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

MARTINA J. A.; DANIOTTI J.L.; MACCIONI H.J.F.

AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC

Lugar: Bethesda, Maryland; Año: 1998 vol. 273 p. 3725 - 3725

D3 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-