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

catalyzes the transfer of sialic acid to Gal ␤ 1,3 N -acetyld -galactosamine (GalNAc) (core-1) in mucin type O-glycosylation, and thus terminates chain extension. A Core-2 branch can also be formed from core-1 by the core-2 ␤ 1,6 N -acetyld -glucosamine transferase ( ␤ 1,6 GlcNAc T) that leads to chain extension. Increased levels of the ␣ 2,3 SAT (O) and decreased levels of the core-2 ␤ 1,6 GlcNAc T are seen in breast cancer cells and correlate with differences in the structure of the O-glycans synthesized (Brockhausen et al., 1995; Lloyd et al., 1996) . Since in mucin type O-glycosylation sugars are added individually and sequentially in the Golgi apparatus, the position of the transferases, as well as their activity, can determine the final structure of the O-glycans synthesized. A cDNA coding for the human ␣ 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) and malignant (T47D) breast epithelial cell lines. Transfectants were analyzed for expression of the enzyme at the level of message and protein, as well as for enzymic activity. In T47D cells, which do not express core-2 ␤ 1,6 GlcNAc T, the increased activity of the sialyltransferase correlated with increased sialylation of core-1 O-glycans on the epithelial mucin MUC1. Furthermore, in MTSV1-7 cells, which do express core-2 ␤ 1,6 GlcNAc T, an increase in sialylated core-1 structures is accompanied by a reduction in the ratio of GlcNAc: GalNAc in the O-glycans attached to MUC1, implying a decrease in branching. Using quantitative immunoelectron microscopy, the sialyltransferase was mapped to the medial-and trans -Golgi cisternae, with some being present in the TGN. The data represent the first fine mapping of a sialyltransferase specifically active in O-glycosylation and demonstrate that the structure of O-glycans synthesized by a cell can be manipulated by transfecting with recombinant glycosyltransferases. I n eukaryotic cells, proteins are synthesized in the ER from where they are transported to different locations, either within the cell or to the plasma membrane. Along the exocytic pathway, proteins undergo various modifications including proteolytic cleavage, glycosylation, and sulfation. Within this pathway there are two main types of glycosylation, N-linked and mucin-type O-linked. The addition of N-linked glycans is initiated in the ER by the addition to asparagine of an oligosaccharide chain via an intermediate lipid carrier. This chain is then modified by trimming and the addition of sugars. In contrast, mucin-type O-linked glycosylation is thought to be initiated in the cis -Golgi cisternae where the first sugar, N -acetyld -galactosamine (GalNAc), 1 is added to the hydroxyl groups of serine and threonine (Roth et al., 1994; Clausen and Bennett, 1996) . The oligosaccharide chains are then built up by the sequential addition of individual sugars, each reaction being catalyzed by a specific enzyme or enzymes (Brockhausen, 1996) . Thus, the final structure of the O-glycans is strongly influenced, not only by the level of activity of an enzyme but also by its position within the Golgi apparatus.