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 absolute values for ␣2,3 sialyltransferase activity in the MTSV1-7 and T47D series of cell lines are not directly comparable as the assays on the two series were performed at different times. and 2 (Fig. 5, a and b) . We have previously shown that peak 1 contains neutral oligosaccharides consisting mainly of Gal␤1-3 GalNAc-ol with a small amount of GalNAc-ol, whereas peak 2 contains monosialylated Gal␤1-3 Gal-NAc-ol (Lloyd et al., 1996) . Strikingly, the ␣2,3 SAT (O) transfected T47D clones show an increase in the proportion of the monosialylated peak (Fig. 5, c and d, peak 2 ; Table II) , and a corresponding reduction of the neutral peak (Fig. 5, c and d, peak 1 ; Table II ). These data indicate that activity of the ␣2,3 sialyltransferase in the transfected cell lines has resulted in the increased sialylation of the Gal␤1,3GalNAc substrate. Further confirmation of the increase in sialylated core-1 comes from FACS ® analysis using PNA lectin, which binds to the unsialylated disaccharide (Gillespie et al., 1993) . Fig. 6 shows that, while the parental T47D (a) and T47D neo transfectant (b) bind the lectin, in the transfected clones PNA binding is totally absent (c and d). However, after removal of sialic acid with neuraminidase treatment, both the transfectants and the untransfected cells show equivalent binding of peanut lectin. As for the T47D series, the binding of PNA seen in the parental MTSV1-7 cell line and the puromycin transfectant (Fig. 6 , e and f) was not evident in the MTSV1-7 transfectants (Fig. 6, g and h) , but it could again be induced by neuraminidase treatment. Thus, the sialyltransferase was also active in the MTSV1-7 transfectants in increasing the level of sialylated core-1. To test whether the ␣2,3 SAT (O) enzyme was affecting side chains that contain GlcNAc (Fig. 1) , the radiolabeled MUC1 precipitates from the MTSV1-7 puro and MTSV1-7 3ST clones were analyzed for hexosamine content. Fig. 7 a shows the elution profiles of the puromycin clone, and Fig. 7 b shows the corresponding profile of one of the sialyltransferase transfectants. It can be clearly seen that, in the ␣2,3 SAT (O) transfectant, there is a marked increase in the proportion of counts eluting in the first peak, which corresponds to galactosamine, compared to the second peak, which corresponds to glucosamine. This reduction in GlcNAc content clearly demonstrates a loss of chain branching and/or extension of the side chains on the MUC1 expressed by the MTSV1-7 transfectants.