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Chronic myeloid leukemia is a hematopoietic stem cell cancer, originated by the perpetually “switched on” activity of the tyrosine kinase Bcr-Abl, leading to uncontrolled proliferation and insensitivity to apoptotic stimuli. The genetic phenotype of myeloid leukemic K562 cells includes the suppression of cytosolic sialidase Neu2. Neu2 transfection in K562 cells induced a marked decrease (-30% and -80%) of the mRNA of the anti-apoptotic factors Bcl-XL and Bcl-2, respectively, and an almost total disappearance of Bcl-2 protein. In addition, gene expression and activity of Bcr-Abl underwent a 35% diminution, together with a marked decrease of Bcr-Abl-dependent Src and Lyn kinase activity. Thus, the antiapoptotic axis Bcr-Abl, Src, and Lyn, which stimulates the formation of Bcl-XL and Bcl-2, was remarkably weakened. The ultimate consequences of these modifications were an increased susceptibility to apoptosis of K562 cells and a marked reduction of their proliferation rate. The molecular link between Neu2 activity and Bcr-Abl signaling pathway may rely on the desialylation of some cytosolic glycoproteins. In fact, three cytosolic glycoproteins, in the range 45–66 kDa, showed a 50–70% decrease of their sialic acid content upon Neu2 expression, supporting their possible role as modulators of the Bcr-Abl complex.

Gangliosides play key roles in cell differentiation, cell-cell interactions, and transmembrane signaling. Sialidases hydrolyze sialic acids to produce asialo compounds, which is the first step of degradation processes of glycoproteins and gangliosides. Sialidase involvement has been implicated in some lysosomal storage disorders such as sialidosis and galactosialidosis. Neu2 is a recently identified human cytosolic sialidase. Here we report the first high resolution x-ray structures of mammalian sialidase, human Neu2, in its apo form and in complex with an inhibitor, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA). The structure shows the canonical six-blade β-propeller observed in viral and bacterial sialidases with its active site in a shallow crevice. In the complex structure, the inhibitor lies in the catalytic crevice surrounded by ten amino acids. In particular, the arginine triad, conserved among sialidases, aids in the proper positioning of the carboxylate group of DANA within the active site region. The tyrosine residue, Tyr334, conserved among mammalian and bacterial sialidases as well as in viral neuraminidases, facilitates the enzymatic reaction by stabilizing a putative carbonium ion in the transition state. The loops containing Glu111 and the catalytic aspartate Asp46 are disordered in the apo form but upon binding of DANA become ordered to adopt two short α-helices to cover the inhibitor, illustrating the dynamic nature of substrate recognition. The N-acetyl and glycerol moieties of DANA are recognized by Neu2 residues not shared by bacterial sialidases and viral neuraminidases, which can be regarded as a key structural difference for potential drug design against bacteria, influenza, and other viruses.

Recombinant human cytosolic sialidase (HsNEU2), expressed in Escherichia coli, was purified to homogeneity, and its substrate specificity was studied. HsNEU2 hydrolyzed 4-methylumbelliferyl alpha-NeuAc, alpha 2-->3 sialyllactose, glycoproteins (fetuin, alpha-acid glycoprotein, transferrin, and bovine submaxillary gland mucin), micellar gangliosides GD1a, GD1b, GT1b, and alpha 2-->3 paragloboside, and vesicular GM3. alpha 2-->6 sialyllactose, colominic acid, GM1 oligosaccharide, whereas micellar GM2 and GM1 were resistant. The optimal pH was 5.6, kinetics Michaelis-Menten type, V(max) varying from 250 IU/mg protein (GD1a) to 0.7 IU/mg protein (alpha(1)-acid glycoprotein), and K(m) in the millimolar range. HsNEU2 was activated by detergents (Triton X-100) only with gangliosidic substrates; the change of GM3 from vesicular to mixed micellar aggregation led to a 8.5-fold V(max) increase. HsNEU2 acted on gangliosides (GD1a, GM1, and GM2) at nanomolar concentrations. With these dispersions (studied in detailed on GM1), where monomers are bound to the tube wall or dilutedly associated (1:2000, mol/mol) to Triton X-100 micelles, the V(max) values were 25 and 72 microIU/mg protein, and K(m) was 10 and 15 x 10(-9) m, respectively. Remarkably, GM1 and GM2 were recognized only as monomers. HsNEU2 worked at pH 7.0 with an efficiency (compared with that at pH 5.6) ranging from 4% (on GD1a) to 64% (on alpha(1)-acid glycoprotein), from 7% (on GD1a) to 45% (on GM3) in the presence of Triton X-100, and from 30 to 40% on GM1 monomeric dispersion. These results show that HsNEU2 differentially recognizes the type of sialosyl linkage, the aglycone part of the substrate, and the supramolecular organization (monomer/micelle/vesicle) of gangliosides. The last ability might be relevant in sialidase interactions with gangliosides under physiological conditions.

