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.
|Titolo:||CELLULAR DYNAMICS OF SIALIDASE NEU3 IN A MODEL OF STABLE INDUCIBLE OVEREXPRESSION IN HELA CELLS|
|Supervisori e coordinatori interni:||BONOMI, FRANCESCO|
|Data di pubblicazione:||9-dic-2010|
|Parole Chiave:||sialidase NEU3 ; lipid rafts|
|Settore Scientifico Disciplinare:||Settore BIO/10 - Biochimica|
|Citazione:||CELLULAR DYNAMICS OF SIALIDASE NEU3 IN A MODEL OF STABLE INDUCIBLE OVEREXPRESSION IN HELA CELLS ; Tutor: Bruno Venerando ; Coordinatore: Francesco Bonomi. - Milano : Università degli studi di Milano. Universita' degli Studi di Milano, 2010 Dec 09. ((23. ciclo, Anno Accademico 2010.|
|Appare nelle tipologie:||Tesi di dottorato|