Membrane association and topology of human sialidase Neu3

RODRIGUEZ WALKER M.; DANIOTTI J.L.

Villa Gral. Belgrano, Córdoba, Argentina

Simposio; Second Argentinian Symposium of Glycobiology GlycoAR 2016; 2016

Local Organizing committee

Plasma membrane (PM)-bound sialidase Neu3 is a key enzyme in the catabolism of glycolipids. However, there is little information concerning its membrane topology and the mechanism of association with the PM. In this study, we performed a detailed biochemical and subcellular characterization of human Neu3. Ectopically expressed Neu3 was active towards its natural substrates GD3 and GD1a. Confocal microscopy analysis showed that Neu3 was mainly localized at the PM, but also in membranes from lysosomes and endosomes. 35% of the enzyme was accessible to cell surface biotinylation, indicating that a fraction of the protein is exposed to the outer leaflet of the PM. However, two different epitopes of Neu3 were accessible by antibodies only after cell permeabilization, indicating that the C-terminus is oriented toward the inner of the cell. Neu3 was not released from the lipid bilayer in presence of high salt concentrations, suggesting that its binding does not depend on electrostatic interactions. 50% of Neu3 had a hydrophobic behavior in Triton X-114 partitioning assay, suggesting a posttranslational modification by lipidation. In fact, we found that Neu3 is S-acylated, representing the first demonstration of a posttranslational modification on Neu3. Given the absence of a signal peptide or hydrophobic stretches in Neu3, we speculate that S-acylation could be operating in its targeting to PM and might have crucial roles for determining its subcellular localization and function.