GM3 alpha 2,8-sialyltransferase (GD3 synthase): Protein characterization and sub-Golgi location in CHO-K1 cells

GD3 synthase (Sial-T2) is a key enzyme of ganglioside synthesis that, in co ncert with GM2 synthase (GalNAc-T), regulates the ratio of a- and b-pathway gangliosides. In this work, we study the sub-Golgi location of an epitope- tagged version of chicken Sial-T2 transfected to CHO-K1 cells. The expresse d protein was enzymatically active both in vitro and in vivo and showed a m olecular mass of similar to 47 or similar to 95 kDa on sodium dodecyl sulfa te-polyacrylamide get electrophoresis in the presence or absence of, respec tively, beta-mercaptoethanol. The 95-kDa form of Sial-T2 was also detected if the protein was retained in the endoplasmic reticulum (ER) due to impair ed glycosylation, indicating that it was formed in the ER. Confocal immunof luorescence microscopy showed Sial-T2 localized to the Golgi complex and, w ithin the organelle, partially co-localizing with the mannose-6-phosphate r eceptor, a marker of the trans-Golgi network (TGN). In cells treated with b refeldin A, a major fraction of Sial-T2 redistributed to the ER, even under controlled expression to control for mislocalization due to protein overlo ading. In experiments of incorporation of sugars into endogenous accepters of Golgi membranes in vitro, GD3 molecules formed by incubation with CMP-Ne uAc were converted to GD2 upon incubation with UDP-GalNAc. These results in dicate that Sial-T2 localizes mainly to the proximal Golgi, although a frac tion is located in the TGN functionally coupled to GalNAc-T. Consistent wit h this, most of the enzyme was in an endoglycosidase H (Endo-H)-sensitive, neuraminidase (NANase)-insensitive form. A minor secreted form lacking simi lar to 40 amino acids was Endo-H-resistant and NANase-sensitive, indicating that the cells were able to process N-glycans to Endo-H-resistant forms. T aken together, the results of these biochemical and immunocytochemical expe riments indicate that in CHO-K1 cells, most Sial-T2 localizes in the proxim al Golgi and that a functional fraction is also present in the TGN.