Littérature scientifique sur le sujet « Sialidase NEU4 »

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-resistant ganglioside analogs having similar biological activities to natural gangliosides are expected to be important probes for clarifying the biological functions of gangliosides. Focusing on difluoromethylene-linked (CF2-linked) and methylene-linked (CH2-linked) α(2,3)sialylgalactose as a core structure of sialidase-resistant ganglioside mimics, we have developed novel, stereocontrolled, and efficient methodologies to synthesize C-sialosides based on Ireland-Claisen rearrangement. These methods were employed to synthesize CF2-linked GM4. The CF2-linked GM4 inhibited human sialidases NEU2 and NEU4 with IC50 values of 754 and 930 μM, respectively, and strongly inhibited human lymphocyte proliferation in the same concentration range as natural GM4.

Neuraminidase, also known as sialidase, is ubiquitous in animals and microorganisms. It is predominantly distributed in the cell membrane, cytoplasmic vesicles, and lysosomes. Neuraminidase generally recognizes the sialic acid glycosidic bonds at the ends of glycoproteins or glycolipids and enzymatically removes sialic acid. There are four types of neuraminidases, named as Neu1, Neu2, Neu3, and Neu4. Among them, Neu1 is the most abundant in mammals. Recent studies have revealed the involvement of Neu1 in several diseases, including cardiovascular diseases, diabetes, cancers, and neurological disorders. In this review, we center the attention to the role of Neu1 in cardiovascular diseases, including atherosclerosis, ischemic myocardial injury, cerebrovascular disease, congenital heart disease, and pulmonary embolism. We also summarize inhibitors from Chinese herbal medicines (CHMs) in inhibiting virus neuraminidase or human Neu1. Many Chinese herbs and Chinese herb preparations, such as Lonicerae Japonicae Flos, Scutellariae Radix, Yupingfeng San, and Huanglian Jiedu Decoction, have neuraminidase inhibitory activity. We hope to highlight the emerging role of Neu1 in humans and potentially titillate interest for further studies in this area.

Based on the human cDNA sequence predicted to represent the NEU4 sialidase gene in public databases, a cDNA covering the entire coding sequence was isolated from human brain and expressed in mammalian cells. The cDNA encodes two isoforms: one possessing an N-terminal 12-amino-acid sequence that is predicted to be a mitochondrial targeting sequence, and the other lacking these amino acids. Expression of the isoforms is tissuespecific, as assessed by reverse transcription–PCR. Brain, muscle and kidney contained both isoforms; liver showed the highest expression, and the short form was predominant in this organ. In transiently transfected COS-1 cells, enzyme activity was markedly increased with gangliosides as well as with glycoproteins and oligosaccharides as substrates compared with the control levels. This differs from findings with other human sialidases. Although the isoforms were not distinguishable with regard to substrate specificity, they exhibited differential subcellular localizations. Immunofluorescence microscopy and biochemical fractionation demonstrated that an exogenously expressed haemagglutinin-tagged long form of NEU4 was concentrated in mitochondria in several human culture cell types, whereas the short form was present in intracellular membranes, indicating that the sequence comprising the N-terminal 12 amino acid residues acts as a targeting signal for mitochondria. Co-localization of the long form to mitochondria was further supported by efficient targeting of the N-terminal region fused to enhanced green fluorescent protein, and by the targeting failure of a mutant with an amino acid substitution in this region. NEU4 is possibly involved in regulation of apoptosis by modulation of ganglioside GD3, which accumulates in mitochondria during apoptosis and is the best substrate for the sialidase.

