1 Ganglioside

Abstract: GM1 ganglioside binding to the crude mitochondrial fraction of rat brain and its effect on (Na+, K+)‐ATPase were studied, the following results being obtained: (a) the binding process followed a biphasic kinetics with a break at 50 nM‐GM1; GM1 at concentrations below the break was stably associated, while over the break it was loosely associated; (b) stably bound GM1 activated (Na+, K+)‐ATPase up to a maximum of 43%; (c) the activation was dependent upon the amount of bound GM1 and was highest at the critical concentration of 20 pmol bound GM1× mg protein‐1; (d) loosely bound GM1 suppressed the activating effect on (Na+, K+)‐ATPase elicited by firmly bound GM1; (e) GM1‐activated (Na+, K+)‐ATPase had the same pH optimum and apparent Km (for ATP) as normal (Na+, K+)‐ATPase but a greater apparent Vmax; (f) under identical binding conditions (2 h, 37°C, with 40 nM substance) all tested gangliosides (GM1, GD1a, GD1b, GT1b) activated (Na+, K+)‐ATPase (from 26–43%); NeuNAc, sodium dodecylsulphate, sulphatide and cerebroside had only a very slight effect. It is suggested that the ganglioside activation of (Na+ ‐K+)‐ATPase is a specific phenomenon not related to the amphiphilic and ionic properties of gangliosides, but due to modifications of the membrane lipid environment surrounding the enzyme.

Promotion of Neuritogenesis in Mouse Neuroblastoma Cells by Exogenous Gangliosides. Relationship Between the Effect and the Cell Association of Ganglioside GM1

Abstract: Ganglioside GM1 promoted neuritogenesis of neuroblastoma cells, neuro‐2a clone, in monolayer culture. GM1 bound to neuro‐2a cells in three distinct forms, one removable by treatment with serum‐containing solutions, one serum‐resistant and labile to trypsin treatment, and one resistant to serum and trypsin treatments. The proportions among the three forms of cell‐associated GM1 varied in relation to duration of exposure to ganglioside, ganglioside concentration in the medium, and number of cells in culture. The form removable by serum was predominant at the initial stages of association and at the highest ganglioside concentrations (over 10−6M); the trypsin‐labile and ‐stable forms tended to increase with increasing cell number and decreasing ganglioside concentration. The neuritogenic effect of GM1 was higher when neuro‐2a cells were incubated for 24 h in the presence of GM1 and fetal calf serum. Under this condition the percentage of neurite‐bearing cells increased from 11% of control to 62% at the optimal ganglioside concentration of 10−4M. The effect was still present, although to a lower extent (from 11% to 28% of neurite‐bearing cells), when cells were first exposed for only 2 h to GM1, then washed and incubated for 24 h in the presence of fetal calf serum. The trypsin‐labile and ‐stable forms of cell‐associated GM1 had a fundamental role in the effect, whereas the form removable by serum was not involved. The preparation of GM1 used was extremely pure (99%) and, in particular, had a peptide contamination, if any, <1:20,000–1:50,000. Therefore the neuritogenic effect can be attributed to ganglioside itself. The results obtained suggest that under the experimental conditions used the stimulation of neuro‐2a cell differentiation by GM1 is related to changes of the plasma membrane properties following association of exogenous GM1 molecules. This would facilitate the spontaneous process of differentiation, or enhance cell responsiveness to differentiating factors present in the serum.

Ganglioside/glycosphingolipid turnover: New concepts

In this review focus is given to the metabolic turnover of gangliosides/glycosphingolipids. The metabolism and accompanying intracellular trafficking of gangliosides/glycosphingolipids is illustrated with particular attention to the following events: (a) the de novo biosynthesis in the endoplasmic reticulum and Golgi apparatus, followed by vesicular sorting to the plasma membrane; (b) the enzyme-assisted chemical modifications occurring at the plasma membrane level; (c) the internalization via endocytosis and recycling to the plasma membrane; (d) the direct glycosylations taking place after sorting from endosomes to the Golgi apparatus; (e) the degradation at the late endosomal/lysosomal level with formation of fragments of sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) nature; (f) the metabolic recycling of these fragments for biosynthetic purposes (salvage pathways); and (g) further degradation of fragments to waste products. Noteworthy, the correct course of ganglioside/glycosphingolipid metabolism requires the presence of the vimentin intracellular filament net work, likely to assist intracellular transport of sphingoid molecules.Out of the above events those that can be quantitatively evaluated with acceptable reliability are the processes of de novo biosynthesis, metabolic salvage and direct glycosylation. Depending on the cultured cells employed, the percentage of distribution of de novo biosynthesis, salvage pathways, and direct glycosylation, over total metabolism were reported to be: 35% (range: 10–90%) for de novo biosynthesis, 7% (range: 5–10%) for direct glycosylation, and 58% (range: 10–90%) for salvage pathways. The attempts made to calculate the half-life of overall ganglioside turnover provided data of unsure reliability, especially because in many studies salvage pathways were not taken into consideration. The values of half-life range from 2 to 6.5 h to 3 days depending on the cells used. Available evidence for changes of ganglioside/glycosphingolipid turnover, due to extracellular stimuli, is also considered and discussed. Published in 2004.

