GM3 α2,8‐Sialyltransferase (GD3 Synthase)

GD3 synthase (Sial‐T2) is a key enzyme of gangliosidesynthesis that, in concert with GM2 synthase (GAlNAc‐T), regulates the ratioof a‐ and b‐pathway gangliosides. In this work, we study the sub‐Golgilocation of an epitope‐tagged version of chicken Sial‐T2 transfected to CHO‐K1cells. The expressed protein was enzymatically active both in vitro and invivo and showed a molecular mass of ∼47 or ∼95 kDa on sodium dodecylsulfate‐polyacrylamide gel electrophoresis in the presence or absence of,respectively, β‐mercaptoethanol. The 95‐kDa form of Sial‐T2 was alsodetected if the protein was retained in the endoplasmic reticulum (ER) due toimpaired glycosylation, indicating that it was formed in the ER. Confocalimmunofluorescence microscopy showed Sial‐T2 localized to the Golgi complexand, within the organelle, partially co‐localizing with themannose‐6‐phosphate receptor, a marker of the trans ‐Golgi network(TGN). In cells treated with brefeldin A, a major fraction of Sial‐T2redistributed to the ER, even under controlled expression to control formislocalization due to protein overloading. In experiments of incorporation ofsugars into endogenous acceptors of Golgi membranes in vitro, GD3 moleculesformed by incubation with CMP‐NeuAc were converted to GD2 upon incubation withUDP‐GalNAc. These results indicate that Sial‐T2 localizes mainly to theproximal Golgi, although a fraction is located in the TGN functionally coupledto GalNAc‐T. Consistent with this, most of the enzyme was in anendoglycosidase H (Endo‐H)‐sensitive, neuraminidase (NANase)‐insensitive form.A minor secreted form lacking ∼40 amino acids was Endo‐H‐resistant andNANase‐sensitive, indicating that the cells were able to process N ‐glycans to Endo‐H‐resistant forms. Taken together, the results of these biochemical and immunocytochemical experiments indicate that in CHO‐K1 cells, most Sial‐T2 localizes in the proximal Golgi and that a functional fraction is also present in the TGN.