Critical role of evolutionarily conserved glycosylation at Asn²¹¹ in the intracellular trafficking and activity of sialyltransferase ST3Gal-II

ST3Gal-II, a type II transmembrane protein, is the main mammalian sialyltransferase responsible for GD1a and GT1b ganglioside biosynthesis in brain. It contains two putative N-glycosylation sites (Asn⁹² and Asn²¹¹). Whereas Asn⁹² is only conserved in mammalian species, Asn²¹¹ is highly conserved in mammals, birds and fish. The present study explores the occupancy and relevance for intracellular trafficking and enzyme activity of these potential N-glycosylations in human ST3Gal-II. We found that ST3Gal-II distributes along the Golgi complex, mainly in proximal compartments. By pharmacological, biochemical and site-directed mutagenesis, we observed that ST3Gal-II is mostly N-glycosylated at Asn²¹¹ and that this co-translational modification is critical for its exit from the endoplasmic reticulum and proper Golgi localization. The individual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn²¹¹ seems to negatively influence the activity of the enzyme using both glycolipid and glycoprotein as acceptor substrates, the single N-glycan mutant at Asn⁹² had only a moderate effect. Lastly, we demonstrated that the N-terminal ST3Gal-II domain containing the cytosolic, transmembrane and stem region (amino acids 1–51) is able to drive a protein reporter out of the endoplasmic reticulum and to retain it in the Golgi complex. This suggests that the C-terminal domain of ST3Gal-II depends on N-glycosylation to attain an optimum conformation for proper exit from the endoplasmic reticulum, but it does not represent an absolute requirement for Golgi complex retention of the enzyme.