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Sialidases remove sialic acid residues from various sialo-derivatives. To gain further insights into the biological roles of sialidases in vertebrates, we exploited zebrafish (Danio rerio) as an animal model. A zebrafish transcriptome- and genome-wide search using the sequences of the human NEU polypeptides as templates revealed the presence of seven different genes related to human sialidases. neu1 and neu4 are the putative orthologues of the mammalian sialidases NEU1 and NEU4 respectively. Interestingly, the remaining genes are organized in clusters located on chromosome 21 and are all more closely related to mammalian sialidase NEU3. They were thus named neu3.1, neu3.2, neu3.3, neu3.4 and neu3.5. Using RT-PCR (reverse transcription-PCR) we detected transcripts for all genes, apart from neu3.4, and whole-mount in situ hybridization experiments show a localized expression pattern in gut and lens for neu3.1 and neu4 respectively. Transfection experiments in COS7 (monkey kidney) cells demonstrate that Neu3.1, Neu3.2, Neu3.3 and Neu4 zebrafish proteins are sialidase enzymes. Neu3.1, Neu3.3 and Neu4 are membrane-associated and show a very acidic pH optimum below 3.0, whereas Neu3.2 is a soluble sialidase with a pH optimum of 5.6. These results were further confirmed by subcellular localization studies carried out using immunofluorescence. Moreover, expression in COS7 cells of these novel zebrafish sialidases (with the exception of Neu3.2) induces a significant modification of the ganglioside pattern, consistent with the results obtained with membrane-associated mammalian sialidases. Overall, the redundancy of sialidases together with their expression profile and their activity exerted on gangliosides of living cells indicate the biological relevance of this class of enzymes in zebrafish.

Sialidase NEU3 is also known as the plasma-membrane-associated form of mammalian sialidases, exhibiting a high substrate specificity towards gangliosides. In this respect, sialidase NEU3 modulates cell-surface biological events and plays a pivotal role in different cellular processes, including cell adhesion, recognition and differentiation. At the moment, no detailed studies concerning the subcellular localization of NEU3 are available, and the mechanism of its association with cellular membranes is still unknown. In the present study, we have demonstrated that sialidase NEU3, besides its localization at the plasma membrane, is present in intracellular structures at least partially represented by a subset of the endosomal compartment. Moreover, we have shown that NEU3 present at the plasma membrane is internalized and locates then to the recycling endosomal compartment. The enzyme is associated with the outer leaflet of the plasma membrane, as shown by selective cell-surface protein biotinylation. This evidence is in agreement with the ability of NEU3 to degrade gangliosides inserted into the plasma membrane of adjacent cells. Moreover, the mechanism of the protein association with the lipid bilayer was elucidated by carbonate extraction. Under these experimental conditions, we have succeeded in solubilizing NEU3, thus demonstrating that the enzyme is a peripheral membrane protein. In addition, Triton X-114 phase separation demonstrates further the hydrophilic nature of the protein. Overall, these results provide important information about the biology of NEU3, the most studied member of the mammalian sialidase family.

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 protein's localization in the plasma membrane.

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10-120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

Granule cells and astrocytes from rat cerebellum were fed in culture with 2 microM ganglioside [Gal-3H]GD1b and then analysed for the presence of carboxyl esters of that ganglioside. Before extraction and purification of gangliosides, cells were treated with NaBH4 under conditions that would allow complete reductive cleavage of carboxyl ester linkages, [Gal-3H]GD1b monolactone and dilactone being used as reference esters of GD1b. These conditions, established by adding harvested cells (250 micrograms of protein) with 0.01-2 nmol of standard [Gal-3H]GD1b monolactone or dilactone and [Gal-3H]GD1b-1ol or -2ol formed respectively, consisted of an NaBH4/cell protein ratio of 2:1 (w/w). Cerebellar granule cells, but not astrocytes, were able to produce a radioactive compound which was identified as GD1b-1ol. The formation of this compound increased with pulse (up to 4 h) and chase (up to 3 h) time after a 2 h pulse and also occurred when ganglioside endocytosis was blocked. It can be concluded that cerebellar granule cells are able to convert ganglioside GD1b into a carboxyl ester form, presumably GD1b monolactone. The natural occurrence of the same GD1b carboxyl ester in cerebellar granule cells was also demonstrated.

The influence of ceramide composition on the rate of GM1 association to HeLa cells has been investigated by incubating the cells in the presence of either native ganglioside or molecular species carrying highly homogeneous long chain base moieties, fractionated from native GM1. The GM1 ganglioside species carrying the unsaturated C18 long chain base moiety proved to have the fastest rate of association, whereas the saturated species carrying 20 carbon atoms had the slowest rate. After having increased the GM1 cell content (65-fold) by incubation with the various ganglioside species, the cells were incubated with cholera toxin and the time course of cyclic AMP accumulation was monitored. Remarkable differences among cells enriched with the various molecular species were found in the duration of the lag time preceding the accumulation of cyclic AMP, the shortest being displayed by the unsaturated C18 species. Moreover, the amount of cyclic AMP accumulated after a given time of incubation with cholera toxin was significantly higher when the C18:1-GM1 species was present than with native GM1. Fluorescence anisotropy experiments, carried out using the probe 1,3-diphenylhexatriene, show that the GM1 ganglioside ceramide moiety was also modifying the cell membrane fluidity of the host.

It was previously shown that sphingomyelin and gangliosides can be biosynthesized starting from sphingosine or sphingosine-containing fragments which originated in the course of GM1 ganglioside catabolism. In the present paper we investigated which fragments were specifically re-used for sphingomyelin and ganglioside biosynthesis in rat liver. At 30 h after intravenous injection of GM1 labelled at the level of the fatty acid ([stearoyl-14C]GM1) or of the sphingosine ([Sph-3H]) moiety, it was observed that radioactive sphingomyelin was formed almost exclusively after the sphingosine-labelled-GM1 administration. This permitted the recognition of sphingosine as the metabolite re-used for sphingomyelin biosynthesis. Conversely, gangliosides more complex than GM1 were similarly radiolabelled after the two treatments, thus ruling out sphingosine re-utilization for ganglioside biosynthesis. For the identification of the lipid fragment re-used for ganglioside biosynthesis, we administered to rats neutral glycosphingolipids (galactosylceramide, glucosylceramide and lactosylceramide) each radiolabelled in the sphingosine moiety or in the terminal sugar residue. Thereafter we compared the formation of radiolabelled gangliosides in the liver with respect to the species administered and the label location. After galactosylceramide was injected, no radiolabelled gangliosides were formed. After the administration of differently labelled glucosylceramide, radiolabelled gangliosides were formed, regardless of the position of the label. After lactosylceramide administration, the ganglioside fraction became more radioactive when the long-chain-base-labelled precursors were used. These results suggest that glucosylceramide, derived from glycosphingolipid and ganglioside catabolism, is recycled for ganglioside biosynthesis.

The effect of gangliosides on membrane permeability was investigated by studying the kinetic properties of cytochrome c oxidase, the activity of which, when the enzyme is reconstituted in phospholipid vesicles, is dependent on membrane permeability to H+ and K+. The experiments indicate that three different gangliosides (GM1, DD1a, GT1b) incorporated into cytochrome c oxidase-containing phospholipid vesicles stimulate enzymic activity, in the absence of ionophores, most probably by disorganizing the bilayer lipid assembly and increasing its permeability to ions. This interpretation was confirmed by fluorescence-spectroscopy experiments in which the rate of passive leakage of carboxyfluorescein entrapped in the vesicles was measured. Cholera toxin, or its isolated B-subunit, added to GM1-containing proteoliposomes inhibited cytochrome c oxidase activity, indicating the lack of formation, under these experimental conditions, of channels freely permeable to H+ or K+.

