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

Western blotting with the 9E10 antibody showed a specific band in the region of 46-48 kD, which corresponds to the myc-tagged ␣2,3 SAT (O). Fig. 3 a shows the blots for the highest expressing MTSV1-7 and T47D transfectants, indicating higher expression of the enzyme in the MTSV1-7 clone. Expression of the protein was also confirmed by FACS ® analysis of permeabilized cells. Fig. 3 b shows the 3STMYC clone of T47D, the MTSV1-7 3STMYC, and the T47D neo and MTSV1-7 puro cell lines. As seen on the Western blots, the expression of ␣2,3 SAT (O) by the T47D clones was considerably weaker than by the MTSV1-7 clones. However, the positive shift in 9E10 staining observed in the T47D transfectants was consistent and significant as the expression of keratin 18 completely overlaps in the T47D neo and T47D transfectants (Fig. 3 b) . Functionality of the expressed enzyme was demonstrated by showing an increase in the specific activity of the enzyme in the transfected clones. Table I shows the activity of the enzyme in cell extracts of the clones in comparison with cells transfected or transduced with vector alone. In the T47D clones, ␣2,3 sialyltransferase activity was elevated about sevenfold compared with T47D neo, whereas the MTSV1-7 clones had increased activity ranging from 19-30-fold in comparison with MTSV1-7puro or wild-type cells. The levels of activity of the enzyme correlated with the levels of expression, (Figs. 2 and 3 ; Table I ). (The levels of enzyme activity of the T47D series of cell lines and the MTSV1-7 series are not comparable since they were done at different times and do not reflect the increased level of activity present in the T47D cells [Brockhausen et al., 1995] ). The overexpression of ␣2,3 sialyltransferase had no obvious effect on the morphology of the Golgi apparatus as shown by electron micrographs from wild-type MTSV1-7 cells (Fig. 4 a) and one of the MTSV1-7 3ST clones (Fig. 4 b) .