Tn glycosylation of the MUC6 protein modulates its immunogenicity and promotes the induction of Th17-biased T cell responses

The Tn antigen (α-GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. This antigen, together with mucins, the major carriers of O-glycosylated tumor antigens in adenocarcinomas, are being evaluated as anti-cancer immunotherapeutic targets. In particular, the MUC6 protein, which is normally expressed only in gastric tissues, has been detected in intestinal, pulmonary, colorectal, and breast carcinomas. To develop anti-cancer vaccines based on the Tn antigen, we produced MUC6 proteins with different Tn density by using mixtures of recombinant ppGalNAc-T1, -T2, and -T7. The obtained glycoproteins were characterized and analyzed for their immunological properties, as compared with the non-glycosylated MUC6. We show that these various MUC6:Tn glycoproteins were well recognized by both MUC6 and Tn-specific antibodies. However, Tn glycosylation of the MUC6 protein strongly affected their immunogenicity by partially abrogating Th1 cell responses, and promoting IL-17 responses. Moreover, the non-glycosylated MUC6 was more efficiently presented than MUC6:Tn glycoproteins to specific T CD4(+) hybridomas, suggesting that Tn glycosylation may affect MUC6 processing or MHC binding of the processed peptides. In conclusion, our results indicate that Tn glycosylation of the MUC6 protein strongly affects its B and T cell immunogenicity, and might favor immune escape of tumor cells.     

Efficient monitoring of enzymatic conjugation reaction by surface-enhanced laser desorption/ionization time of flight mass spectrometry for process optimization

Efficient analysis of bioconjugation reactions is one the most challenging task for optimizing and eventually achieving the reproducible production of large amount of conjugates. In particular, the complexity of some reaction mixtures precludes the use of most of the existing methods, because of the presence of large amounts of contaminants. As an alternative method, we used surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) for monitoring an in vitro enzymatic transglycosylation of N-acetylgalactosamine (GalNAc) residues to a recombinant mucin protein MUC6. For this reaction, catalyzed by the uridine 5'-diphospho-N-acetylgalactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts), we used either a recombinant ppGalNAc-T1 or a mixture of ppGalNAc-Ts contained in MCF7 tumor cell extracts. In the present study, we show that SELDI-TOF MS offers unique advantages over the traditional methodologies. It is a rapid, accurate, sensitive, reproducible, and very convenient analytical method for monitoring the course of a bioconjugation, even in heterogeneous samples such as cell extracts. SELDI-TOF MS proved very useful for optimizing the reaction parameters of the transglycosylation and for achieving the large scale preparation of Tn antigen-glycosylated mucins for antitumor immunotherapy applications.     

UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase-6 as a new immunohistochemical breast cancer marker.

Mucin O-glycosylation is characterized in cancer by aberrant expression of immature carbohydrate structures (Tn, T, and sialyl-Tn antigens). The UDP-N-acetyl-D-galactosamine: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-T) family enzymes regulate the initial steps of mucin O-glycosylation and could be responsible for the altered glycosylation observed in cancer. Considering that we recently found the ppGalNAc-T6 mRNA expressed in breast carcinomas, we produced a highly specific monoclonal antibody (MAb T6.3) to assess the expression profile of ppGalNAc-T6 protein product in breast tissues. The expression of ppGalNAc-T6 by breast carcinoma cells was confirmed on MCF-7 and T47D cell lines. In formalin-fixed tissues, ppGalNAc-T6 expression was observed in 60/74 (81%) breast cancers, 21/23 (91.3%) adjacent ductal carcinoma in situ (DCIS), 4/20 benign breast lesions (2/2 sclerosing adenosis and 2/13 fibroadenoma), and in 0/5 normal breast samples. We observed a statistically significant association of ppGalNAc-T6 expression with T1 tumor stage. This fact, as well as the observation that ppGalNAc-T6 was strongly expressed in sclerosing adenosis and in most DCIS, suggests that ppGalNAc-T6 expression could be an early event during human breast carcinogenesis. Considering that an abnormal O-glycosylation greatly contributes to the phenotype and biology of breast cancer cells, ppGalNAc-T6 expression could provide new insights about breast cancer glycobiology.     

