Abstract
The alpha2,3 sialyltransferase, alpha2,3 SAT (O), catalyzes the transfer of sialic acid to Galbeta1,3 N-acetyl-D-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 beta1,6 N-acetyl-d-glucosamine transferase (beta1,6 GlcNAc T) that leads to chain extension. Increased levels of the alpha2,3 SAT (O) and decreased levels of the core-2 beta1,6 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 alpha2,3 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 beta1,6 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 beta1,6 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.
Highlights
In 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
The construct was transfected into the breast carcinoma cell line, T47D, and two stable cell lines were selected in the presence of gentimycin
The ␣2,3 sialyltransferase studied here is relevant to the study of breast cancer since the activity of the enzyme is elevated in breast cancer cell lines (Brockhausen et al, 1995)
Summary
Proteins are synthesized in the ER from where they are transported to different locations, either within the cell or to the plasma membrane. The key glycosyltransferases involved in the changes seen in breast malignancies are the ␣2,3 sialyltransferase (EC 2.4.99.4), which adds sialic acid to Gal1,3 GalNAc (core-1), and the 1,6 N-acetyl-d-glucosamine (GlcNAc) transferase (EC 2.4.1.102), which forms core-2 from core-1 and is crucial for chain branching (Fig. 1) (Kuhns et al, 1993). These enzymes use the same substrate, and their effect is to terminate or initiate chain branching leading to extension, respectively. 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
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