Sialidases or neuraminidases are glycohydrolytic enzymes removing sialic acid residues from glycoproteins and glycolipids. They are widely distributed in nature, from microorganisms to vertebrates. In mammals, four sialidases with different subcellular localization and biochemical features have been described: a lysosomal sialidase (NEU1), a cytosolic sialidase (NEU2), and two membrane-associated sialidases (NEU3 and NEU4). NEU4, the most recently identified member of the human sialidase family, is found in two forms, long and short, differing in the presence of a 12 amino acids sequence at the N-terminus of the protein. Contradictory data are present in the literature about the subcellular distribution of these enzymes, and their membrane anchoring mechanism is still unclear. First of all, in this work we investigated NEU4 long and NEU4 short membrane anchoring mechanism and their subcellular localization in COS-7 and HeLa cells. As observed by solubilization and cross-linking experiments, NEU4 long and short are extrinsic membrane proteins, probably associated to the lipid bilayer through protein-protein interactions. These results are in accordance with primary structure analysis that did not evidence any transmembrane sequence, nor the presence of any membrane binding motifs. Subcellular localization studies, performed through confocal immunofluorescence and subcellular fractionation, showed that human NEU4 is a membrane-bound enzyme; in particular, the long form of NEU4 localizes in mitochondria, while the short one is mainly associated with the endoplasmic reticulum. In addition, a finer submitochondrial fractionation and a protease treatment of intact mitochondria and mitoplasts provided evidence for NEU4 long location in the outer mitochondrial membrane. Moreover, primary structure analysis showed the presence of a proline-rich region which is unique to NEU4, having no counterpart in any other human sialidase. Deletion mutants lacking this loop showed subcellular distributions similar to those of wild-type proteins in COS-7 cells, suggesting that this region does not directly affect the association of NEU4 to the membranes. We subsequently hypothesized an involvement in the interaction with signaling pathway components. Studies in collaboration with the Department of Medical Chemistry, Biochemistry and Biotechnology (L.I.T.A.) of the University of Milano evaluated the effect of NEU4 long transfection in human neuroblastoma SK-N-BE cell line. Similarly, we produced stable SK-N-BE clones transfected with the mutated form of NEU4 long (N4LnoP). The mRNA level of both NEU4 and the other human sialidases were checked in both wild-type and mutated NEU4 clones by RT-PCR and real-time PCR. In addition, only the wild-type form of NEU4 long was able to alterate the sialoglycoprotein profile and significantly enhance the proliferative ability of SK-N-BE cells, suggesting that the Pro-rich region of NEU4 is involved in both these phenomena. Subsequently, in order to study the function of NEU4 in SK-N-BE cell line, we analyzed the effect of NEU4 expression also under retinoic acid induced differentiating conditions. Our results show that retinoic acid treatment increases the expression of NEU4, either wild-type or mutated, due to the presence of RA response elements (RARE) in the CMV promoter. In addition, both morphological change analysis and neurite outgrowth quantification were consistent with acetylcholinesterase activity data, indicating a role for NEU4 long and its Pro-rich region in the early phases of retinoic acid induced neuronal differentiation process. Since potential Akt and Erk1 kinase motifs were found in NEU4 proline-rich region, activation of both phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase (MAPK) signaling pathways were studied in stable SK-N-BE clones. Our results demonstrate that the expression of NEU4, both wild-type and mutated, does not significantly affect retinoic acid induced activation of both Akt and Erk1/2 pathways in SK-N-BE cells, suggesting that NEU4 could be located in a downstream place in these signaling pathways. As confirmed by immunoprecipitation experiments, NEU4 long interacts with Akt kinase in SK-N-BE cells. Conversely, the lacking of the proline-rich region impaired interaction between NEU4 and Akt, indicating that the formation of NEU4-Akt complex occurs through the proline-rich region. On the contrary, no interactions between NEU4 and Erk1/2 kinase were observed, suggesting that NEU4 is not a substrate of this kinase. Finally, treatment with LY294002 PI3K inhibitor demonstrates that PI3K/Akt signaling pathway is required for neuronal differentiation induced by retinoic acid in this neuroblastoma cell line. On the whole, these data suggest that NEU4 long is a downstream component of Akt signaling pathway required for RA induced neuronal differentiation in SK-N-BE cells.

Abnormal glycosylation is known to be associated with cancer malignancy. In the cell, this process is finely regulated by many enzymes, including sialidases or neuraminidases; these are glycohydrolases widely distributed in nature that remove sialic acid residues from glycoproteins and glycolipids. In mammals, four sialidases with different subcellular localizations and biochemical features have been described: a lysosomal sialidase (NEU1), a cytosolic sialidase (NEU2), a plasma membrane-associated sialidase (NEU3) and a mitochondrial sialidase (NEU4). Studies performed over the last decade have focused on the involvement of sialylation in the progression of cancer and moreover on the association of sialidase deregulation to the tumorigenic transformation. In particular, recent studies on the Japanese population have shown that sialidase NEU3 is often deregulated in colorectal cancer and also that it co-immunoprecipitates with the epidermal growth factor receptor (EGFR), the molecular target of the most recent therapies based on monoclonal antibodies. In collaboration with the Istituto Nazionale dei Tumori of Milan (Italy) (IRCCS) and with the Istituto Cantonale di Patologia of Locarno (Switzerland) we recruited a cohort of 85 Caucasian patients resected for a colorectal cancer. By real-time PCR experiments we observed a deregulation of the mitochondrial sialidase transcripts and, on the contrary, an up regulation of NEU3 mRNA in