N-glycosylation of sialyltransferase ST3Gal-II is critical for intracellular trafficking and activity

RUGGIERO F.M.; VILCAES A.; IGLESIAS-BARTOLOMÉ R.; DANIOTTI J.L.

Villa Gral. Belgrano, Córdoba

Simposio; Second Argentinian Symposium of Glycobiology GlycoAR 2016; 2016

Local organizing comitee

The sialyltransferase ST3Gal-II is a type II integral membrane protein with a short cytoplasmic domain, a transmembrane region and a luminally oriented C-terminal domain. It is the main sialyltransferase responsible for the in vivo synthesis of gangliosides GD1a and GT1b. In this study, we demonstrated that N-glycosylation of ST3Gal-II at Asn211 is necessary for proper endoplasmic reticulum (ER) exiting, Golgi Complex (GC) localization and in vitro activity. A chimeric protein containing the N-terminal, non-glycosylated, region of ST3Gal-II fused to a fluorescent tag (ST3Gal-II-FT) was able to drive the reporter out of the ER and to retain it at the GC. An N-glycosylation mutant of ST3Gal-II, which is usually retained at the ER, was rescued by ST3Gal-II-FT and localized at the GC, probably by the formation of ST3Gal-II homocomplexes at the ER. To this regards, a dimer was detected in absence of 2-mercaptoethanol, which disappeared under reduction conditions with the concomitant increment of the monomer fraction. Furthermore, the formation of the dimer persisted when the protein is retained at the ER by pharmacological treatments or point mutation at Asn211. Finally, when the Cys residue on position 3 of ST3Gal-II-FT is substituted by Ala, the dimer is no longer detected. This result reveals a novel contribution of the Cys present in the cytoplasmic domain of ST3Gal-II on its dimeric homoassociation via disulfide bridges.