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

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

Recombinant MUC1 mucin with a breast cancer-like O-glycosylation produced in large amounts in Chinese-hamster ovary cells

“…In T47D, only core 1-based structures were found, consistent with the earlier finding that T47D have lost the ability to make core 2 glycans, as they lack important core 2 β6-GlcNAc-transferases [9]. In MCF-7, this enzyme is still present, explaining the synthesis of core 2-based structures, but the presence of the α2,3-sialyltransferase ST3Gal-I, which adds sialic acid in the α2,3 position of the Gal in Galβ1,3GalNAc, prevents to some extent the conversion into core 2 structures [9,14]. The MCF-7 cell line is representative of a differentiated breast carcinoma in that it shows polarized expression of MUC1 MUC1 TR glycopeptides were generated by partial deglycosylation of purified MUC1-IgG glycoproteins and subsequent cleavage with the endoprotease Clostripain.…”

“…The glycoprotein reacted with all three mAbs, the weakest reaction being that of mAb HMFG-1 (Figure 4), which is negatively affected by the presence of sialic acids [26]. The binding of the mAbs SM3 and HMFG-2 is inhibited instead by long O-glycan chains, for mAb SM3 more specifically by the presence of GlcNAc in the β1-6 position of the core GalNAc [14]. Both SM3 and HMFG-2 were found to react well with the CHO-K1-produced MUC1.…”

“…The cancer-derived MUC1 also has, in some cases, a higher occupancy of the O-glycosylation sites in the TRs (tandem repeats) compared with MUC1 from normal cells [12]. The altered O-glycan profile of tumour MUC1 can be partly explained by the enhanced expression of the sialyltransferase ST3Gal-I in breast cancer cells [13,14], which competes with the core 2-forming enzyme (C2GnT1) for their common core 1 substrate (Galβ1-3GalNAc).…”

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

“…As shown schematically in Fig. 10B (step 4), the Core 1 structure is generated by the addition of a galactose residue to the core GalNAc, and the Core 2 trisaccharide can be formed subsequently by the action of Core2GlcNAcT-I (step 5) which has been localized to the cis/medial compartment of the Golgi (55). Sulfation of the Core 2 GlcNAc residue must occur at this point (step 6), before further elaboration with poly-LacNAc extensions or the sLe x capping group.…”

“…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.…”