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© The Rockefeller University Press, 0021-9525/1997//1229 $5.00
The Journal of Cell Biology, Volume 137, Number 6, , 1997 1229-1241

Article

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



Caroline Whitehouse*, Joy Burchell*, Stephen Gschmeissner{ddagger}, Inka Brockhausen§, Kenneth O. Lloyd||, and Joyce Taylor-Papadimitriou*

* Epithelial Cell Biology Laboratory, {ddagger} Electron Microscopy Unit, Imperial Cancer Research Fund, Lincoln's Inn Fields, London WC2A 3PX, UK; § Department of Biochemistry, Hospital for Sick Children and University of Toronto, Toronto M5G 1X8, Canada; and || Sloan-Kettering Institute, New York 10021

The {alpha}2,3 sialyltransferase, {alpha}2,3 SAT (O), catalyzes the transfer of sialic acid to Galβ1,3 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 β1,6 N-acetyl-d-glucosamine transferase (β1,6 GlcNAc T) that leads to chain extension. Increased levels of the {alpha}2,3 SAT (O) and decreased levels of the core-2 β1,6 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 {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) 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 β1,6 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 β1,6 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.

1. Abbreviations used in this paper: GalNAc, N-acetyl-d-galactosamine; GlcNAc, N-acetyl-d-glucosamine; GalT, β1-4 galactosyltransferase; HPAEC, high performance anion exchange chromatography; PNA, peanut agglutinin.

Please address all correspondence to Joyce Taylor-Papadimitriou, Imperial Cancer Research Fund, P.O. Box 123, Lincoln's Inn Fields, London WC2A 3PX, United Kingdom.


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