tumor tissues compared to paired normal mucosa. Moreover, by comparing NEU3 and EGFR mRNA levels, we observed a statistically significant correlation, suggesting that the increase in EGFR expression could be associated with NEU3 increment. Vice versa, no correlation was observed between the overexpression of NEU3 sialidase and mutations in KRAS, BRAF, PIK3CA and PTEN diagnostic markers. Experiments performed on colorectal cancer cell lines have demonstrated that overexpression of wild type NEU3 enhanced EGFR activation, compared to colon normal mucosa CCD841 cell line, irrespectively of mRNA and protein levels, mutational and gene status of the receptor in all the cell lines tested. The only exception was represented by SW620 cells, that are commonly used as EGFR negative control. Moreover, Western Blots of wild type NEU3 overexpressing cells revealed increased EGFR and ERK1/2 phosphorylation in SW480 colorectal cell line and also in DIFI cells, which represents the best cellular model to study the EGFR pathway, while mRNA and total EGFR protein contents remained constant. On the contrary, we could not detect any EGFR activation in cells overexpressing a totally inactive mutant of NEU3. In addition we performed MTT based test in transfected cells. Western blot analyses showed a significant increase of cell viability, only upon overexpression of wild type NEU3, the inactive mutant being completely ineffective in this respect. Having demonstrated that the human NEU3 sialidase is more strongly anchored to the membrane its murine counterpart, we proved, not only by the lectin binding assay but also by mass spectrometry, that this sialidase directly modified the sialylation level of EGFR extracellular domain. Moreover we also showed that NEU3 overexpression modulated the response to the pharmacological treatment with Cetuximab. The overexpression of the active form of NEU3 sialidase lead to a significant increase in cell viability in all tested cell lines, also under pharmacological treatment with Cetuximab, with the exception of SW48 cells that presented a hyperactivating mutation in the tyrosine kinase domain of EGFR, causing the receptor to act independently from the dimerization. Our data suggest that, in the cell lines in which EGFR acts correctly as a dimer, sialidase overexpression caused an increment of viability even in those presenting hyperactivating mutations in the downstream pathways, that influence the efficacy of the therapy. We decided to extend the analysis of the deregulation of human sialidases to other types of cancer, in order to identify and deepen the knowledge of common variations of these enzymes also in the Western population. On the whole, by demonstrating the role of sialidase NEU3 in CRC, our work strongly suggests that this enzyme might be taken into consideration as a new effective molecular marker for CRC diagnosis and treatment. Furthermore, these data confirm the need of further studies concerning the role played by sialidases as a defining factor in cancer progression, opening up potential applications in diagnosis and therapy.

The G9 gene, located within the MHC class III region of man and mouse, has been shown to encode a 46kDa sialidase, optimally active at pH 4.6. The genomic localization of G9 indicates that it corresponds to the Neu1 gene, which was mapped to the S region of the H-2 complex (the murine MHC class III region) by genetic linkage studies and analysis of Neu1 phenotypes in mice, and has been shown to modulate the sialic acid content of several liver lysosomal enzymes. Three alleles of Neu1 have been defined. The most common allele, Neu1^b, is associated with normal levels of liver sialidase activity, whilst a small number of mouse strains, e.g. the SM/J strain, that carry the Neu1^a allele, show significantly reduced sialidase activity. In humans, reduced sialidase activity leads to the lysosomal storage disorder, sialidosis, characterised by developmental and neurological abnormalities. Characterisation of the substrate specificity of the G9 sialidase, expressed and purified from Drosophila S2 cells, showed that the enzyme hydrolyses the synthetic substrate 4MU-NANA. However, little or no activity was seen towards a number of natural substrates, including gangliosides, or towards the synthetic substrate, fetuin. It has been established that the G9 sialidase is unique within the sialidase family, in that it requires association with another lysosmal protein protective protein cathepsin A (PPCA) in order to be optimally active. Co-expression of G9 and PPCA in both mammalian and Drosophila expression systems has been attempted in order to accurately characterise the substrate specificity of the G9 sialidase. The reduced sialidase activity within the SM/J mouse makes this a good model to investigate the role of G9 in the immune response. SM/J mice are known to be approximately 20% smaller in size than normal mice, however, it was observed that the thymus size of the SM/J mouse was ~60% smaller than that of an age matched C57BL/6 control. Characterisation of the B- and T-cells of the spleen and thymus of SM/J mice showed variations in the populations of these cells between the two mice strains, with the most significant difference observed in the CD4⁺ thymic T-cell population. Analysis of antisera taken from SM/J and C57BL/6 control mice after primary and secondary immune responses showed that the levels of antibody isotypes compared in the two strains were similar except for IgG1, which was present at significantly reduced levels in the sera of SM/J mice. Deficiency in hexosaminidase A results in the ganglioside storage disorder, Tay-Sachs disease. HexA^-/- mice were generated as a model for Tay-Sachs disease, however, a catabolic bypass, mediated by the lysosomal sialidase, G9, (which occurs in mice but not humans) allows the Tay-Sachs disease model to escape disease onset in the first year of life, in contrast to the progression of the disease in human sufferers. In order to generate a more accurate disease model, the sialidase deficient mouse strain, SM/J has been crossed with the existing Tay-Sachs disease model. This crossing should substantially reduce the sialidase activity that allows the mice to escape onset of Tay-Sachs and thus lead to a disease model more comparable to human Tay-Sachs sufferers.