Thomsen-Friedenreich antigen expression in gastric carcinomas is associated with MUC1 mucin VNTR polymorphism

Aberrant glycosylation of mucins is a common phenomenon associated with oncogenic transformation. We investigated the association between expression of the tumor-associated antigens T, Tn, and sialyl-Tn and polymorphism in the length of the MUC1 mucin tandem repeat in a series of gastric carcinomas. We further evaluated the relevance of MUC1 tandem repeat length on the expression of these tumor-associated carbohydrate antigens (TACAs) using a gastric carcinoma cell line model expressing recombinant MUC1 constructs carrying 0, 3, 9, and 42 repeats. Gastric carcinomas showed a high prevalence of Tn and sialyl-Tn antigens, whereas T antigen was less frequently expressed. The expression of T antigen was significantly higher in gastric carcinomas from patients homozygous for MUC1 large tandem repeat alleles. No significant associations were found for Tn and sialyl-Tn antigens. This novel association was reinforced by the gastric carcinoma cell line model experiments, where de novo expression of T antigen was detected in clones transfected with larger VNTR regions. Our results indicate that polymorphism in the MUC1 tandem repeat influences the expression of TACAs in gastric cancer cells and may therefore allow the identification of subgroups of patients that develop more aggressive tumors expressing T antigen.     

Nuclear magnetic resonance-based dissection of a glycosyltransferase specificity for the mucin MUC1 tandem repeat

The human glycoprotein MUC1 mucin plays a critical role in cancer progression. Breast, ovarian, and colon cancer cells often display unique cell-surface antigens corresponding to aberrantly glycosylated forms of the MUC1 tandem repeat. In this report, (15)N- and (13)C-labeled forms of a recombinant MUC1 construct containing five tandem repeats were used as substrates to define the order and kinetics of addition of N-acetylgalactosamine (GalNAc) moieties by a recombinant active form of the human enzyme UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase I (ppGalNAc-T1; residues 40-559). Heteronuclear NMR experiments were performed to assign resonances associated with the two serines (Ser5 and Ser15) and three threonines (Thr6, Thr14, and Thr19) present in the 20-residue long MUC1 repeat. The kinetics and order of addition of GalNAc moieties (Tn antigen) on the MUC1 construct by human ppGalNAc-T1 were subsequently dissected by NMR spectroscopy. Threonine 14 was shown to be rapidly glycosylated by ppGalNAc-T1 with an initial rate of 25 microM/min, followed by Thr6 (8.6 microM/min). The enzyme also modified Ser5 at a slower rate (1.7 microM/min), an event that started only after the glycosylation of Thr14 and Thr6 side chains was mostly completed. Ser15 and Thr19 remained unglycosylated by ppGalNAc-T1. Corresponding O-glycosylation sites within all five tandem repeats were simultaneously modified by ppGalNAc-T1, suggesting that each repeat behaves as an independent substrate unit. This study demonstrated that the hydroxyl oxygens of Thr14 and to a lesser extent Thr 6 represent the two dominant substrates modified by ppGalNAc-T1 within the context of a complex MUC1 peptide substrate. More importantly, the availability of defined isotopically labelled MUC1 glycopeptide substrates and the relative simplicity of their NMR spectra will facilitate the analysis of other transferases within the O-glycosylation pathways and the rational design of tumor-associated MUC1 antigens.     

Mucin-type O-glycosylation in Mesocestoides vogae (syn. corti)