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40-70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137–145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from eitherl-[3H]serine or [3H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

Cultured fibroblasts from normal subjects and from subjects affected by Salla disease, characterized by the lack or misfunction of the membrane carrier responsible for the egress of sialic acid from lysosomes, were fed with ganglioside GM3 labeled at the sialic acid acetyl group, [Neu5Ac-3H]GM3, or at C-3 of sphingosine (Sph), [Sph-3H]GM3, or at C-1 of stearoyl chain, [stearoyl-14C]GM3. After a 15-h pulse the total amount of cell-bound GM3 corresponded to about 2% of the endogenous ganglioside content. Cells were then subjected to a 72-h chase, and the radioactive products from both ganglioside catabolism and salvage processes of catabolic fragments were measured. These data indicated that about 50% of the cell-bound ganglioside underwent metabolic processing, suggesting a ganglioside half-life of 2-3 days. [Neu5Ac-3H] formed from [Neu5Ac-3H]GM3 degradation was mostly re-cycled for the biosynthesis of gangliosides and sialoglycoproteins, only a minor part being degraded to [3H]water, which constituted only 1.6% of total metabolite linked radioactivity. [Sph-3H] from the [Sph-3H]GM3 degradation was partly re-cycled for the biosynthesis of gangliosides, neutral glycosphingolipids and sphingomyelin, and partly (about 20% of the total metabolite linked radioactivity) degraded to [3H]water. In Salla fibroblasts metabolic processing of [Neu5Ac-3H]GM3 produced large amounts of free [3H]Neu5Ac, and a reduced incorporation of radioactivity into glycoconjugates (as compared to normal cells). However, the accumulation of free Neu5Ac was not accompanied by an increase of tritiated water. LacCer and Cer from [stearoyl-14C]GM3 catabolism were found to accumulate in Salla fibroblasts, an indication that the enzymes of glycosphingolipid metabolism were affected by the impairment of Neu5Ac egress from lysosomes. Particularly relevant was the accumulation of ceramide which was hardly detectable in control cells.

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 [3H]sphingomyelin or [3H]sphingosine or L-[3H]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, beta-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 C2-ceramide or bacterial sphingomyelinase) inhibited cell proliferation and stimulated neurite outgrowth; dihydro-analogues of sphingosine, ceramide, and C2-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.

A new ganglioside containing an inner ester linkage was extracted from adult brain specimens, obtained at the time of surgery on 51-70-year-old subjects, purified, and analyzed. It contains glucose, galactose, N-acetylgalactosamine, an N-acetylneuraminic acid in the molar ratio 1:2:1:2, but, on ion-exchange chromatography, behaves as a monosialoganglioside. Structural analyses showed its basic neutral glycosphingolipid core to be ganglio-N-tetraose ceramide, carrying a disialosyl residue on the 3-position of internal galactose. Sialidase degradation and chemical analysis of the products obtained after alkaline treatments suggested one sialic acid residue to be involved in an ester linkage. Fast atom bombardment-mass spectrometry indicated the presence of an inner ester linkage between the carboxyl group of the external sialic acid residue and a hydroxyl group of the internal one. On these bases, the new ganglioside can be assumed to be a GD1b in lactonic form. This ganglioside is present only in trace amounts in the brain of infants, but its content increases with age, reaching a value of 3.5% of total sialic acid in 51-70-year-old subjects.

We have studied the substrate specificities of a non-specific activator protein on the enzymatic hydrolyses of the following compounds: GM1 and GM2, as well as several of their derivatives including oligosaccharides, GgOse3Cer-II3-sulfate and LacCer-II3-sulfate, Gb-Ose3Cer and GbOse4Cer, three neolacto-series glycosphingolipids, and two non-ceramide glycolipids. Our results show that this activator protein has a broad spectrum of activity and exhibits the properties of a nonspecific natural detergent. The evidence of non-specificity was the ability of this activator protein to stimulate the hydrolyses of glycolipids, regardless of glycosphingolipids or non-ceramide glycolipids, carried out by glycosidases from animals, plants, and microorganisms. Its activity was, however, limited to substrates that had a lipid moiety. The oligosaccharide of GM1 and deacetyl-fatty acid free GM1 (II3-NeuGg-Ose4-sphingosine) were hydrolyzed by beta-galactosidase in the absence of this activator protein.