Sialidases or neuraminidases (EC 3.2.1.18) are enzymes widely distributed in nature, from viruses to vertebrates. They play a key role in the metabolism of sialoglycoconjugates. In particular, they are able to remove sialic acid residues from gangliosides, sialoglycoproteins and sialoligosaccharides. It is becoming more and more evident that the partial desialylation of senescent erythrocyte membrane sialoglycoconjugates is a primary or preliminary signal for erythrophagocytosis. The removal of sialic acid from the sialoglycoconjugates is assumed to be promoted by the sialidases present on the membranes. The presence of two sialidases in human erythrocyte membranes has been previously reported. One acts optimally at acidic pH (4.2-4.7) and the other one at neutral pH. In order to identify the exact nature of these two sialidases and the enzyme–membrane leaflet interaction, we incubated the erythrocyte membranes with different solutions. We obtained interesting results when we treated the membranes with 0.1 M sodium carbonate pH 11.5. In literature, it is reported that this treatment is one of the methods used to discriminate between peripheral and integral cell membrane proteins. We demonstrated that sodium carbonate caused the partial release of the acidic sialidase from erythrocyte membranes, whereas the neutral one remained totally membrane-associated. Our results suggest that the alkaline pH value alone is responsible for the extraction of the acidic enzyme from the membrane and that this protein behaves as a peripheral protein. The analysis of the biochemical and kinetic characteristics allowed us to define the extracted acidic sialidase suitable for purification. Moreover, in order to obtain more information about the nature of the acidic sialidase released from the erythrocyte membranes by sodium carbonate treatment, we performed an analysis of the erythrocyte membranes, on the extracted and non-extractable proteins, by immunoblotting using anti-Neu1 antibody. Our results show the presence of sialidase Neu1 on erythrocyte membranes and the capacity of the sodium carbonate treatment to induce the partial solubilization of Neu1 from the membranes. This represents the first evidence of the presence of Neu1 on human erythrocyte membranes and suggests that the erythrocyte acidic sialidase may be identified with sialidase Neu1.

Human melanoma has been shown to be marked by an atypical cellular ganglioside profile and by the upregulation of plasma membrane sialidase NEU3. In this PhD thesis, I analyzed NEU3 silencing effects on the two human primary melanoma cell lines, named L3 and L6. These cell lines were stably transfected with a lentiviral vector. Previous results already demonstrated that NEU3 silenced melanoma cells reduced their migration potential and their growth in soft agar medium. Based on these data, my PhD work was focused towards the molecular features and signaling pathways alterations induced by NEU3 silencing in primary melanoma cell lines. Whole genome microarray analysis revealed the presence of differentially expressed genes above all associated with migration, motility, and control of cell death (G0 biological processes enrichment analysis). Some of these genes were validated by Real Time PCR: MAL, SEMA 3B, SEMA 3C and SEMA 5A. NEU3 silenced clones of both cell lines, 3C, 6A and 6B underwent to the upregulation of MAL, SEMA 3B and 3C. In contrast, SEMA 5A was downregulated. Different expression of markers related to epithelial mesenchymal transitions (N and E cadherin, MITF, vimentin, claudin 1, zo 1) was also revealed in NEU3 silenced clones. This proved the acquisition of a different molecular phenotype after NEU3 silencing that could explain the less motile properties. Moreover, we analyzed the activation of signaling pathways involved in these processes and we found a less activation of PI3K/AKT/PRAS40 axis and p38 kinases. Significantly, both these signaling pathways are involved in melanoma migration and differentiation control. All these results suggest that NEU3 upregulation could enhance melanoma malignancy by altering specific signaling pathways that are involved in cell motility and cell differentiation and thus NEU3 could be a novel target for treating melanoma.

