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Kinetic and spatial interrelationships between ganglioside glycosyltransferases and O-acetyltransferase(s) in human melanoma cells.
The melanoma-associated disialogangliosides 9(7)-O-acetyl-GD3 and 9(7)-O-acetyl-GD2 have been structurally well characterized. However, the compartmentalization and sequence of action of the biosynthetic activities responsible for synthesizing these molecules remain obscure. Here, we have studied the spatial and temporal interrelationships among the activities responsible for the later stages of ganglioside biosynthesis and those for O-acetylation in cultured human melanoma cells. First, brefeldin A treatment was used to separate biosynthetic steps into compartments distal or proximal to the transport block imposed by the drug. In keeping with prior reports, GM2/GD2 synthase was consistently rendered inaccessible to its acceptors GM3 and GD3. In contrast, the effect on GD3 biosynthesis was cell line-specific. Synthesis of GD3 was nearly abrogated in two lines, while it accumulated in a third line. This indicates that the spatial organization of ganglioside processing activities can vary even between similar cell lines. However, in all cell lines studied, the ratio of 9(7)-O-acetyl-GD3 to GD3 was not changed by brefeldin A, indicating that the majority of ganglioside O-acetyltransferase activity is co-localized with GD3 biosynthetic activity in the same Golgi subcompartment(s). As an alternative approach, Golgi-enriched fractions from melanoma cells were incubated with radiolabeled and nonlabeled nucleotide sugars or acetyl-CoA. In these preparations, biosynthesis is dependent upon the co-localization of appropriate sugar nucleotide transporters, glycosyltransferases, and acceptors that are endogenously present within intact topologically correct compartments. Incubations with CMP-Neu5Ac and acetyl-CoA corroborated the results with brefeldin A, co-localizing ganglioside O-acetyltransferase activity in compartments where GD3 biosynthesis takes place. Analyses with CMP-Neu5Ac and UDP-GalNAc showed that GD2 and GD3 synthesis occur in partially overlapping compartments. Labeling with acetyl-CoA and UDP-GalNAc indicated that although labeled acetate can be transferred from acetyl-CoA directly to GD2, ganglioside O-acetyltransferase activity does not substantially overlap with the biosynthetic compartment(s) for GD2. Instead, O-acetyl-GD3 appears to be co-localized with the compartment of GD2 biosynthesis and serves as an acceptor for GD2 synthase. Thus, both 9-O-acetyl-GD3 and GD2 can be precursors of 9-O-acetyl-GD2, but apparently in distinct compartments.
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