A Mediator Role of Ceramide in the Regulation of Neuroblastoma Neuro2a Cell Differentiation

Current studies indicate that ceramide is involved in the regulation of important cell functions, namely cell growth, differentiation, and apoptosis. In the present study, the possible role of ceramide in the differentiation of neuroblastoma Neuro2a cells was investigated. The following results were obtained. (a) Ceramide content of Neuro2a cells, induced to differentiate by retinoic acid (RA) treatment rapidly increased after addition of RA, was maintained at high levels in RA-differentiated cells and returned to the starting levels with removal of RA and reversal of differentiation; under the same conditions, the sphingosine content remained unchanged. (b) After a short pulse with [H]sphingomyelin or [H]sphingosine or L-[H]serine, the metabolic formation of ceramide was markedly higher and more rapid in RA-differentiated than undifferentiated cells. (c) Inhibitors of ceramide biosynthesis (Fumonisin B1, β-chloroalanine and L-cycloserine) diminished the extent of the differentiating effect of RA and concomitantly Cer content decreased. (d) The activity of neutral sphingomyelinase increased after addition of RA, maintained high levels in RA-differentiated cells, and returned to the initial levels with removal of RA. (e) Experimental conditions that cause an elevation of ceramide content (treatment with sphingosine or ceramide or C-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C-ceramide had no effect on differentiation. (f) treatment with Fumonisin B1 completely inhibited sphingosine-induced differentiation. These data suggest a specific bioregulatory function of ceramide in the control of Neuro2a cell growth and differentiation and pose the general hypothesis of a mediator role of ceramide in the differentiation of cells of neural origin.

Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the proteins localization in the plasma membrane.

Interactions of GM1 Ganglioside with Crude Rat Brain Neuronal Membranes

The binding of GM1, ganglioside to crude preparations of rat brain neuronal membranes was studied, the following results being obtained: (a) the binding process followed a biphasic kinetics, which displayed a break at 0.07–0.08 X 10−6m GM1, concentration; (b) the features of the binding process at GM1, concentrations below the break and, over the break, above 10‐6m appeared to be different. Below the break the process proceeded slowly and brought a stable and irreversible association of GM1, molecules to the membranes. Over 10‐6m the process was much more rapid and caused GM1, molecules to interact in such a way that they were releasable by washing and could exchange with newly added free ganglioside; (c) the two binding processes displayed the characteristics of a saturation phenomenon; (d) in both cases, GM1, taken up was freely available to galactose oxidase, indicating that the oligosaccharide chains protrude from the membrane surface. We postulate that GM1, occurs, below and above the break, in different physical forms, each of them having a different mechanism of interaction with the membrane. Above 10‐6m GM1, interacts as micelles, and the basis of the micelle‐membrane inter action is a fusion process. Below the break, in the 10−8–10−7m range, the binding is the result of hydrophobic interactions between sites on the membrane and the hydrophobic portion of individual ganglioside molecules, most likely in the monomeric form. Toffano G. et al. Interactions of GM1, ganglioside with crude rat brain neuronal membranes. J. Neurochem.35, 861–866 (1980).

Sialidases in Vertebrates : A Family Of Enzymes Tailored For Several Cell Functions*

This review summarizes the recent research development on vertebrate sialidase biology. Sialic acid-containing compounds play important roles in many physiological processes, including cell proliferation, apoptosis and differentiation, control of cell adhesion, immune surveillance, and clearance of plasma proteins. In this context, sialidases, the glycohydrolases that remove the terminal sialic acid at the non-reducing end of various glycoconjugates, perform an equally pivotal function. Sialidases in higher organisms are differentially expressed in cells and tissues/organs, with particular subcellular distribution and substrate specificity: they are the lysosomal (NEU1), the cytosolic (NEU2), and plasma membrane- and intracellular-associated sialidases (NEU3 and NEU4). The molecular cloning of several mammalian sialidases since 1993 has boosted research in this field. Here we summarize the results obtained since 2002, when the last general review on the molecular biology of mammalian sialidases was written. In those few years many original papers dealing with different aspects of sialidase biology have been published, highlighting the increasing relevance of these enzymes in glycobiology. Attention has also been paid to the trans-sialidases, which transfer sialic acid residues from a donor sialoconjugate to an acceptor asialo substrate. These enzymes are abundantly distributed in trypanosomes and employed to express pathogenicity, also in humans. There are structural similarities and strategic differences at the level of the active site between the mammalian sialidases and trans-sialidases. A better knowledge of these properties may permit the design of better anti-pathogen drugs.

Salvage pathways in glycosphingolipid metabolism

In this review, the focus is on the role of salvage pathways in glycosphingolipid, particularly, ganglioside metabolism. Ganglioside de novo biosynthesis, that begins with the formation of ceramide and continues with the sequential glycosylation steps producing the oligosaccharide moieties, is briefly outlined in its enzymological and cell-topological aspects. Neo-synthesized gangliosides are delivered to the plasma membrane, where their oligosaccharide chains protrude toward the cell exterior. The metabolic fate of gangliosides after internalization via endocytosis is then described, illustrating: (a) the direct recycling of gangliosides to the plasma membrane through vesicles gemmated from sorting endosomes; (b) the sorting through endosomal vesicles to the Golgi apparatus where additional glycosylations may take place; and (c) the channelling to the endosomal/lysosomal system, where complete degradation occurs with formation of the individual sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) components of gangliosides. The in vivo and in vitro evidence concerning the metabolic recycling of these components is examined in detail. The notion arises that these salvage pathways, leading to the formation of gangliosides and other glycosphingolipids, sphingomyelin, glycoproteins and glycosaminoglycans, represent an important saving of energy in the cell economy and constitute a relevant event in overall ganglioside (or glycosphingolipid, in general) turnover, covering from 50% to 90% of it, depending on the cell line and stage of cell life. Sialic acid is the moiety most actively recycled for metabolic purposes, followed by sphingosine, hexosamine, galactose and fatty acid. Finally, the importance of salvage processes in controlling the active concentrations of ceramide and sphingosine, known to carry peculiar bioregulatory/signalling properties, is discussed.