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

Abstract

The α2,3 sialyltransferase, α2,3 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 α2,3 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 α2,3 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.

The Relative Activities of the C2GnT1 and ST3Gal-I Glycosyltransferases Determine O-Glycan Structure and Expression of a Tumor-associated Epitope on MUC1

“…Although the expression of the C2GnT1 enzyme has been found to be decreased or absent in most breast cancer cell lines (24,14), reasonable levels of the mRNA coding for this enzyme can be found in a high proportion of primary breast cancers (19). This is in contrast to the level of expression of ST3Gal-I mRNA, which is consistently elevated in primary breast cancers (19).…”

“…However, of the cloned ␣2,3-sialyltransferases enzymes, ST3Gal-I appears to play the major role in the sialylation of core 1 structures on MUC1 in breast, as deduced from enzyme specificity and tissue distribution (16 -18). Significantly, the ST3Gal-I enzyme has been found to be elevated in breast cancer cell lines and primary breast cancers (14,19).…”

“…It now appears that glycosyltransferases are not confined exclusively to specific Golgi compartments but show a more diffuse distribution among compartments (13). For example, although the expression of the sialyltransferase ST3Gal-I 1 is highest in the medial/transGolgi, some expression can be seen in the cis Golgi (14).…”

Golgi Localization of Carbohydrate Sulfotransferases Is a Determinant of L-selectin Ligand Biosynthesis

“…At saturating levels, highly expressed enzymes may distribute into Golgi subcompartments they would not otherwise occupy, thereby gaining access to novel substrates (52)(53)(54)(55). To exclude the possibility that dramatic differences in expression levels account for the distinct substrate preferences of GlcNAc6ST-1, -2, and -3 chimeras, we analyzed protein levels in the stable CHO cell lines by Western blot using anti-GFP serum.…”

Overexpression of MUC1 reconfigures the binding properties of tumor cells

“…6,9 -12 Levels of expression and types of posttranslational modifications of MUC1 by transformed epithelial cells often differ from forms produced by corresponding normal epithelia. [13][14][15][16][17][18][19] The appearance of novel oligosaccharide structures on MUC1 expressed by tumors (compared with normal epithelia) or differential glycosylation at different positions on the MUC1 core protein may confer new properties of adhesion that contribute to the ability of tumor cells to metastasize. For example, certain glycoforms of MUC1 have been shown to associate with intracellular adhesion molecule 1 (ICAM-1), 20 and it is predicted that certain oligosaccharide structures present on MUC1, such as sialyl Lewis A (sLe a ) or sialyl Lewis X (sLe x ) 15,21 interact with different lectin-like molecules 22 that influence general properties of cell adhesion.…”