A metabolic recycling of N-acetylgalactosamine (GalNAc), liberated from exogenous GM2 ganglioside [nomenclature of Svennerholm (1964) J. Lipid Res. 5, 145-155; IUPAC-IUB recommendations (1977) Lipids 12, 455-468], is demonstrated in rat liver. After the injection of a GM2 ganglioside isotopically radiolabelled on the terminal GalNAc residue ([GalNAc-3H]GM2), the liver retained a large amount of radioactivity distributed among: (1) a glycoprotein/glycosaminoglycan fraction, (2) a ganglioside fraction; and (3) a free-sugar fraction. Furthermore, volatile radioactivity was also found. The relative incorporation in the above fractions was time-dependent. The glycoprotein/glycosaminoglycan fraction contained radioactivity that was located on the GalNAc and GlcNAc residues. The ganglioside fraction was composed of two main families: gangliosides formed by a recycling of the liberated GalNAc, and gangliosides derived by direct utilization of the administered GM2. The free-sugar fraction contained mainly GalNAc. We suggest that GalNAc, after being released in the course of intra-lysosomal ganglioside catabolism, crosses the lysosomal membrane and passes into the cytosol, where the part not degraded is re-utilized for the biosynthesis of the different glycoconjugate classes.

In order to assess metabolic recycling of sialic acid, GM1 ganglioside [nomenclature of Svennerholm (1964) J. Lipid. Res. 5, 145-155; IUPAC-IUB Recommendations (1977) Lipids 12, 455-468], 14C-radiolabelled at the acetyl group of sialic acid, was intravenously injected into Wistar rats, and the presence of radioactive sialic acid in liver sialoglycolipids (gangliosides) and sialoglycoproteins was ascertained. A time-course study (20 min-72 h) showed that the radioactivity present in the liver distributed in the following fractions, with reciprocal proportion varying with time: the protein (glycoprotein) fraction, the ganglioside fraction and the diffusible fraction, which contained low-Mr compounds, including sialic acid. Ganglioside-linked radioactivity gradually decreased with time; protein-linked radioactivity appeared soon after injection (20 min), reached a maximum around 20 h, then slowly diminished; diffusible radioactivity provided a sharp peak at 4 h, then rapidly decreased till disappearing after 40 h. The behaviour of bound radioactivity in the individual liver gangliosides was as follows: (a) rapid diminution with time in GM1, although with a lower rate at the longer times after injection; (b) early appearance (20 min) with a peak at 1 h, followed by continuous diminution, in GM2; (c) early appearance (20 min), peak at 1 h, diminution till 4 h, followed by a plateau, in GM3; (d) appearance at 60 min, maximum around 40 h and slow diminution thereafter, in GD1a, GD1b and GT1b. A detailed study, accomplished at 40 h after injection, demonstrated that almost all radioactivity present in the protein fraction was released by mild acid treatment and recovered in purified sialic acid; most of radioactive glycoprotein-bound sialic acid was releasable by sialidase action. In addition, the radioactivity present in the different gangliosides was exclusively carried by sialic acid and present in both sialidase-resistant and sialidase-labile residues. Only in the case of GD1a was the specific radioactivity of sialidase-resistant sialic acid superior to that of sialidase-releasable sialic acid. The results obtained lead to the following conclusions: (a) radioactive GM3 and GM2 were produced by degradation of GM1 taken up; GM3 originated partly by a process of neosynthesis; (b) radioactive GM1 consisted in part of residual exogenous GM1 and in part of a neosynthetized product; (c) radioactive GD1a originated in part by direct sialylation of GM1 taken up and in part by a neosynthetic process; (d) radioactive GD1b and GT1b resulted only from neosynthesis.(ABSTRACT TRUNCATED AT 400 WORDS)

