CELLULAR DYNAMICS OF SIALIDASE NEU3 IN A MODEL OF STABLE INDUCIBLE OVEREXPRESSION IN HELA CELLS
Tesi di Dottorato
Data di Pubblicazione:
2010
Citazione:
CELLULAR DYNAMICS OF SIALIDASE NEU3 IN A MODEL OF STABLE INDUCIBLE OVEREXPRESSION IN HELA CELLS / L. Dileo ; Tutor: Bruno Venerando ; Coordinatore: Francesco Bonomi. Universita' degli Studi di Milano, 2010 Dec 09. 23. ciclo, Anno Accademico 2010.
Abstract:
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
Tipologia IRIS:
Tesi di dottorato
Keywords:
sialidase NEU3 ; lipid rafts
Elenco autori:
L. Dileo
Link alla scheda completa: