New Insights into Molecular Organization of Human Neuraminidase-1: Transmembrane Topology and Dimerization Ability.

Pascal Maurice,Manuel Dauchez,Beatrice Romier,Stéphanie Baud,Sébastien Blaise,Eduard V Bocharov,Maxime Ghirardi,Laurent Duca,Laurent Debelle,Laurent Martiny,Laetitia Gorisse,Olga V Bocharova,Andrey S Kuznetsov,Charlotte Kawecki,Olivier Bocquet,Roman G Efremov

doi:10.1038/srep38363

Abstract

Neuraminidase 1 (NEU1) is a lysosomal sialidase catalyzing the removal of terminal sialic acids from sialyloconjugates. A plasma membrane-bound NEU1 modulating a plethora of receptors by desialylation, has been consistently documented from the last ten years. Despite a growing interest of the scientific community to NEU1, its membrane organization is not understood and current structural and biochemical data cannot account for such membrane localization. By combining molecular biology and biochemical analyses with structural biophysics and computational approaches, we identified here two regions in human NEU1 - segments 139–159 (TM1) and 316–333 (TM2) - as potential transmembrane (TM) domains. In membrane mimicking environments, the corresponding peptides form stable α-helices and TM2 is suited for self-association. This was confirmed with full-size NEU1 by co-immunoprecipitations from membrane preparations and split-ubiquitin yeast two hybrids. The TM2 region was shown to be critical for dimerization since introduction of point mutations within TM2 leads to disruption of NEU1 dimerization and decrease of sialidase activity in membrane. In conclusion, these results bring new insights in the molecular organization of membrane-bound NEU1 and demonstrate, for the first time, the presence of two potential TM domains that may anchor NEU1 in the membrane, control its dimerization and sialidase activity.

Highlights

Storage disorders is the progressive accumulation of sialylated glycoproteins, glycopeptides and oligosaccharides in lysosomes of many cell types, as well as excretion of sialyloligosaccharides in body fluids
Prior permeabilization of COS-7 cells increased the percentage of HA-positive cells (Fig. 2H). These results demonstrate that monomers and presumably dimers of Neuraminidase 1 (NEU1) are present at the membrane, in the absence of any detectable association with PPCA, and suggest that both extremities of NEU1 are oriented towards the cytosol
NEU1 is localized in lysosomes, where its catalytic activity requires its close association with PPCA and β-galactosidase

Summary

Introduction

Storage disorders is the progressive accumulation of sialylated glycoproteins, glycopeptides and oligosaccharides in lysosomes of many cell types, as well as excretion of sialyloligosaccharides in body fluids. A plasma membrane-bound NEU1, controlled by the phosphorylation of its C-terminus, has been reported[8]. NEU1 has been shown to be required for signal transduction and elastogenesis through the elastin receptor complex[9,10,11] and to be involved in the modulation of insulin receptor signaling[12,13], regulation of integrin beta 414, TLR415, Trk A16, PDGF-BB and IGF receptors[17], EGF and MUC1 receptors[18] and more recently CD3119. NEU1 emerges as a catabolic enzyme and as a key actor involved in cell signaling regulation[20]. How NEU1 translocates to the plasma membrane in association with PPCA, or not, is not known. Given the critical roles played by membrane-bound NEU1, the present study was designed to characterize the organization and assembly of human NEU1 in membrane using a combination of biology-based, biophysical and computational approaches

Methods

Results

Conclusion