Augusto Preti

Recent Development in Mammalian Sialidase Molecular Biology

This review summarizes the recent research development on mammalian sialidase molecular cloning. Sialic acid–containing compounds are involved in several physiological processes, and sialidases, as glycohydrolytic enzymes that remove sialic acid residues, play a pivotal role as well. Sialidases hydrolyze the nonreducing, terminal sialic acid linkage in various natural substrates, such as glycoproteins, glycolipids, gangliosides, and polysaccharides. Mammalian sialidases are present in several tissues/organs and cells with a typical subcellular distribution: they are the lysosomal, the cytosolic, and the plasma membrane–associated sialidases. Starting in 1993, 12 different mammalian sialidases have been cloned and sequenced. A comparison of their amino acid sequences revealed the presence of highly conserved regions. These conserved regions are shared with viral and microbial sialidases that have been characterized at three-dimensional structural level, allowing us to perform the molecular modeling of the mammalian proteins and suggesting a monophyletic origin of the sialidase enzymes. Overall, the availability of the cDNA species encoding mammalian sialidases is an important step leading toward a comprehensive picture of the relationships between the structure and biological function of these enzymes.

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.

Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts

Ceramide is a key lipid molecule necessary to regulate some cellular processes, including apoptosis and cell differentiation. In this context, its production has been shown to occur via sphingomyelin hydrolysis or sphingosine acylation. Here, we show that in human fibroblasts, plasma membrane ceramide is also produced from ganglioside GM3 by detachment of sugar units. Membrane‐bound glycosylhydrolases have a role in this process. In fact, the production of ceramide from GM3 has been observed even under experimental conditions able to block endocytosis or lysosomal activity, and the overexpression of the plasma membrane ganglioside sialidase Neu3 corresponded to a higher production of ceramide in the plasma membrane. The increased activity of Neu3 was paralleled by an increase of GM3 synthase mRNA and GM3 synthase activity. Neu3‐overexpressing fibroblasts were characterized by a reduced proliferation rate and higher basal number of apoptotic cells in comparison with wild‐type cells. A similar behavior was observed when normal fibroblasts were treated with exogenous C2‐ceramide.—Valaperta, R., Chigorno, V., Basso, L., Prinetti, A., Bresciani, R., Preti, A., Miyagi, T., and Sonnino, S. Plasma membrane production of ceramide from ganglioside GM3 in human fibroblasts. FASEB J. 20, E450–E461 (2006)

Sialidase NEU3 is a peripheral membrane protein localized on the cell surface and in endosomal structures

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.

Overexpression of cytosolic sialidase Neu2 induces myoblast differentiation in C2C12 cells.

Cytosolic sialidase Neu2 has been implicated in myoblast differentiation. Here we observed a significant upregulation of Neu2 expression during differentiation of murine C2C12 myoblasts. This was evidenced both as an increase in Neu2 mRNA steady‐state levels and in the cytosolic sialidase enzymatic activity. To understand the biological significance of Neu2 upregulation in myoblast differentiation, C2C12 cells were stably transfected with the rat cytosolic sialidase Neu2 cDNA. Neu2 overexpressing clones were characterized by a marked decrement of cell proliferation and by the capacity to undergo spontaneous myoblast differentiation also when maintained under standard growth conditions. This was evidenced by the formation of myogenin‐positive myotubes and by a significant decrease in the nuclear levels of cyclin D1 protein. No differentiation was on the contrary observed in parental and mock‐transfected cells under the same experimental conditions. The results indicate that Neu2 upregulation per se is sufficient to trigger myoblast differentiation in C2C12 cells.

Synthesis, spectral properties and enzymatic hydrolysis of fluorescent derivatives of cerebroside sulfate containing long-wavelength-emission probes

Fluorescent derivatives of cerebroside sulfate (sulfogalactosyl ceramide, sulfatide) containing long-wavelength-emission fluorophores were synthesized. For this purpose a procedure was developed for preparing a cerebroside 3-sulfate derivative with an amino group on the terminal carbon atom of its fatty acyl residue. The latter compound has been used to prepare cerebroside 3-sulfate, coupled to lissamine-rhodamine, fluoresceine, eosine and NBD. The spectroscopic properties of these compounds, in different solvent systems and when incorporated into micelles of a non-ionic detergent or liposomes of a phospholipid, are reported. Incubation of these respective sulfatides with a human leukocyte preparation, resulted in the formation of the corresponding fluorescent cerebrosides.

Cytosolic sialidase Neu2 upregulation during PC12 cells differentiation.

The enzymatic activity of cytosolic sialidase Neu2 was found to increase transiently only during differentiation, whereas was undetectable in untreated PC12 cells. These data suggest a possible involvement of cytosolic sialidase Neu2 in differentiation of PC12 cells.