The pathways of metabolic processing of exogenously administered GM1 ganglioside in rat liver was investigated at the subcellular level. The GM1 used was 3H-labelled at the level of long-chain base ([Sph(sphingosine)-3H]GM1) or of terminal galactose ([Gal-3H]GM1). The following radioactive compounds, derived from exogenous GM1, were isolated and chemically characterized: gangliosides GM2, GM3, GD1a and GD1b (nomenclature of Svennerholm [(1964) J. Lipid Res. 5, 145-155] and IUPAC-IUB Recommendations [(1977) Lipids 12, 455-468]); lactosylceramide, glucosylceramide and ceramide; sphingomyelin. GM2, GM3, lactosylceramide, glucosylceramide and ceramide, relatively more abundant shortly after GM1 administration, were mainly present in the lysosomal fraction and reflected the occurrence of a degradation process. 3H2O was also produced in relevant amounts, indicating complete degradation of GM1, although no free long-chain bases could be detected. GD1a and GD1b, relatively more abundant later on after administration, were preponderant in the Golgi-apparatus fraction and originated from a biosynthetic process. More GD1a was produced starting from [Sph-3H]GM1 than from [Gal-3H]GM1, and radioactive GD1b was present only after [Sph-3H]GM1 injection. This indicates the use of two biosynthetic routes, one starting from a by-product of GM1 degradation, the other implicating direct sialylation of GM1. Both routes were used to produce GD1a, but only the first one for producing GD1b. Sphingomyelin was the major product of GM1 processing, especially at the longer times after injection, and arose from a by-product of GM1 degradation, most likely ceramide.

Ganglioside GM1, 3H-labelled at the level of terminal galactose or of sphingosine, was intravenously injected into Swiss albino mice and some steps in its metabolic fate in the liver were investigated. After administration of [3H]sphingosine-labelled GM1 all major liver gangliosides [GM3, GM2, GM1, GD1a-(NeuAc,NeuGl)] became radioactive, the radioactivity residing in all cases on the sphingosine moiety. The specific radioactivity was highest in GM1, which carried about 53% of the radioactivity incorporated into gangliosides, followed by GM2, with 34.5% of incorporated radioactivity, GM3 and GD1a-(NeuAc,NeuGl), both with about 5% of incorporated radioactivity. After administration of [3H]galactose-labelled GM1 the only radioactive gangliosides present in the liver were GM1 and GD1a-(NeuAc,NeuGl), the former carrying about 95% of the total ganglioside-incorporated radioactivity, the latter about 3%. Both gangliosides were radioactive exclusively in the terminal galactose residue. According to these results exogenously administered GM1, after being taken up by the liver, is mainly degraded to GM2 and GM3, a part being, however, sialylated to GD1a-(NeuAc,NeuGl). All this suggests that exogenous GM1 may be involved in the metabolic routes of endogenous liver gangliosides.

The ganglioside patterns in the liver of different inbred and hybrid strains of mice were investigated. The inbred strains were Balb/cAnNCr1BR, C57BL/6NCr1BR, DBA/2NCr1BR. C3H/HeNCr1BR; the hybrid strain was the Swiss albino. The following major gangliosides were found to be present in mouse liver: GM3-NeuAc; GM3-NeuGl, GM2 [a mixture of one species carrying N-acetylneuraminic acid (NeuAc) and one carrying N-glycollylneuraminic acid (NeuGl)], GM1 and GD1a-(NeuAc,NeuGl). The qualitative and quantitative patterns of liver gangliosides were markedly different in the various inbred strains of mice; in Balb/cAnNCr1BR strain, ganglioside GM2 was preponderant (99.2% of total ganglioside content); in C57BL/6NCr1BR, the major ganglioside was GM2 (90.4%), followed by GM3-NeuAc (5.6%) and GM3-NeuGl (4.0%); in DBA/2NCr1BR, GM2 accounted for 77.1%, GD1a-(NeuAc,NeuGl) 18.9% and GM1 3.1% of gangliosides; in C3H/HeNCr1BR, GM2 constituted 50.6%, GM1 22.8% and GD1a-(NeuAc,NeuGl) 22.1%. In the hybrid Swiss albino mice, liver ganglioside composition markedly varied from one animal to another, GM3-NeuGl, GM2 and GD1a-(NeuAc,NeuGl) being the predominant gangliosides in the various cases.

