Influence of N-glycosylation and N-glycan trimming on the activity and intracellular traffic of GD3 synthase.

GD3 synthase (ST8Sia I) transfers a sialic acid in alpha-2-->8 linkage to the sialic acid moiety of GM3 to form the ganglioside GD3. The cDNAs of GD3 synthases predict several putative N-glycosylation sites. In this work we have examined the occupancy of these sites in a chicken GD3 synthase and how they affect its activity and intracellular traffic. COS-7 cells were transfected with an influenza virus hemagglutinin (HA) epitope-tagged form of GD3 synthase (GD3 synthase-HA). Cells acquired GD3 synthase activity, cell surface GD3 immunoexpression, and GD3 synthase-HA immunoreactivity in the Golgi complex. In Western blots, a main GD3 synthase-HA band of 47 kDa was detected, which was radioactive upon metabolic labeling with [2-3H] mannose. Tunicamycin prevented the incorporation of [2-3H]mannose into GD3 synthase-HA, blocked the enzyme activity, and promoted a reduction of the enzyme molecular mass of 6-7 kDa. Timed deglycosylation with N-glycosidase F showed that all three potential N-glycosylation sites of GD3 synthase-HA were glycosylated. The deglycosylated forms were enzymatically more unstable than the native form. Tunicamycin treatment of cells led to retention of GD3 synthase-HA immunoreactivity in the endoplasmic reticulum (ER). Castanospermine and deoxynojirimycin, inhibitors of the ER-processing enzymes alpha-glucosidases I and II, also prevented the exit from the ER but did not essentially affect the enzyme specific activity. 1-Deoxymannojirimycin and swainsonine, inhibitors of mannosidases, did not affect either the enzyme activity or the Golgi localization. Results indicate that (a) N-glycosylation is necessary for GD3 synthase to attain and to maintain a catalytically active folding, and for exiting the ER; and (b) N-glycan trimming in the ER, while not required for enzyme activity, is necessary for proper trafficking of GD3 synthase to the Golgi complex.