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

Abstract

NEU3 sialidase has been shown to be a key player in many physio-and pathological processes, including cell differentiation, cellular response to hypoxic stress, and carcinogenesis. The enzyme, peculiarly localized on the outer leaflet of the plasma membrane, has been shown to be able to remove sialic acid residues from the gangliosides present on adjacent cells, thus creating cell to cell interactions. Nonetheless, herein we report that the enzyme localization is dynamically regulated between the plasma membrane and the endosomes, where a substantial amount of NEU3 is stored with low enzymatic activity. However, under opportune stimuli, NEU3 is shifted from the endosomes to the plasma membrane, where it greatly increases the sialidase activity. Finally, we found that NEU3 possesses also the ability to interact with specific proteins, many of which are different in each cell compartment. They were identified by mass spectrometry, and some selected ones were also confirmed by cross-immunoprecipitation with the enzyme, supporting NEU3 involvement in the cell stress response, protein folding, and intracellular trafficking.

Human Sialidase Neu3 is S-Acylated and Behaves Like an Integral Membrane Protein

“…suggested that Neu3 behaves as a peripheral membrane protein associated with the outer leaflet of the plasma membrane 16 . However, this topological distribution of Neu3 is not fully consistent with the results recently obtained by other laboratories and ours, mainly concerning Neu3 sialidase protein interactors 11 , 12 , 17 , post-translational modifications (this work) and regulation of clathrin-mediated endocytosis 9 . In the present study, we provide a comprehensive analysis of Neu3 membrane topology and propose an alternative view of its association with membranes.…”

“…In fact, it has been recently demonstrated that Neu3 can directly desialylate EGFR glycoprotein and in this way, modulates its activation 15 . In addition, although Neu3 is generally thought to be mainly localized at the plasma membrane, other laboratories and ours have reported that Neu3 is also present in membranes from endosomal compartments 9 , 16 , where it interacts with key proteins involved in protein folding and intracellular trafficking 17 .…”

“…It has been recently described that Neu3 possesses the ability to interact with specific proteins at the plasma membrane and endosomes, with Neu3 interactors being identified by mass spectrometry and some of these being also confirmed by cross-immunoprecipitation with the enzyme 17 . Among the proteins identified that interact with Neu3 are flotillin-1, flotillin-2, clathrin heavy chain-1 (CLH1), sortin nexin-9 (SNX9) and Sec23A/Sec23B.…”

NEU3 sialidase role in activating HIF-1α in response to chronic hypoxia in cyanotic congenital heart patients

“…Moreover, the expression of SP1/SP3, transcription factors, which are known to be activated under hypoxia and to bind the NEU3 promoter region, both showed an increase of about 1.3 folds in cyanotic patients as compared to controls (Figs 3B and 3C). As the active form of the sialidase NEU3 has been recently shown to be present at the level of plasma membranes [20], the separation of cytosol and membranes protein fractions was performed on auricle samples from cyanotic and acyanotic patients. Analysis of NEU3 expression by Western Blot revealed a 1.7 fold increase of NEU3 protein level in the membranes of cyanotic samples, as compared to controls, whereas the cytosol fractions did not show any significant differences, as expected (Figs 3D and 3E).…”

G3BP1-linked mRNA partitioning supports selective protein synthesis in response to oxidative stress

“…G3BP1 is a pleiotropic protein with diverse biological functions ( 7 , 8 ). Apart from its role as a major SG nucleating protein ( 9 ), G3BP1 localizes to mitochondria ( 10–12 ), endosomes ( 13 ) and nucleus ( 14 ), where it has largely unknown functions. G3BP1 contains low-complexity (LC), or intrinsically disordered (ID), regions necessary for dimerization ( 15 , 16 ), underlying its ability to function as a nucleating factor for SG assembly.…”