Sialidases are glycohydrolytic enzymes that remove sialic acid residues from gangliosides and sialoglycoproteins. In mammals there are four isoenzymes, Neu1, Neu2, Neu3, Neu4, which differ in their subcellular localization and substrate specificity. Neu3 in particular is a peripheral membrane protein localized on the extracellular leaflet of cellular plasma membrane and it is present also in the early and recycling endosome (1). Neu3 shows an high specificity toward gangliosides, moreover it is able to modify the ganglioside composition of cell plasma membrane of adjacent cells (2). Modulating ganglioside content, Neu3 appears to be involved in important cellular processes such as proliferation, differentiation and transmembrane signalling. At the moment, cellular dynamics of Neu3 are still little known, in particular the rate of Neu3 protein attended on plasma membrane and in endosome compartment, like as the possibility of Neu3 recruitment on plasma membrane in response to EGF or FBS stimuli. To investigate these aspects, we have employed a model of MmNeu3 overexpression in HeLa cells using an inducible mammalian expression system (Tet-Off Gene Expression System). In this approach a tetracycline- controlled transactivator (tTA) activates transcription of sialidase in absence of doxycycline (dox); when dox is added to the culture medium MmNeu3 gene promoter is turned off. Experimental Procedures 1. HeLa cells were cultured in Dulbecco’s Modified Eagle Medium with high glucose supplemented with 10% (v/v) fetal bovine serum (FBS), and 4mM glutamine. To obtain a cell line with a stable inducible expression of murine sialidase Neu3, cells were subjectd to a double consecutive transfection. At first HeLa cells were transfected with regulator plasmid pTet-Off, encoding the tetracycline-controlled transactivator (tTA) doxycycline-dependent, and resistant clones were selected in presence of G418. Then, HeLa tTA cells were stable transfected with response plasmid which contained Neu3 gene under control of the tetracycline-response element (TRE). Resistant clones were selected in presence of puromycin. To follow MmNEU3 expression the enzyme has an HA (hemagglutinin) epitope tag and is expressed as GFP fusion protein. Mock cells (HeLa tTA2 pac) were transfected with response plasmid carrying only puromycin resistance. HeLa tTA2 pac were cultured in Dulbecco’s Modified Eagle Medium with high glucose supplemented with 10% (v/v) fetal bovine serum (FBS), 4mM glutamine, 0.5 µg/ml puromycin and 0.25 mg/ml G418. HeLa tTA2 MmNeu3-HA-GFP were cultured in the same medium but with the addition of 1 ng/ml Doxycycline (dox) to keep down the expression of MmNeu3. MmNEU3 gene promoter was turned on removing dox from culture medium. 2. The enzymatic activity of Neu3 was determined using 4-MU-Neu5Ac as substrate. Assays were performed in triplicate with 0.1 mM 4-MU-Neu5Ac, 30 µg of total protein of HeLa tTA2 pac and HeLa tTA2 MmNeu3-HA-GFP cells treated or not with dox (1 ng/ml), in the presence of 12.5 mM sodium-citrate/phosphate buffer pH 3.8 for 30 min at 37°C. 3. To analyze sphingolipid pattern HeLa tTA2 pac and HeLa tTA2 MmNeu3-HA-GFP cells treated or not with dox were labelled with [3-3H] sphingosine (2.5 × 10-9M) with 2 h pulse followed by a 48 h chase. Following total lipid extraction and partitioning, gangliosides and neutral sphingolipids were separated by HPTLC and analysed by radiochromatoscanning. 4. Western Blot analyses were performed on HeLa tTA2 pac and HeLa tTA2 MmNeu3-HA-GFP cells. Primary antibodies were used as follows: anti-EGFR (Cell Signalling), anti-phospho-EGFR (Tyr 1148) (Calbiochem), anti-HA (Sigma), anti-caveolin 1 (Santa Cruz Biotechnology), anti-Transferrin Receptor (TfR, Invitrogen), anti-AKT 1/2/3 (Santa Cruz Biotechnology). 5. HeLa tTA2 MmNeu3-HA-GFP cells at confluence were stained overnight in serum-free medium and then stimulated for 10 minutes with 100 ng/ml of epidermal growth factor (EGF) (Sigma), or for 2-4 hours with 10% (v/v) FBS, in presence or absence of dox in culture medium at difference time (6, 14, 24 h). 6. After or during dox removal, HeLa tTA2 MmNeu3-HA-GFP cells were treated with 0.1 µg/ml Brefeldin A (BFA) at different time (18, 24, 26 h). Then, cells were harvested by scraping and subjected to ultracentrifugation on OptiPrep density gradient. 