Thèses sur le sujet « Sialidase NEU4 »

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

The hypoxic condition determines several functional consequences that ultimately lead to cellular death and irreversible damage to cardiac myocytes. Under hypoxic condition, cells activate several protective pathways; among them, HIF-1α plays a key role in controlling cellular response to hypoxia at molecular level. However, HIF-1α regulatory mechanisms are extremely complex. On these premises, the present work was based on the hypothesis that NEU3 sialidase, a glycolytic enzyme ubiquitously expressed over the plasmatic membrane, can have a regulatory activity on HIF-1α expression in hypoxic/ischemic cardiac myocytes. The experiments performed in this in-vitro model allowed us to draw the following conclusions: 1) Endogenous NEU3 sialidase expression and activity are up-regulated in murine skeletal muscle cells (C2C12) upon oxygen starvation, leading to a signaling cascade resulting in the activation of HIF-1α. 2) Moreover, induced overexpression of NEU3 significantly increases HIF-1α expression and cell resistance to hypoxic stress, whereas NEU3 silencing causes the opposite effects and renders myoblasts more susceptible to apoptosis. 3) The hypoxia-driven activation of NEU3 sialidase can activate the EGFR prosurvival signaling pathway by controlling the content of ganglioside GM3. Furthermore, we demonstrated that NEU3 overexpression causes a reduction of ganglioside GM3, which is known to block EGFR autophosphorylation. Then resulted were extended from skeletal muscle to cardiac myocytes, particularly aiming to ascertain the role of NEU3 in activating the human cardiomyocyte response to hypoxia. Particularly, we evaluated if NEU3 activation occurred in human cardiomyocytes using two different models: 1) A model of acute cardiac ischemia achieved during aortic cross-clamp time and extracorporeal circulation in adult patient submitted to cardiac surgical procedures. 2) A model of chronic hypoxia in neonates and young patients operated for cyanogen congenital cardiac defects. In the acute model of cardiac ischemia, we harvested a sample of right atrial appendage just before and after aortic cross-clamping, during routine adult cardiac surgery procedure. However, no significant activation of NEU3 and HIF-1α was evident in cardiac sample harvested before and after aortic cross-clamping. In our opinion there are several possible explanations for the lack of NEU3 and HIF-1α increased expression in the cardiac surgery model. First, it is possible that in the in-vivo setting the mean aortic cross-clamp time was too short (mean time = 79 minutes) to elucidate the same response that we observed in the in vitro model, where the cells were incubated under hypoxic conditions for at least 12 hours. Secondly, and most important in our opinion, the technique of myocardial protection, especially cardioplegic arrest and hypothermia, by protecting the myocardium from the ischemic injury could have limited NEU3 and HIF-1α expression in our samples. To overcome these limitation, in the final part of my PhD program we evaluated HIF-1α and NEU3 expression in a human in-vivo model of chronic cardiac hypoxia, studying patients affected by cyanotic cardiac defects submitted to surgical correction. In this model of chronic hypoxia, we observed a significant increase in NEU3 expression and activity in cyanotic patients. Furthermore, a significant increase of EGFR was observed, supporting the hypothesis that this signaling pathway is upregulated by the sialidase NEU3. Indeed we observed an increase in expression of genes downstream of EGFR, both related to cellular proliferation (ERK and p38) and to apoptosis resistance (AKT and p70S6K). Finally we observed a significant activation of HIF-1α and of its downstream genes. Another important aspect of cellular adaptation to hypoxia is the metabolic switch between oxidative and glycolytic metabolism, the so-called “Pasteur effect”. In the present study we found that the glycolytic enzymes Glucose transporter Glut1, the Aldolase and the GAPDH were significantly enhanced in the cyanotic group which in turn demonstrates that the myocardium of patients affected by cyanogen cardiac defects is metabolic adapted to chronic hypoxia. In conclusion, the results of this PhD project support the hypothesis of a physiological role of NEU3 in mediating cellular response to hypoxic stress. It is interesting to underline that NEU3 activation is mediated by ganglioside GM3 on cellular membrane. Indeed, an increase in NEU3 level determines a reduction of GM3, which is a well know inhibitor of EGF receptor. On these premises, to mimic the effects of NEU3 activation, it could be possible to inhibit GM3 synthesis, in example by the selective inhibition of the sialyltransferase involved in the last passage of its synthesis. In this direction, our laboratory is performing some experiments with small chemical molecules, designed for blocking selectively the GM3 synthesis with the aim of activating the endogenous response to hypoxic stress.

Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

Background: Lysosomal storage diseases (LSD) often manifest with cherry red macular spots. Diagnosis is based on clinical features and specific biochemical and enzymatic patterns. In uncertain cases, genetic testing with next generation sequencing can establish a diagnosis, especially in milder or atypical phenotypes. We report on the diagnostic work-up in a boy with sialidosis type I, presenting initially with marked cherry red macular spots but non-specific urinary oligosaccharide patterns and unusually mild excretion of bound sialic acid. Methods: Biochemical, enzymatic and genetic tests were performed in the patient. The clinical and electrophysiological data was reviewed and a genotype-phenotype analysis was performed. In addition a systematic literature review was carried out. Case report and results: Cherry red macular spotswere first noted at 6 years of age after routine screening myopia. Physical examination, psychometric testing, laboratory investigations aswell as cerebralMRIwere unremarkable at 9 years of age. So far no clinical myoclonic seizures occurred, but EEG displays generalized epileptic discharges and visual evoked potentials are prolonged bilaterally. Urine thin layer chromatography showed an oligosaccharide pattern compatible with different LSD including sialidosis, galactosialidosis, GM1 gangliosidosis or mucopolysaccharidosis type IV B. Urinary bound sialic acid excretion was mildly elevated in spontaneous and 24 h urine samples. In cultured fibroblasts, α-sialidase activity was markedly decreased to b1%; however, bound and free sialic acid were within normal range. Diagnosis was eventually established by multigene panel next generation sequencing of genes associated to LSD, identifying two novel, compound heterozygous variants in NEU1 gene (c.699CNA, p.S233R in exon 4 and c.803ANG; p.Y268C in Exon 5 in NEU1 transcriptNM_000434.3), leading to amino acid changes predicted to impair protein function. Discussion: Sialidosis should be suspected in patients with cherry red macular spots, even with non-significant urinary sialic acid excretion. Multigene panel next generation sequencing can establish a definite diagnosis, allowing for counseling of the patient and family.

Thymoquinone (TQ), a volatile oil component of black seed oil (derived from Nigella sativa), has been shown to have various biological effects including disease treatment and prevention. TQ is believed to share similar properties to the benzoquinones already in use as therapeutic drugs. Based on previous reports on the anti-inflammatory properties of black seed oil and TQ, it was originally hypothesized that TQ would inhibit lipopolysaccharide (LPS)-induced cellular sialidase activity in an anti-inflammatory manner. Sialidase activity was tested on live mouse bone marrow derived primary macrophage cells, BMC-2 macrophage cells, human embryonic kidney epithelial (HEK293) cells and human fibroblast cells using an assay that measures the cleavage of the sialidase specific fluorescent substrate 2’-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (4-MUNANA). The cleavage of 4-MUNANA causes the release of free 4-methylumbelliferone, which fluoresces at 450nm (blue) after excitation at 365nm. Unexpectedly, TQ induced sialidase activation in all three cell lines and wild type primary macrophage cells. TQ was unable to induce sialidase activity in primary macrophage cells isolated from Neu4 knockout mice suggesting that the TQ activates Neu4 sialidase enzyme. TQ-induced sialidase activity in these live cells was found to occur through intermediate GPCR-associated guanine nucleotide Gαi subunit and matrix metalloproteinase 9 (MMP9) by using specific inhibitors. In addition, TQ was found to induce sialidase activity in Toll-like receptor-deficient HEK293 cells. These latter data suggested that TQ may be activating GPCR Gαi and MMP9 signaling associated with Neu4 sialidase independent of TLRs. It is proposed that TQ-induced sialidase activity may activate Toll-like receptors in macrophage cells and the subsequent production of pro-inflammatory cytokines in the absence of LPS. Immunocytochemical staining of BMC-2 cells shows that TQ induced NFκB activation. NFκB activation was confirmed with electrophoretic mobility shift assay (EMSA) and western immunoblotting techniques. Cytokine arrays were used to test the pro-inflammatory cytokine response induced in mice by 5 hour treatment of TQ, compared to LPS. Mice treated with TQ exhibited an increase in IL-1β, IL-6 and TNF-α production, similar to LPS treatment. Taken together, the findings in these studies suggest that TQ is a novel ligand for Neu4 sialidase activation which consequently induces pro-inflammatory cytokine responses.
Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-04-21 17:38:10.413

The epidermal growth factor receptor (EGFR) exists as a single, highly glycosylated subunit receptor on the plasma membrane of a cell. Upon ligand binding to its extracellular domain, the EGFR dimerizes with an adjacent receptor. This results in activation of the EGFR’s intracellular tyrosine kinase domain, and consequently, autophosphorylation of specific tyrosine residues on the receptor’s cytoplasmic tail. Adaptor proteins bind to these phosphorylated tyrosine residues and transduce the message internally, initiating a multitude of signalling cascades which stimulate cell growth, division, and movement. Despite all that has been elucidated regarding the activation and signalling pathways of the EGFR, the parameters controlling dimerization and activation remain unknown. Recently, Neu1 sialidase, an enzyme which cleaves α-2,3-linked sialic acids from glycosylated substrates, has been implicated as a critical mediator of TrkA receptor activation. Upon activation, the sialidase desialylates the external receptor glycosylation, removing a physical barrier which was formerly hindering receptor dimerization, and thus, receptor activation. Based on the known sialylation of EGFR glycosylation, as well as the demonstrated importance of receptor glycosylation in EGFR activation, we hypothesized that the EGFR may be activated by a similar mechanism. Here, we report an identical membrane signalling paradigm initiated by epidermal growth factor (EGF) binding to EGFR to rapidly induce Neu1 sialidase activity in live NIH3T3-EGFR cells but not in live Neu1-deficient human fibroblast cells. Furthermore, we report that Neu1 sialidase activity is required for EGFR activation, supported by the finding that tyrosine phosphorylation is inhibited in EGF-stimulated NIH3T3-hEGFR cells which have been pretreated with both broad-range (oseltamivir phosphate) and specific (anti-Neu1 neutralizing antibody) sialidase inhibitors. MMP-9 plays a role in the initiation of Neu1 sialidase post-ligand binding, as pre-treatment of NIH3T3-hEGFR cells with specific MMP-9 inhibitor prior to EGF stimulation blocks membrane sialidase activity as well as tyrosine phosphorylation. Of critical importance to this schematic is the finding that both Neu1 and MMP-9 co-immunoprecipitate with EGFR on the plasma membrane of both naïve and EGF-stimulated NIH3T3-hEGFR cells. Together, these findings reveal a novel EGFR activation mechanism in which cross-talk between Neu1 and MMP-9 plays a vital role in EGF-induced receptor activation.
