High Density O-Glycosylation on Tandem Repeat Peptide from Secretory MUC1 of T47D Breast Cancer Cells

Abstract

The site-specific O-glycosylation of MUC1 tandem repeat peptides from secretory mucin of T47D breast cancer cells was analyzed. After affinity isolation on immobilized BC3 antibody, MUC1 was partially deglycosylated by enzymatic treatment with ␣-sialidase/␤-galactosidase and fragmented by proteolytic cleavage with the Arg-C-specific endopeptidase clostripain. The PAP20 glycopeptides were isolated by reversed phase high pressure liquid chromatography and subjected to the structural analyses by quadrupole time-offlight electrospray ionization mass spectrometry and to the sequencing by Edman degradation. All five positions of the repeat peptide were revealed as O-glycosylation targets in the tumor cell, including the Thr within the DTR motif. The degree of substitution was estimated to average 4.8 glycans per repeat, which compares to 2.6 glycosylated sites per repeat for the mucin from milk (Mü ller, S., Goletz, S., Packer, N., Gooley, A. A., Lawson, A. M., and Hanisch, F.-G. (1997) J. Biol. Chem. 272, 24780-24793). In addition to a modification by glycosylation, the immunodominant DTR motif on T47D-MUC1 is altered by amino acid replacements (PAPGSTAPAAHGVTSAPESR), which were revealed in about 50% of PAP20 peptides. The high incidence of these replacements and their detection also in other cancer cell lines imply that the conserved tandem repeat domain of MUC1 is polymorphic with respect to the peptide sequence.Due to the structural complexity of O-linked glycans, this characteristic posttranslational modification of mucin peptides is a polygenic regulated phenomenon and hence is prone to multiple, differentiation-dependent alterations. According to numerous reports, mucin O-glycosylation can now be regarded as a diagnostically relevant indicator of tumor-associated changes that are characterized by 1) the de novo expression of novel glycotopes the ectopic or incompatible expression of carbohydrate blood groups, or 2) by deletion/truncation of glycan chains (1).Also, the widely distributed epithelial mucin MUC1 has been described to be aberrantly processed in cancer cells (2-4). In breast cancer, the nonexpression of the core2 enzyme, Gal␤1-3GalNAc/␤-6-N-acetylglucosaminyltransferase (5), leads to the truncation of polylactosamine-type chains found on the lactation-associated mucin (6) and to the accumulation of core-type chains (2-4). The preponderance of sialylated core1-trisaccharide on carcinoma-associated MUC1, which can be regarded as a biosynthetic dead end product, has been shown to originate from the simultaneous up-regulation and overexpression of Gal␤1-3GalNAc/␣-3-sialyltransferase (7). Moreover, not only the chain length of the glycans but also their density has been described to be reduced on breast cancer cell-specific MUC1 (4).Reduced glycosylation has been assumed to permit the immune system access to the peptide core of the tumor-associated mucin (8). The preferred target site for most peptide-specific mouse antibodies generated to the tumor mucin, to synthetic variable number of tandem repeats (V...

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

“…We observed no difference between the proteins with 32, 16 or 1.7 TRs, indicating that the occupancy was determined by the cells rather than by the number of potential O-glycan sites. The average occupancy obtained for MUC1-IgG from T47D, 4.2 glycans per TR, was similar to that obtained from CHO-K1 cells, but somewhat lower than that previously reported for endogenous MUC1 from T47D (4.8 per TR) [12]. It is difficult to know whether this difference is statistically significant, but it is possible that the IgG tail in the present construct prevents the complete action of the polypeptide-GalNAc-transferases that initiates the synthesis of the O-glycan chains, maybe by moving the protein faster through the Golgi apparatus.…”

“…MUC1 TR glycopeptides were generated by a protocol modified from Müller et al [12]. MUC1-IgG (30-50 µg) from CHO-K1 or T47D cells was bound to 100 µl of anti-mouse Ig-agarose beads (Sigma-Aldrich) and deglycosylated using sequential incubations at 37 • C with 50 m-units of Vibrio cholerae neuraminidase (Roche) in 0.05 M sodium acetate, pH 5.5, with 4 mM CaCl 2 and 100 µg/ml BSA for 22 h and 50 m-units of β-galactosidase (from bovine testes; Sigma-Aldrich), in 0.1 M citrate phosphate buffer, pH 5.0, for 22 h. An additional incubation (after the neuraminidase treatment) with α1-2-fucosidase (1 m-unit) and α1-3,4-fucosidase (1 m-unit) in 25 mM sodium phosphate, pH 5.0, for 20 h was performed for the T47D-produced protein.…”

“…In contrast, MUC1 expressed by breast carcinoma cells has an increased proportion of shorter, non-branched, core 1-based O-glycans with a higher sialic acid content [9][10][11]. 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).…”

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

“…The loss of the SM3 binding to MUC1 is apparently due to the core 2 branch masking the polypeptide PDTRP motif recognized by SM3 (10). This steric hindrance by the core 2 branch of access to the VNTR region of the MUC1 polypeptide could also explain the approximate 2-fold reduction in actual number of O-glycans attached to MUC1 in normal cells compared with T47D cells (6). In this case, access for the GalNAc transferases adding the first sugar to serines and threonines would be blocked.…”

“…The aberrant glycosylation has been well documented in breast cancers where it has been shown (4,5) that the addition of shorter O-glycans leads to the exposure of peptide epitopes in the tandem repeats, which are normally masked, and the appearance of novel carbohydrate epitopes. Recent data suggest that not only the composition but the number of O-glycans added to MUC1 is altered in breast cancer (6). Thus, the cancer-associated glycoprotein is antigenically distinct from the normally processed mucins (7).…”