alpha-L-Fucosidase, prepared in highly purified form (Mr 70 000-74 000) from Octopus hepatopancreas, was able to hydrolyse a fucose-containing ganglioside, namely Fuc-GM1 (II3NeuAc,IV2Fuc-GgOse4-Cer). The enzyme showed an irregular kinetic behaviour (v/[S] and v/[E] relationships following sigmoidal curves) when working on micellar Fuc-GM1 (Mr of the micelle 500 000), but obeyed regular hyperbolic kinetics when acting on low-Mr substances. It was observed that, on incubation with micellar Fuc-GM1 under the conditions used for the enzyme assay, Octopus alpha-L-fucosidase produced a ganglioside-enzyme complex that was catalytically inactive. This complex had an Mr exceeding 500 000 and a ganglioside/protein ratio of 4:1 (w/w), which is consistent with a stoichiometric combination of one ganglioside micelle with two enzyme molecules. Inactivation of alpha-L-fucosidase by formation of the corresponding complexes was also obtained with micellar gangliosides GM1 (II3NeuAc-GgOse4-Cer), GD1a (II3NeuAc,IV3NeuAc-GgOse4-Cer) and GT1b [II3(NeuAc)2,IV3-NeuAc-GgOse4-Cer], which are not substrates for the enzyme, indicating that the ganglioside micelles per se act as enzyme inhibitors. However, alpha-L-fucosidase easily forms a Fuc-GM1-alpha-L-fucosidase complex, displaying regular Michaelis-Menten kinetics. Therefore the anomalous behaviour exhibited by alpha-L-fucosidase on micellar Fuc-GM1 is likely due to formation of the complex, which separates the fucosyl linkage from the active site of the complexed enzyme, but makes it available to the enzyme in the free form.

G(d1a), G(d1b) and G(t1b) gangliosides were dispersed in the following membrane-mimicking systems: (a) homogeneous micelles; (b) mixed micelles with G(m1) ganglioside (which is resistant to the enzyme action), Triton X-100 or bovine serum albumin; (c) small unilamellar vesicles of egg phosphatidylcholine. The effect of dispersion on sialic acid release by Vibrio cholerae sialidase was studied. As reference substrates freely interacting with the enzyme the lipid-free carbohydrates of G(d1a) and 3'-sialosyl-lactose were employed. The apparent V(max.) of the enzyme was, with all the gangliosides, dependent on the type of ganglioside dispersion. It was lowest for homogeneous micelles and mixed micelles with ganglioside G(m1), and increased about 6-fold for ganglioside/bovine serum albumin lipoprotein micelles, 15-fold for mixed-ganglioside/Triton X-100 micelles (optimal molar ratio 1:7.5) and 30-fold for phosphatidylcholine vesicles containing 2.5 mol% ganglioside (this proportion was optimal for enzyme activity on the vesicles). For ganglioside G(d1a), the activity on Triton X-100 mixed micelles and on mixed vesicles was even greater (3- and 6-fold respectively) than that displayed on G(d1a) lipid-free carbohydrate. With each of the used gangliosides the apparent K(m) values were very similar values for homogeneous micelles and vesicular dispersions, but showed marked increases for Triton X-100 mixed micelles, approaching the values exhibited by reference oligosaccharides. Triton X-100 micelles and phosphatidylcholine vesicles did not appreciably alter the kinetics of sialidase action on 3'-sialosyl-lactose and on G(d1a) lipid-free carbohydrate, indicating that the above effects are dependent on the intrinsic characteristics of the membrane-like systems containing gangliosides.