7. Lipid rafts of HeLa tTA2 MmNeu3-HA-GFP cell lysates were separated by a discontinued OptiPrep density gradient. Briefly cell lysates were adjusted to a final density of 40% of OptiPrep Density Gradient Medium (Sigma), placed in a centrifuge tube and overload with a discontinuous OptiPrep density gradient from 30% to 5%. Samples were then centrifuged at 170000 ×g for 4 h, after which eight fractions of 1.5 ml (except for the first fraction that was of 1.2 ml) were collected from the top to the bottom of the gradient. Aliquots of the OptiPrep gradient fractions were analysed for their protein concentration, sialidase content and activity and subjected to Western-blot analysis. Results and Discussion First, we have characterized the cellular model, in particular we have assessed the time course of expression and enzymatic activity of Neu3 after promoter turning off/on. Furthermore, we have analyzed sphingolipid pattern and cell signalling modification during variation of Neu3 expression. When promoter was turning on by removal of dox, sphingolipid pattern analysis showed a significative decrease of GM3 (-60%) and GD1a (-75%) compared to mock cells, whereas we observed an increase of GM1 and lactosylceramide. To analyze MmNeu3 localization and cellular dynamics we have employed a discontinuous OptiPrep density gradient. The fractions were tested for Neu3 sialidase activity, and for HA, caveolin-1, TfR proteins by immunoblot analysis. At the beginning of expression (6h) the enzyme was only associated with no-DRM regions of cell membranes, whereas after 24h, when Neu3 expression was at the 50 % of maximum level, about 50 % of MmNeu3 protein resided in DRM, and 50 % in no-DRM. When MmNeu3 promoter was turned on after FBS withdrawal, sialidase distribution was modified, also 100 ng/ml EGF stimulation for 10 minutes of HeLa N3 cells determined an increased of MmNeu3 protein in DRM regions, consequently sialidase activity and protein rate increased in DRM region after EGF stimulus. BFA action determines the disassembly of the Golgi apparatus, in this way it blocks the transport of many proteins from Golgi to plasma membrane. BFA treatment during 24 h MmNeu3 expression changed sialidase distribution in DRM regions. 1. Zanchetti G. et al., Biochem. J. 2007, 408, 211-219. 2. Papini N. et al., J. Biol. Chem. 2004, 279, 16989-16995.

THE ROLE OF SIALIDASE NEU3 IN THE CARDIAC RESPONSE TO ISCHEMIA AND REPERFUSION INJURY Maria Elena Canalia,b, Marco Piccolia, Andrea Ghiroldia, Federica Cirilloa, and Luigi Anastasiaa,b a Laboratorio delle cellule staminali e ingegneria tissutale, IRCCS Policlinico San Donato, piazza Malan 2, 20097 San Donato Milanese, Milano, Italia; email: maria.canali@unimi.it b Dipartimento di Scienze Biomediche per la Salute, Università degli Studi di Milano, via Luigi Mangiagalli 31, 20133, Milano, Italia; L'infarto miocardico acuto (AMI) è una delle cause più comuni di morte in tutto il mondo. Le strategie di riperfusione sono le procedure salvavita più utilizzate per il trattamento dell'AMI, ma inducono anche l’insorgenza del cosiddetto danno da ischemia e riperfusione (IRI), con conseguente sviluppo di insufficienza cardiaca. Molti sforzi sono stati fatti per chiarire i meccanismi molecolari coinvolti nell'IRI e, in questo contesto, l'attivazione delle chinasi pro-sopravvivenza, nonché il fattore inducibile dell'ipossia (HIF-1α), sono stati riconosciuti come regolatori chiave della risposta cellulare all'IRI. Al riguardo, abbiamo recentemente identificato un nuovo meccanismo di attivazione dei HIF-1α mediato dalla sialidasi NEU3, che è stato in grado di aumentare la resistenza delle cellule muscolari allo stress ipossico. Pertanto, lo scopo di questo studio è stato quello di valutare se NEU3 potesse avere un ruolo anche nel contrastare il danno da ischemia e riperfusione. A tal fine, NEU3 è stato sovraespresso nei cardiomioblasti di ratto H9C2, i quali sono stati trasfettati con un plasmide contenete la sequenza di NEU3 per sovraesprimere l'enzima. Sorprendentemente, le cellule NEU3 overesprimenti hanno mostrato un tasso di proliferazione e sopravvivenza, così come un tasso di attivazione di HIF-1α e delle chinasi pro-sopravvivenza Akt ed Erk significativamente maggiore rispetto ai controlli a seguito dell'IRI. È interessante notare che il trattamento con gli inibitori di Akt ed Erk, nonché con gli inibitori di NEU3 (DANA e LR332) ha completamente annullato gli effetti benefici mediati dall'enzima, supportando il possibile coinvolgimento della sialidasi nel contrastare l'IRI attraverso l'attivazione delle chinasi pro-sopravvivenza. Inoltre, abbiamo studiato anche il possibile coinvolgimento di NEU3 nella regolazione del processo di fibrosi cardiaca, una risposta fisiologica alla lesione del tessuto cardiaco, caratterizzata dalla deposizione di proteine della matrice extracellulare da parte di miofibroblasti attivati. I nostri studi hanno dimostrato che la sovraespressione della sialidasi NEU3 è sufficiente per ridurre il transdifferenziamento dei fibroblasti a miofibroblasti attraverso la diminuzione del contenuto cellulare di GM3.
