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

FERNANDO M. RUGGIERO; ALDO A. VILCAES; RAMIRO IGLESIAS-BARTOLOME; JOSE L. DANIOTTI

BIOCHEMICAL JOURNAL

PORTLAND PRESS LTD

Lugar: Londres; Año: 2015 vol. 469 p. 83 - 95

0264-6021

ST3Gal-II, a type II transmembrane protein, is the mainmammalian sialyltransferase responsible for GD1a and GT1bganglioside biosynthesis in brain. It contains two putative Nglycosylationsites (Asn92 and Asn211). Whereas Asn92 is onlyconserved in mammalian species, Asn211 is highly conservedin mammals, birds and fish. The present study exploresthe occupancy and relevance for intracellular trafficking andenzyme activity of these potential N-glycosylations in humanST3Gal-II. We found that ST3Gal-II distributes along the Golgicomplex, mainly in proximal compartments. By pharmacological,biochemical and site-directed mutagenesis, we observed thatST3Gal-II is mostly N-glycosylated at Asn211 and that thisco-translational modification is critical for its exit from theendoplasmic reticulum and proper Golgi localization. Theindividual N-glycosylation sites had different effects on ST3Gal-II enzymatic activity. Whereas the N-glycan at position Asn211seems to negatively influence the activity of the enzyme usingboth glycolipid and glycoprotein as acceptor substrates, thesingle N-glycan mutant at Asn92 had only a moderate effect.Lastly, we demonstrated that the N-terminal ST3Gal-II domaincontaining the cytosolic, transmembrane and stem region (aminoacids 1?51) is able to drive a protein reporter out of theendoplasmic reticulum and to retain it in the Golgi complex.This suggests that the C-terminal domain of ST3Gal-II dependson N-glycosylation to attain an optimum conformation for properexit from the endoplasmic reticulum, but it does not representan absolute requirement for Golgi complex retention of theenzyme.