A Transfected Sialyltransferase That Is Elevated in Breast Cancer and Localizes to the medial/trans-Golgi Apparatus Inhibits the Development of core-2–based O-Glycans

Whitehouse, Caroline; Burchell, Joy; Gschmeissner, Stephen; Brockhausen, Inka; Lloyd, Kenneth O.; Taylor-Papadimitriou, Joyce

Since in O-glycosylation sugars are added individually and sequentially, the position of an enzyme in the Golgi apparatus relative to other enzymes active in the synthesis of O-glycans will influence the final structure. The conclusion from the studies reported here is that the ␣2,3 sialyltransferase is located in the medial and trans cisternae of the Golgi apparatus with some of this enzyme also being present in the TGN. To localize the protein by immuno-EM, the cDNA was tagged with an immunoreactive epitope (a sequence from the myc gene), and cells were transfected with the tagged gene. The inclusion of the myc epitope (10 amino acids) at the extreme COOH terminus (which in this case is in the lumen of the Golgi compartment) has previously been shown to have no effect on the localization of glycosyltransferases (Rabouille et al., 1995; Nilsson et al., 1993; Munro and Pelham, 1986) . In our studies, the tag clearly had no obvious effect on the folding of the protein as the ␣2,3 SAT (O) was highly active, not only in cell extracts (Table I) but also in the intact transfected cells, as shown by the increased sialylation of the MUC1 glycoprotein (Figs. 5, 6, and 7). Thus, anomalous localization because of misfolding is unlikely. In addition, the sequences that have been shown to be involved in the localization of glycosyltransferases were identical in the endogenous and transfected genes, making it likely that the coded proteins were directed to the same compartment of the Golgi apparatus. The location of the sialyltransferase to within more than one compartment of the Golgi apparatus supports the view that the Golgi cisternae are characterized by their different mixtures of these enzymes, not by discrete types (Nilsson et al., 1993; Rabouille et al., 1995) .