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

The position of the ␣ 2,3 sialyltransferase in the Golgi apparatus will determine to some degree whether it can compete directly with core-2 ␤ 1,6 GlcNAc T for the Gal ␤ 1,3 GalNAc substrate. It is therefore important to precisely map its distribution within the Golgi apparatus. cDNAs coding for the ␣ 2,3 sialyltransferase have been isolated from porcine submaxillary glands (Gillespie et al., 1992) , murine brain (Lee et al., 1993) , and more recently from human placenta (Chang et al., 1995) . It is therefore now possible to directly locate the transferase by transfecting a cDNA tagged with a sequence encoding an immunoreactive epitope (Nilsson et al., 1993) . We have tagged the cDNA coding for the human ␣ 2,3 SAT (O) with an epitope from the myc gene, and the construct has been used to transduce or transfect two mammary epithelial cell lines, both of which express the MUC1 mucin. The T47D cell line was derived from a metastatic breast carcinoma (Keydar et al., 1979) , does not express core-2 ␤ 1,6 GlcNAc T, even at the level of mRNA (Brockhausen et al., 1995) , and produces MUC1 carrying short O-glycans (Lloyd et al., 1996) . The MTSV1-7 cell line was derived from normal human milk epithelial cells (Bartek et al., 1991) and shows many characteristics of normal cells (Shearer et al., 1992) , including the ability to add core-2-based O-glycans to the MUC1 mucin (Lloyd et al., 1996) . In the MTSV1-7 cell line, the transfected ␣ 2,3 SAT (O) has been localized to the medial-and trans -Golgi cisternae, with some enzyme being detected in the TGN. We have also demonstrated that transfection of a cell with the ␣ 2,3 SAT (O) results in increased sialylation of the surface protein, the MUC1 mucin, thus allowing the manipulation of O-linked glycosylation. In the MTSV1-7 transfectants, the increase in sialylated core-1 is accompanied by a decrease in the GlcNAc content of the O-glycans attached to MUC1, suggesting a reduction in the synthesis of core-2-based structures.