Characterization of human sialidase Neu3 membrane association

DANIOTTI, JOSÉ LUIS; RODRIGUEZ WALKER, MACARENA

Córdoba

Congreso; LII Reunión anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2016

SAIB

Plasma membrane (PM)-bound sialidase Neu3 is a key enzyme in the catabolism of glycoconjugates. Given the absence of a signal peptide or transmembrane stretches in Neu3, there is little information about its mechanism of PM association. Hence, the aim of this work was to provide more details on Neu3 membrane anchoring mechanism. Microscopy analysis showed that Neu3 was mainly localized at the PM and endosomes. 35% of the enzyme was accessible to cell surface biotinylation and two different epitopes were accessible by antibodies only after cell permeabilization. Protease digestion assays resulted in the appearance of a protected fragment of Neu3. Together, these results indicated that the C-terminus of Neu3 is exposed to the cytosol while other fraction is exposed to the extracellular space. By different approaches, we showed that Neu3 interacts with itself via disulfide bridges and with other proteins, most of them facing the cytosol. Binding of Neu3 to the lipid bilayer was independent on electrostatic interactions and 55% of Neu3 had a hydrophobic behavior in TX-114 assay, suggesting a posttranslational modification by lipidation. In fact, we found that Neu3 is S-acylated, representing the first demonstration of a posttranslational modification. These results provide a comprehensive analysis of Neu3 topology and allow us to propose a model of its association with the PM.