Characterization of human sialidase Neu3 membrane association

RODRIGUEZ WALKER M.; DANIOTTI J.L.

Córdoba

Congreso; 52 Reunión anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB); 2016

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB)

Plasma membrane (PM)-bound sialidase Neu3 is a key enzyme in the catabolism of glycoconjugates. However, there is little information concerning its membrane topology and the mechanism of association with biomembranes. Hence, the aim of this work was to further contribute on our knowledge on this matter. 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 recognized by antibodies only after cell permeabilization. Protease digestion assays on intact cells resulted in the appearance of a protected fragment of Neu3. Together, these results indicated that Neu3 exposes its C-terminus toward the cytosol while other fraction is exposed to the extracellular milieu. 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.