Uptake and metabolism of a fluorescent sulfatide analogue in cultured skin fibroblasts

The sulfatide fluorescent analogue N-lissamine rhodaminyl-(12-aminododecanoyl) cerebroside 3-sulfate was administered in the form of albumin complex to normal human skin fibroblasts and its metabolic fate was investigated. Ceramide, galactosylceramide, glucosylceramide, sphingomyelin and free acid, all containing the fluorophore lissamine rhodamine, have been synthesized as reference standards for the identification of the metabolic products. Ceramide appeared to be the main metabolic product present both in cell extract and medium, followed by galactosylceramide and sphingomyelin. Fluorescence microscopy of cells showed a marked perinuclear fluorescence.

Insulin‐like growth factor 1 signaling regulates cytosolic sialidase Neu2 expression during myoblast differentiation and hypertrophy

Cytosolic sialidase (neuraminidase 2; Neu2) is an enzyme whose expression increases during myoblast differentiation. Here we show that insulin‐like growth factor 1 (IGF1)‐induced hypertrophy of myoblasts notably increases Neu2 synthesis by activation of the phosphatidylinositol 3‐kinase/AKT/mammalian target of rapamycin (P13K/AKT/mTOR) pathway, whereas the proliferative effect mediated by activation of the extracellular regulated kinase 1/2 (ERK1/2) pathway negatively contributed to Neu2 activity. Accordingly, the differentiation L6MLC/IGF‐1 cell line, in which the forced postmitotic expression of insulin‐like growth factor 1 stimulates a dramatic hypertrophy, was accompanied by a stronger Neu2 increase. Indeed, the hypertrophy induced by transfection of a constitutively activated form of AKT was able to induce high Neu2 activity in C2C12 cells, whereas the transfection of a kinase‐inactive form of AKT prevented myotube formation, triggering Neu2 downregulation. Neu2 expression was strictly correlated with IGF‐1 signaling also in C2 myoblasts overexpressing the insulin‐like growth factor 1 binding protein 5 and therefore not responding to endogenously produced insulin‐like growth factor 1. Although Neu2‐transfected myoblasts exhibited stronger differentiation, we demonstrated that Neu2 overexpression does not override the block of differentiation mediated by PI3 kinase and mTOR inhibitors. Finally, Neu2 overexpression did not modify the ganglioside pattern of C2C12 cells, suggesting that glycoproteins might be the target of Neu2 activity. Taken together, our data demonstrate that IGF‐1‐induced differentiation and hypertrophy are driven, at least in part, by Neu2 upregulation and further support the significant role of cytosolic sialidase in myoblasts.

Occurrence in brain lysosomes of a sialidase active on ganglioside.

A lysosomal preparation, obtained from brain ho‐mogenate of 17‐day‐old C57BL mice by centrifugation on a self‐generating Percoll linear density gradient, showed relative specific activity (RSA) values for typical lysosomal enzymes of 40–120 and for mitochondria, plasma membrane, and cytosol markers of much lower than 1, a result indicating a high degree of homogeneity. The lysosomal preparation contained a sialidase activity that was assayed radiometrically with ganglioside [3H]GDla and fluorimetrically with 4‐methylumbelliferyl‐α‐D‐N‐acetylneuraminic acid (MUB‐NeuAc). The properties of the lysosomal enzyme were compared with those of the plasma membrane‐bound sialidase contained in a purified synaptosomal plasma membrane fraction that was prepared from the same homogenate and assayed with the same substrates. The optimal pH was 4.2 for the lysosomal and 5.1 for the plasma Membrane‐bound enzyme. The apparent Km values for GDl a and MUB‐NeuAc were 1.5 × 10‐5 and 4.2 × 10‐5M, respectively, for the lysosomal enzyme and 2.7 × 10‐4 and 6.3 × 10‐5M for the plasma membrane‐bound one. Triton ×‐ 100 had a predominantly inhibitory effect on the lysosomal enzyme, whereas it strongly activated the plasma membrane‐bound one. The lysosomal enzyme was highly unstable on storage and freezing and thawing cycles, whereas the plasma membrane‐bound one was substantially stable. The RSA value of the lysosomal sialidase in the lysosomal fraction closely resembled that of authentic lysosomal enzymes, whereas the RSA value of plasma membrane‐bound sialidase in the plasma membrane fraction was very similar to that of typical plasma membrane markers. It is thus evident that the sialidase present in the lysosomal fraction is an authentic lysosomal enzyme distinct and different from the sialidase contained in the plasma membrane. The lysosomal sialidase affected other ganglio‐sides, like GDlb and GM3. These data constitute the first direct evidence for the presence in brain lysosomes of a sialidase activity on gangliosides and contribute to a better knowledge of ganglioside breakdown and turnover in the brain.