Characterization of human sialidase NEU4: role of the proline-rich region in signal transduction

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.