THE ROLE OF SIALIDASE NEU3 IN THE CARDIAC RESPONSE TO ISCHEMIA AND REPERFUSION INJURY Maria Elena Canalia,b, Marco Piccolia, Andrea Ghiroldia, Federica Cirilloa, and Luigi Anastasiaa,b a Laboratory of Stem Cells for Tissue Engineering, IRCCS Policlinico San Donato, piazza Malan 2, 20097 San Donato Milanese, Milan, Italy; email: maria.canali@unimi.it b Department of Biomedical Sciences for Health, University of Milan, via Luigi Mangiagalli 31, 20133, Milan, Italy; Acute myocardial infarction (AMI) is one of the most common causes of death worldwide. Reperfusion strategies are the most used life-saving procedures for AMI treatment but they also induce ischemia/reperfusion injury (IRI), ultimately resulting in development of heart failure. Many efforts have been made to clarify the molecular mechanisms involved in IRI. In this context, the activation of pro-survival kinases, as well as the hypoxia inducible factor (HIF-1α), have been recognized as key steps in the cellular response to IRI. Along this line, we recently identified a novel mechanism of HIF-1α activation mediated by sialidase NEU3, which ultimately increased muscular cells resistance to hypoxic stress. Thus, aim of this study was to assess whether NEU3 could play a role in reducing IRI. To this purpose, NEU3 was overexpressed in H9C2 rat cardiomyocytes and were transfected with NEU3 plasmid to overexpress the enzyme. Remarkably, NEU3 overexpressing cells showed a significantly increased proliferation rate and survival, as well as the activation of HIF-1α and pro-survival kinases Akt and Erk after IRI, as compared to controls. Interestingly, treatment with Akt and Erk inhibitors, as well as with NEU3 inhibitors (DANA and LR332) reverted the beneficial effects mediated by the enzyme, supporting the possible involvement of NEU3 in counteracting IRI through the activation of pro-survival kinases. Moreover, we investigated also the possible involvement of NEU3 in regulating the process of cardiac fibrosis, a physiological response to cardiac tissue injury, characterized by the deposition of extracellular matrix proteins by activated myofibroblasts. Interestingly, we demonstrated that the overexpression of the sialidase NEU3 is sufficient to reduce the fibroblasts-myofibroblasts conversion by reducing the cellular content of GM3.

Sialidases are glycohydrolytic enzymes that remove sialic acid residues from gangliosides, sialoglycoproteins and oligosaccharides. The most recently identified member of the human sialidase family is Neu4 and is found in two forms, NEU4 long (Neu4L) and NEU4 short (Neu4S), differing in the presence of a 12-amino-acid sequence at the N-terminus (1-2). Overall, Neu4 is scarcely expressed in adult tissues, in contrast to fetal tissues, where its biological role is currently unclear. We evidenced that the more aggressive neuroblastoma cell lines, a tumor derived from the neural crest, characteristically show a high expression of Neu4L. Here, we demonstrated that this feature is connected to the distorted control of proliferation and differentiation in neuroblastoma. In order to understand Neu4L role in neuroblastoma, we stably over-expressed Neu4L in human neuroblastoma cells, SK-N-BE. The first important consequence was a marked acceleration of proliferation rate assessed by increase of 3[H]thymidine incorporation (+ 36%), by cell count for up to 4 days (+36%), by MTT test for up 4 days (+26) and by soft agar assay. Moreover, Neu4L over-expressing cells were impaired to differentiate under FBS depletion culture, as we assessed following the formation and the length of neurites, MTT assay and the content of src, as neuronal marker. These phenotypical modifications were associated to a marker activation of Wnt/-Catenin pathway, a well-known regulator of proliferation and self-renewal in many cell types. We assessed an increased content of intracellular, not phosphorylated -Catenin (+30%) in Neu4L over-expressing SK-N-BE cells. In parallel, the main inhibitor of Wnt pathway, GSK3, decreased its expression as mRNA (-30%), while c-myc, cyclin D2 and axin2, which have been identified as target genes of -Catenin (3), underwent a significant increase, as mRNA content, in Neu4L over-expressing SK-N-BE cells. In order to further confirm the activation of the Wnt canonical pathway, we transiently transfected the TOP-Flash luciferase vector into mock and Neu4L over-expressing cells, as gene reporter assay responsive to -Catenin. We detected an increased activation of luciferase in Neu4L over-expressing cell (+52%), confirming our previous results. In order to confirm the greater ability of control cells to differentiate, we studied the neuronal marker , v-src. During the process of differentiation, we observed that Neu4L over-expressing cells had a lower content of this protein in contrast with control cells. Moreover, we identified the precise localization of Neu4L in SK-N-BE cells. We observed that Neu4L mainly localized in mitochondria (40%) and in endoplasmatic reticulum (60%). The direct substrates of Neu4L, in SK-N-BE cells, seem to be soluble glycoproteins in the range of 60 kDa; on the base of literature data, it could be hypothesized that one of these glycoproteins could belong to the Wnt family or to inhibitor-glycoproteins interacting with Wnt signaling. Therefore, we provided the evidence that Neu4L promotes the activation of Wnt/-Catenin signaling in neuroblastoma, inducing proliferation and inhibiting differentiation at the same time, enhancing self-renewal of malignant neuroblasts. 1 Monti E et al. (2004) Genomics 83: 445–53 2 Yamaguchi K et al. (2005) Biochem J 390: 85–93 3 Clevers H et al. (2006) Cell 127; 469-480.