Thesis (Master, Microbiology & Immunology) -- Queen's University, 2011-06-26 12:04:40.486

Receptor glycosylation is critical in receptor-ligand interactions in immune cells, but the exact role of glycosylation in receptor activation upon ligand binding has not been elucidated. In neuronal cells, we have shown that when neurotrophic factors bind their respective Trk tyrosine kinase receptors, receptor activation and subsequent neurotrophin-mediated signaling is dependent upon the induction and activity of an endogenous sialidase enzyme. In this thesis, we report that toll-like receptor (TLR) activation upon ligand binding is similarly dependent on the induction of a cellular sialidase, which we have identified as Neu1 sialidase, which specifically targets and hydrolyses alpha-2,3-linked sialic acid residues on the receptor. Blocking Neu1 sialidase activity with specific inhibitor Tamiflu detrimentally impacts ligand-induced TLR4/MyD88 interaction, NFkappaB activation and TLR-mediated effector responses like nitric oxide and pro-inflammatory cytokine production. Diminished cytokine production is also seen in vivo in Neu1-deficient mice. We propose a mechanism for the induction of Neu1 sialidase, upon ligand binding to TLR, that involves the activation of heterotrimeric G-alpha protein-dependent G-protein coupled receptor (GPCR) signaling to activate a matrix metalloproteinase (MMP) enzyme, likely MMP-9. It is suggested that MMP-(9) targets the cell surface elastin receptor complex of Neu1/protective protein cathepsinA/elastin binding protein (EBP), which potentially catalytically activates Neu1. In addition, we report an association between Neu1 and TLR2, TLR3 and TLR4 on the plasma membrane that has not previously been described. The idea that the multiple functionality and diversity of TLRs and TLR-mediated signaling may be an immunologic paradigm capable of explaining all human disease is provocative but plausible. Certainly, the structural integrity of TLRs, their ligand interactions and activation are essential for immunological protection. Thus, understanding the molecular mechanism of Neu1 sialidase regulation of TLR activation will provide important opportunities for disease control through TLR manipulation. The future directions of this research will also open a new area of glycobiology research (the glycomics of innate immune responses) and will widen the scope for the development of novel therapeutic drugs to combat infections and inflammatory diseases.
Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2009-01-26 12:33:32.743

The molecular mechanism(s) by which Toll-like receptors become activated are not well understood. For the majority of TLR receptors, dimerization is a prerequisite to facilitate MyD88-TLR complex formation and subsequent cellular signaling to activate NF-κB. However, the parameters controlling interactions between the receptors and their ligands still remain poorly defined. Previous reports have identified that neuraminidase-1 (NEU1) is an important intermediate in the initial process of TLR ligand induced receptor activation and subsequent cell function. What we do not yet understand is how NEU1 is activated following TLR ligand binding. In this thesis, the findings disclose a receptor signaling paradigm involving a process of receptor ligand-induced GPCR-signaling via neuromedin-B (NMBR) Gα-proteins, matrix metalloproteinase-9 (MMP-9) activation, and the induction of Neu1 activation. Central to this process is that NEU1–MMP-9-NMBR complex is associated with TLR-4 receptors on the cell surface of naive primary macrophages and TLR-expressing cell lines. Ligand binding to the receptor initiate GPCR-signaling via GPCR Gα subunit proteins and MMP-9 activation to induce NEU1. Activated NEU1 targets and hydrolyzes sialyl α-2-3-linked to β-galactosyl residues at the ectodomain of TLRs, enabling the removal of steric hindrance to receptor association, activation of receptors and cellular signaling. Furthermore, a novel glycosylation model is uncovered for the activation of nucleic acid sensing intracellular TLR-7 and TLR-9 receptors. It discloses an identical signaling paradigm as described for the cell-surface TLRs. NEU1 and MMP9 cross-talk in alliance with neuromedin-B receptors tethered to TLR-7 and -9 receptors at the ectodomain is essential for ligand activation of the TLRs and pro-inflammatory responses. However, the mechanism(s) behind this GPCR and TLR cross-talk has not been fully defined. Here, GPCR agonists mediate GPCR-signaling via membrane Gα subunit proteins to induce NEU1 and MMP-9 cross-talk at the TLR ectodomain on the cell surface. This molecular organizational GPCR signaling platform is proposed to be an initial processing stage for GPCR agonist-induced transactivation of TLRs and subsequent cellular signaling. Collectively, these novel findings radically redefine the current dogma(s) governing the mechanism(s) of the interaction of TLRs and their ligands, which may provide important pioneering approaches to disease intervention strategies.
Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2013-04-30 12:23:42.429

The signaling pathways of tyrosine kinase Trk receptors and their downstream biological effects are well known, but the parameters controlling the interactions between the receptors and their natural ligands still remain to be defined. Recent published reports from our laboratory indicate that nerve growth factor (NGF)-induced TrkA receptor activation is dependent on a membrane cellular sialidase. This sialidase activity specifically targets and hydrolyzes sialyl α-2, 3-linked β-galactosyl residues resulting in the desialylation and activation of the receptor. These findings support a novel hypothesis that places mammalian sialidase(s) in a cycle of activation of these receptors by their natural ligand. Taken together, they also predict a prerequisite desialylation of Trk receptors caused by a sialidase on the cell surface enabling the removal of a steric hindrance to receptor dimerization. Until now, the sialidase associated with neurotrophin-treated live Trk-expressing cells has not been identified. The molecular mechanism(s) of sialidase activation by neurotrophin factors binding to their receptors also remains unknown. In this thesis, the novel role of Neu1 sialidase in the activation of ligand-induced TrkA and TrkB receptors has been identified. It has been reported for the first time that Neu1 is already in complex with naïve and ligand-induced Trk receptors. In addition, a membrane sialidase mechanism initiated by NGF binding to TrkA has been indentified. It suggests a potentiation of GPCR-signaling via membrane Gαi subunit proteins and matrix metalloproteinase-9 (MMP-9) activation to induce Neu1 sialidase activation in live TrkA- and TrkB-expressing cells and primary neurons. These results establish a unique mode of regulation of Trk receptors by their natural ligand and define a new function for Neu1 sialidase. Preliminary data indicate that members of the family of tyrosine kinase receptors like epidermal growth factor receptor (EGFR) and insulin receptor are also under the same regulatory control of Neu1 sialidase. Recent reports from the laboratory have indicated that ligand-induced activation of the highly glycosylated Toll-like receptors, TLR-2,-3 and -4 is also dependent on Neu1 sialidase on the cell surface. Taken all together, the findings in this thesis uncover a Neu1 and MMP-9 cross-talk on the cell surface which is critically essential for neurotrophin-induced Trk tyrosine kinase receptor activation and neuron function.
Thesis (Ph.D, Microbiology & Immunology) -- Queen's University, 2010-04-26 11:44:51.418

Tesis (Doctora en Ciencias Químicas) - - Universidad Nacional de Córdoba. Facultad de Ciencias Químicas, 2017
Resumen: La sialidasa asociada a membranas Neu3 está involucrada en el catabolismo de glicoconjugados, especialmente de gangliósidos, e interviene en numerosos procesos biológicos tales como diferenciación, proliferación, migración y adhesión celular, regulando principalmente vías de transducción de señales. Dado que el mecanismo de asociación de Neu3 con membranas biológicas no se conoce, uno de los objetivos de este trabajo fue proveer más información sobre ese aspecto. Se encontró que Neu3 localiza principalmente en membrana plasmática, y también en endomembranas de compartimientos endosomales. Utilizando diferentes metodologías se demostró que el extremo C-terminal de la proteína está expuesto hacia el lado citosólico, y que otra porción de Neu3 está expuesta hacia el espacio extracelular, sugiriendo que la sialidasa posee las características de una proteína transmembrana. Sin embargo, análisis in silico y modelado molecular predijeron que la sialidasa no contiene segmentos α-hélices transmembrana en su secuencia, y que comparte la estructura de β-propeller, típica de las sialidasas virales y bacterianas. En la búsqueda de modificaciones post-traduccionales de Neu3, se encontró que la misma está S-acilada, y dado que la S-acilación está restringida al lado citosólico de las membranas, este descubrimiento apoya la idea de que la sialidasa contiene un dominio expuesto al citosol. Aunque aún queda por determinar exactamente cómo esta enzima atraviesa la bicapa lipídica, la primera parte de este estudio provee una nueva visión sobre la topología de Neu3 y se discuten algunas posibles