Abstract
In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based. We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope. Of the three enzymes known to convert core 1 to core 2, by the addition of GlcNAc to GalNAc in core1 C2GnT1 is the dominant enzyme expressed in normal breast tissue. Expression of C2GnT1 is low or absent in around 50% of breast cancers, whereas expression of ST3Gal-I is consistently increased. Mapping of ST3Gal-I and C2GnT1 within the Golgi pathway showed some overlap. To examine functional competition, the enzymes were overexpressed in T47D cells, which normally make core 1-based structures, have no detectable C2GnT1 activity and express the SM3 epitope. Overexpression of C2GnT1 resulted in loss of binding of SM3 to MUC1, accompanied by a decrease in the GalNAc/GlcNAc ratio, indicative of a switch to core 2 structures. Transfection of a C2GnT1 expressing line with ST3Gal-I restored SM3 binding and reduced GlcNAc incorporation into MUC1 O-glycans. Thus, even when C2GnT1 is expressed, the O-glycans added to MUC1 become core 1-dominated structures, provided expression of ST3Gal-I is increased as it is in breast cancer.
Highlights
In breast cancer, the O-glycans added to the MUC1 mucin are core 1- rather than core 2-based
We have analyzed whether competition by the glycosyltransferase, ST3Gal-I, which transfers sialic acid to galactose in the core 1 substrate, is key to this switch in MUC1 glycosylation that results in the expression of the cancer-associated SM3 epitope
In situ hybridization analysis of primary breast cancers has shown that C2GnT1 levels vary widely among breast carcinomas, whereas levels of ST3Gal I mRNA are consistently increased [19]
Summary
Materials—Biotinylated Arachis hypogaea Peanut agglutinin (PNA) was from Vector laboratories Ltd. (Peterborough, UK). ST3Gal-I DNA (tagged with an Myc epitope at the C terminus) and C2GnT cDNA, subcloned into the pBabe puro vector, was transfected into the amphotrophic packaging cell line AM12. All incubations were carried out in the presence of 0.02% sodium azide and on wet ice. After a 30-min incubation, cells were washed three times in growth medium containing 0.02% sodium azide. After washing three times in growth medium containing 0.02% sodium azide, cells were resuspended in 300 l of phosphatebuffered saline, and 10,000 cells were analyzed by a Becton Dickinson FACScan. Western Blotting—300 g of cell lysate were incubated with or without 1.0 unit of C. perfringens sialidase in 50 mM sodium acetate buffer, pH 5.0, in a 37 °C water bath overnight. After 3 additional washes, the bound lectin-streptavidin-horseradish peroxidase was detected using enhanced chemiluminescence (ECL) and light-sensitive film
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