Jl. Daniotti et al., GM3 alpha 2,8-sialyltransferase (GD3 synthase): Protein characterization and sub-Golgi location in CHO-K1 cells, J NEUROCHEM, 74(4), 2000, pp. 1711-1720
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.