Sialidase NEU3 Dynamically Associates to Different Membrane Domains Specifically Modifying Their Ganglioside Pattern and Triggering Akt Phosphorylation

Abstract

Lipid rafts are known to regulate several membrane functions such as signaling, trafficking and cellular adhesion. The local enrichment in sphingolipids and cholesterol together with the low protein content allows their separation by density gradient flotation after extraction with non-ionic detergent at low temperature. These structures are also referred to as detergent resistant membranes (DRM). Among sphingolipids, gangliosides play important roles in different biological events, including signal transduction and tumorigenesis. Sialidase NEU3 shows high enzymatic specificity toward gangliosides. Moreover, the enzyme is present both at the cell surface and in endosomal structures and cofractionates with caveolin. Although changes in the expression level of NEU3 have been correlated to different tumors, little is known about the precise distribution of the protein and its ability in modifying the ganglioside composition of DRM and non-DRM, thus regulating intracellular events. By means of inducible expression cell system we found that i) newly synthesized NEU3 is initially associated to non-DRM; ii) at steady state the protein is equally distributed between the two membrane subcompartments, i.e., DRM and non-DRM; iii) NEU3 is degraded via the proteasomal pathway; iv) the enzyme specifically modifies the ganglioside composition of the membrane areas where it resides; and v) NEU3 triggers phosphorylation of Akt, even in absence of exogenously administered EGF. Taken together our data demonstrate that NEU3 regulates the DRM ganglioside content and it can be considered as a modulator of Akt phosphorylation, further supporting the role of this enzyme in cancer and tumorigenesis.

Ganglioside GM3 as a gatekeeper of obesity‐associated insulin resistance: Evidence and mechanisms

“…Moreover, the ability of oleate alone to enhance insulin sensitivity in L6 myotubes concurred with increased NEU3 protein. Together, these findings support a positive role for NEU3 sialidase in facilitating insulin signal transduction, potentially through its reported ability to co‐localise with caveolin proteins in membrane microdomains[54,55].…”

NEU3 Sialidase Protein Interactors in the Plasma Membrane and in the Endosomes

“…However, it has been demonstrated that they are also present in the endosomal compartment, in the phagosomes, in the Golgi, and in the exosomes (33)(34)(35)(36). Previously, we demonstrated that NEU3 participates in a complex cell trafficking between the endosomes and the membrane, and, as it reaches the plasma membrane, it is initially associated with non-detergent-resistant membrane areas, but eventually it partially translocates to detergent-resistant membranes, reaching an even distribution (4). Therefore, the interaction with Flotillin1 could be instrumental for the mobilization of NEU3 in the cell membrane.…”

“…It is topologically associated with the outer leaflet of the plasma membrane, where its natural gangliosidic substrates are present (2). The localization of NEU3 on the plasma membrane has been recently defined to be dynamic, because the enzyme is distributed between detergent-resistant membranes and nondetergent-resistant membranes, in a cholesterol-dependent manner, suggesting its presence in supramolecular organized structures (2,4). Moreover, NEU3 could be found also in vesicular structures corresponding to the endosomes (2).…”

Overexpression of sialidase NEU3 increases the cellular radioresistance potential of U87MG glioblastoma cells

“…Units/ml Penicillin, 100 mg/ml Streptomycin and 10% (v/v) Fetal Bovine Serum (FBS) (GIBCO). Cells were stably transfected with pcDNA3.1-mNEU3eHA [13], while Mock cells were obtained by transfection with pcDNA3.1 empty vector. Transfectants were grown in complete medium supplemented with 0.5 mg/ml G418.…”

“…Up-regulation of NEU3 has been correlated to different human tumors such as colon, renal, prostate and ovarian cancers [12]. Overexpression of NEU3 in HeLa cells induces the phosphorylation of Akt even in the absence EGF [13] and a recent study has demonstrated that NEU3 can also directly act on EGFR glycan(s), resulting in the activation of the receptor [14]. In the last few years many scientific papers demonstrated the involvement of the enzyme in oncogenic transformation mediated by EGFR [10,15].…”