A neuraminidase-1 (Neu1) participa da regulação do catabolismo de sialoglicoconjugados nos lisossomos. A deficiência congênita da Neu1 é a base da sialidose, doença neurossomática grave associada a deformidades osteoesqueléticas, hipotonia e fraqueza muscular. Camundongos com deficiência de Neu1 (Neu1-/-) desenvolvem uma forma atípica de degeneração muscular caracterizada por expansão da matriz extracelular (MEC), invasão das fibras musculares por fibroblastos, fragmentação do citoplasma, formação vacuolar e atrofia muscular. Apesar de a degeneração muscular estar bem caracterizada nestes animais, a miogênese ainda não havia sido estudada. O objetivo desta pesquisa foi avaliar o envolvimento da Neu1 no processo de regeneração muscular, após aplicação de cardiotoxina (CTX) em camundongos Neu1-/-, em comparação com controles normais. A CTX foi administrada no músculo tibial anterior direito e os animais foram eutanasiados por deslocamento cervical 1, 3, 5, 7, 10, 14, 21 e 28 dias após a lesão. Os músculos foram analisados através de histologia; medição da área transversa das fibras musculares centronucleadas; verificação do potencial proliferativo celular por quantificação de marcação de BrdU; imunoistoquímica para inflamação, fibras regenerativas e fibrose; e expressão gênica e proteica de fatores de transcrição musculares. Os dados foram comparados estatisticamente e as variações significativas devem apresentar p <= 0,05. Nos animais com deficiência de Neu1, o processo inflamatório (especialmente a reação macrofágica) e o potencial proliferativo estavam aumentados nas fases iniciais, acompanhados da hiperexpressão de Pax7. Observamos atraso na maturação muscular caracterizado por maior expressão de miosina embrionária em estágios mais tardios da regeneração. Os genes MyoD e MyoG estavam com expressão aumentada no período de 5 a 10 dia após a lesão, embora a expressão destas proteínas estivesse reduzida. Ao final da regeneração, houve maior deposição de reticulina na MEC, indicando processo fibrótico. A Neu1 parece atuar em todos os estágios da regeneração muscular, desde a fase aguda da lesão através do controle da proliferação celular, até a maturação muscular e estágios finais em que regularia a deposição de componentes da MEC
Neuraminidase-1 (Neu1) participates in sialoglycoconjugates catabolism in lysosomes. Congenital Neu1 deficiency is the basis of sialidosis, a severe neurosomatic disorder associated with osteoskeletal deformities, hypotonia and muscle weakness. Mice with Neu1 deficiency (Neu1-/-) develop an atypical form of muscle degeneration characterized by abnormal fibroblast proliferation and expanded extracellular matrix (ECM), invasion of muscle fibers by fibroblast, cytosolic fragmentation, vacuolar formation and muscle atrophy. Despite muscle degeneration is well characterized in these animals, myogenesis has not been studied so far. The aim of this study was to evaluate the involvement of Neu1 in muscle regeneration process after cardiotoxin (CTX) injection in Neu1-/- mice and normal controls. CTX was applied in the right tibialis anterior muscle, and the animals were euthanized by cervical dislocation 1, 3, 5, 7, 10, 14, 21 and 28 days after injury. The muscles were analyzed through histology; cross-sectional area of regenerative muscle fibers; quantification of BrdU labeling; immunohistochemistry labelling for inflammation, regenerative fibers, and fibrosis; and gene and protein expression of muscle transcription factors. The data were compared and variances considered statistically significant in case p <= 0.05. In animals with Neu1 deficiency, both inflammatory process (mainly macrophagic response) and proliferative potential were increased in the initial stages, accompanied by overexpression of Pax7. We observed delay in muscle maturation characterized by higher expression of embryonic myosin later in muscle regeneration. MyoD and MyoG genes were overexpressed from 5 to 10 days after injury, though the expression of these proteins was reduced. At the end of muscle regeneration, reticulin deposition in ECM was increased, indicating fibrotic process. Neu1 seems to participate in all stages of muscle regeneration, since acute injury phase through the control of cell proliferation, towards muscle maturation, and at the final stages when it would regulate the deposition of ECM components