configuraciones. Por otro lado, se estudiaron aspectos funcionales de la enzima no descriptos con anterioridad, como ser su participación en eventos endocíticos. Se observó que células que sobreexpresan Neu3 endocitan menos transferrina (Tf), un típico ejemplo de molécula que ingresa a la célula por endocitosis mediada por clatrina (CME). Dicha disminución en la endocitosis de Tf se observó también en células con reducido nivel de expresión de glicoesfingolípidos, sugiriendo que el efecto observado es independiente de la acción de Neu3 sobre gangliósidos. La disminución en la internalización de Tf, como así también de otras moléculas que ingresan por CME, pudo ser explicada debido a una distribución subcelular alterada del adaptador de clatrina AP-2, el cual se observó formando agregados en células que sobreexpresan Neu3. Por el contrario, clatrina, el fosfoinosítido PIP2 y caveolina mostraron una distribución normal. En conjunto estos resultados sugieren un rol específico de Neu3 en CME.
Abstract: Membrane-bound sialidase Neu3 is involved in the catabolism of glycoconjugates, especially gangliosides, and plays crucial roles in numerous biological processes, such as differentiation, proliferation, migration and cell adhesion, regulating transmembrane signaling. Since the mechanism of its association with membranes is still not completely understood, the aim of this work was to provide further information regarding this aspect. Human Neu3 was found to be associated with the plasma membrane and endomembranes from endosomal compartments, and it was not released from the lipid bilayer under conditions that typically release peripheral membrane proteins. By different experimental approaches, it was demonstrated that its C-terminus is exposed to the cytosol while another portion of the protein is exposed to the extracellular space, suggesting that Neu3 possesses the features of a transmembrane protein. However, in silico analysis and homology modeling predicted that the sialidase does not contain any α-helical transmembrane segment and shares the same β-propeller fold typical of viral and bacterial sialidases. Additionally, it was found that Neu3 is S-acylated, and since this post-translational modification is restricted to the cytosolic side of membranes, this finding strongly supports the idea that Neu3 may contain a cytosolic-exposed domain. Although it remains to be determined exactly how this sialidase crosses the lipid bilayer, the first part of this study provides new insights about membrane association and topology of Neu3. On the other hand, some previously unknown functional aspects of the enzyme, such as its involvement in endocytic events, were studied in this work. The ectopic expression of Neu3 led to a drastic decrease in the internalization of transferrin (Tf), the archetypical cargo for clathrin-mediated endocytosis (CME). This reduction was still observed in cells with reduced expression of glycosphingolipids, suggesting that the observed effect is independent of Neu3 activity toward gangliosides. This decrease in the internalization of Tf and also of other molecules entering by CME could be explained by an altered subcellular distribution of the clathrin adaptor AP-2, which was observed to be aggregated in cells overexpressing Neu3. In contrast, clathrin, the phosphoinositide PIP2 and caveolin showed a normal distribution. Together these results suggest a specific and novel role of Neu3 in CME.
Fil: Rodríguez Walker, Macarena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas; Argentina
Fil: Daniotti, José Luis. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.
Fil: Genti de Raimondi, Susana Del Valle. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.
Fil: Genti de Raimondi, Susana del Valle. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.
Fil: Alvarez, María Elena. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Química Biológica; Argentina.
Fil: Alvarez, María Elena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Química Biológica de Córdoba; Argentina.
Fil: Touz, María Carolina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación Médica Mercedes y Martín Ferreyra; Argentina.
Fil: González-Baró, María del Rosario. Universidad Nacional de La Plata; Argentina.
Fil: González-Baró, María del Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner"; Argentina.