No abstract

Sialyl-Tn is a carbohydrate antigen overexpressed in several epithelial cancers, including breast cancer, and usually associated with poor prognosis. Sialyl-Tn is synthesized by a CMP-Neu5Ac:GalNAcalpha2,6-sialyltransferase: CMP-Neu5Ac: R-GalNAcalpha1-O-Ser/Thr alpha2,6-sialyltransferase (EC 2.4.99.3) (ST6GalNAc I), which transfers a sialic acid residue in alpha2,6-linkage to the GalNAcalpha1-O-Ser/Thr structure. However, established breast cancer cell lines express neither ST6GalNAc I nor sialyl-Tn. We have previously shown that stable transfection of MDA-MB-231, a human breast cancer cell line, with ST6GalNAc I cDNA induces sialyl-Tn antigen (STn) expression. We report here the modifications of the O-glycosylation pattern of a MUC1-related recombinant protein secreted by MDA-MB-231 sialyl-Tn positive cells. We also show that sialyl-Tn expression and concomitant changes in the overall O-glycan profiles induce a decrease of adhesion and an increase of migration of MDA-MB-231. Moreover, STn positive clones exhibit an increased tumour growth in severe combined immunodeficiency (SCID) mice. These observations suggest that modification of the O-glycosylation pattern induced by ST6GalNAc I expression are sufficient to enhance the tumourigenicity of MDA-MB-231 breast cancer cells.

Sixteen research groups participated in the ISOBM TD-4 Workshop in which the reactivity and specificity of 56 monoclonal antibodies against the MUC1 mucin was investigated using a diverse panel of target antigens and MUC1 mucin- related synthetic peptides and glycopeptides. The majority of antibodies (34/56) defined epitopes located within the 20-amino acid tandem repeat sequence of the MUC1 mucin protein core. Of the remaining 22 antibodies, there was evidence for the involvement of carbohydrate residues in the epitopes for 16 antibodies. There was no obvious relationship between the type of immunogen and the specificity of each antibody. Synthetic peptides and glycopeptides were analyzed for their reactivity with each antibody either by assay of direct binding (e.g. by ELISA or BiaCore) or by determining the capacity of synthetic ligands to inhibit antibody binding interactions. There was good concordance between the research groups in identifying antibodies reactive with peptide epitopes within the MUC1 protein core. Epitope mapping tests were performed using the Pepscan analysis for antibody reactivity against overlapping synthetic peptides, and results were largely consistent between research groups. The dominant feature of epitopes within the MUC1 protein core was the presence, in full or part, of the hydrophilic sequence of PDTRPAP. Carbohydrate epitopes were less easily characterized and the most useful reagents in this respect were defined oligosaccharides, rather than purified mucin preparations enriched in particular carbohydrate moieties. It was evident that carbohydrate residues were involved in many epitopes, by regulating epitope accessibility or masking determinants, or by stabilizing preferred conformations of peptide epitopes within the MUC1 protein core. Overall, the studies highlight concordance between groups rather than exposing inconsistencies which gives added confidence to the results of analyses of the specificity of anti-mucin monoclonal antibodies.

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...

Since there is no consensus sequence directing the initial GalNAc incorporation into mucin peptides, Oglycosylation sites are not reliably predictable. We have developed a mass spectrometric sequencing strategy that allows the identification of in vivo O-glycosylation sites on mucin-derived glycopeptides. Lactation-associated MUC1 was isolated from human milk and partially deglycosylated by trifluoromethanesulfonic acid to the level of core GalNAc residues. The product was fragmented by the Arg-C-specific endopeptidase clostripain to yield tandem repeat icosapeptides starting with the PAP motif. PAP20 glycopeptides were subjected to sequencing by post-source decay matrix-assisted laser desorption ionization mass spectrometry or by solid phase Edman degradation to localize the glycosylation sites. The masses of C-or N-terminal fragments registered for the mono-to pentasubstituted PAP20 indicated that GalNAc was linked to the peptide at Ser 5 ,Thr 6 (GSTA) and Thr 14 (VTSA) but contrary to previous in vitro glycosylation studies also at Thr 19 and Ser 15 located within the PDTR or VTSA motifs, respectively. Quantitative data from solid phase Edman sequencing revealed no preferential glycosylation of the threonines. These discrepancies between in vivo and in vitro glycosylation patterns may be explained by assuming that O-glycosylation of adjacent peptide positions is a dynamically regulated process that depends on changes of the substrate qualities induced by glycosylation at vicinal sites.Post-translational modification of proteins by glycosylation has extensively been studied in the case of N-linked glycans, and accordingly, rules for the substitution of asparagine residues by dolichol phosphate-linked glycans are well established (1). While N-glycosylation is directed by the consensus peptide motif Asn-X-(Ser/Thr), no strict sequence dependence is known for the initiation of O-glycosylation. Currently, there are several approaches to the identification of O-glycosylation sites: studies on in vitro O-glycosylation of synthetic peptides (2-4) or studies on in vivo processed mucin-type glycoproteins (5-7).The relative merits of both approaches have been discussed (8).The results obtained so far agree with the statement that there are no clear-cut motifs for the addition of GalNAc at Ser or Thr residues; however, the non-random patterns of O-glycosylation suggest influences of flanking sequences (2). Wang et al. (3) proposed different motifs for threonine and serine glycosylation, and the studies of O'Connell et al. (2) revealed critical positions in the vicinity of putative O-glycosylation sites. The obviously less specific GalNAc addition to Ser/Thr residues compared with N-glycosylation is reflected also in the existence of at least four distinct species of UDP-GalNAc/peptide Nacetylgalactosaminyltransferase(s) (GalNAc-transferase) for which different substrate specificities have been established (9). Accordingly, the differentiation and organ localization of a cell should determine its characteristic equipment ...