A TRANSFECTED SIALYLTRANSFERASE THAT IS ELEVATED IN BREAST-CANCER ANDLOCALIZES TO THE MEDIAL TRANS-GOLGI APPARATUS INHIBITS THE DEVELOPMENT OF CORE-2-BASED O-GLYCANS/

The alpha 2,3 sialyltransferase, alpha 2,3 SAT (O), catalyzes the tran sfer of sialic acid to Gal beta 1,3 N-acetyl-D-galactosamine (GalNAc) (core-1) in mucin type O-glycosylation, and thus terminates chain exte nsion. A Core-2 branch can also be formed from core-1 by the core-2 be ta 1,6 N-acetyl-D-glucosamine transferase (beta 1,6 GlcNAc T) that lea ds to chain extension. Increased levels of the alpha 2,3 SAT (O) and d ecreased levels of the core-2 beta 1,6 GlcNAc T are seen in breast can cer cells and correlate with differences in the structure of the O-gly cans synthesized (Brockhausen et al., 1995; Lloyd et al., 1996). Since in mucin type O-glycosylation sugars are added individually and seque ntially in the Golgi apparatus, the position of the transferases, as w ell as their activity, can determine the final structure of the O-glyc ans synthesized. A cDNA coding for the human alpha 2,3 SAT (O) tagged with an immunoreactive epitope from the myc gene has been used to map the position of the glycosyltransferase in nontumorigenic (MTSV1-7) an d malignant (T47D) breast epithelial cell lines. Transfectants were an alyzed for expression of the enzyme at the level of message and protei n, as well as for enzymic activity. In T47D cells, which do not expres s core-2 beta 1,6 GlcNAc T, the increased activity of the sialyltransf erase correlated with increased sialylation of core-1 O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do expres s core-2 beta 1,6 GlcNAc T, an increase in sialylated core-1 structure s is accompanied by a reduction in the ratio of GlcNAc: GalNAc in the O-glycans attached to MUC1, implying a decrease in branching. Using qu antitative immuno-electron microscopy, the sialyltransferase was mappe d to the medial- and trans-Golgi cisternae, with some being present in the TGN. The data represent the first fine mapping of a sialyltransfe rase specifically active in O-glycosylation and demonstrate that the s tructure of O-glycans synthesized by a cell can be manipulated by tran sfecting with recombinant glycosyltransferases.