Protein glycosylation is an important post-translational modification underlying host-parasite interactions, which may determine the outcome of infection. Although Mesocestoides vogae represents an important model for investigating the various aspects of cestode biology, virtually no information is available about the structure and synthesis of glycans in this parasite. In this work, focused on the initiation pathway of mucin-type O-glycosylation in M. vogae, we characterized O-glycoproteins bearing the simple mucin-type cancer-associated Tn and sialyl-Tn antigens, and the expression and activity of ppGalNAc-T, the key enzyme responsible for the first step of mucin-type O-glycosylation. Using immunohistochemistry, Tn and sialyl-Tn antigens were detected mainly in the tegument (microtriches) and in parenchymal cells. Tn expression was also observed in lateral nerve cords. Both Tn and sialyl-Tn antigens were detected in in vitro cultured parasites. Based on their electrophoretic mobility, Tn- and sialyl-Tn-bearing glycoproteins from M. vogae were separated into several components of 22 to 60 kDa. The observation that Tn and sialyl-Tn glycoproteins remained in the 0.6N perchloric acid-soluble fraction suggested that they could be good candidates for characterizing mucin-type glycosylation in this parasite. O-glycoproteins were purified and initially characterized using a proteomic approach. Immunohistochemical analysis of the tissue distribution of ppGalNAc-T revealed that this enzyme is expressed in the sub-tegumental region and in the parenchyma of the parasite. In M. vogae cultured in vitro, ppGalNAc-T was mainly detected in the suckers. Using a panel of 8 acceptor substrate synthetic peptides, we found that M. vogae ppGalNAc-T preferentially glycosylate threonine residues, the best substrates being peptides derived from human mucin MUC1 and from Trypanosoma cruzi mucin. These results suggest that M. vogae might represent a useful model to study O-glycosylation, and provide new research avenues for future studies on the glycopathobiology of helminth parasites.     

Mucins in the pathogenesis of breast cancer: implications in diagnosis, prognosis and therapy

Mucins are high molecular weight, multifunctional glycoproteins comprised of two structural classes-the large transmembrane mucins and the gel-forming or secreted mucins. The primary function of mucins is to protect and lubricate the luminal surfaces of epithelium-lined ducts in the human body. Recent studies have identified a differential expression of both membrane bound (MUC1, MUC4 and MUC16) and secreted mucins (MUC2, MUC5AC, MUC5B and MUC6) in breast cancer tissues when compared with the non-neoplastic breast tissues. Functional studies have also uncovered many unique roles of mucins during the progression of breast cancer, which include modulation in proliferative, invasive and metastatic potential of tumor cells. Mucins function through many unique domains that can form complex association with various signaling molecules including growth factor receptors and intercellular adhesion molecules. While there is growing information about mucins in various malignancies including breast cancer, no focused review is there on the expression and functional roles of mucins in breast cancer. In this present review, we have discussed the differential expression and functional roles of mucins in breast cancer. The potential of mucins as diagnostic and prognostic markers and as therapeutic targets in breast cancer have also been discussed.     

Mucin-type O-glycosylation in Fasciola hepatica: characterisation of carcinoma-associated Tn and sialyl-Tn antigens and evaluation of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity

Simple mucin-type cancer-associated O-glycan structures, such as the Tn antigen (GalNAc-O-Ser/Thr), are expressed by certain helminth parasites. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. The aim of this work was to study the initiation pathway of mucin-type O-glycosylation in Fasciola hepatica, performing a biochemical and immunohistochemical characterisation of Tn and sialyl-Tn antigens, and evaluating the ppGaNTase activity, which catalyses the first step in O-glycan biosynthesis. Using ELISA, both Tn and sialyl-Tn antigens were detected predominantly in the somatic and deoxycholate extracts. Immunofluorescence analysis revealed that Tn antigen is preferentially expressed in testis, while sialyl-Tn glycoproteins were more widely distributed, being present in parenchymal cells, basal membrane of the tegument, and apical surface of epithelial cells lining the caeca. On the basis of their electrophoretic mobility, Tn glycoproteins were resolved as six components of 10, 37, 76, 125, 170 and 205 kDa, and sialyl-Tn components showed an apparent molecular mass of 28 and 32 kDa, and two broad bands of 90-110 and 170-190 kDa. The observation that only the 76 kDa Tn-glycoprotein remained in the 0.6 N perchloric acid-soluble fraction suggests that it could be a good candidate for mucin characterisation in this parasite. The ppGaNTase activity showed its maximal activity at pH 7-7.5 and 37 degrees C, showing that Mn(2+) was the best divalent cation activator. Using a panel of nine synthetic peptides as acceptor substrates, we found that F. hepatica ppGaNTase was able to glycosylate both threonines and serines, the best substrates being the peptides derived from the tandem repeat region of human mucins (MUC2 and MUC6), and from Trypanosoma cruzi and Trypanosoma brucei glycoproteins. The results reported here constitute the first evidence on O-glycosylation pathways in F. hepatica, and may help to identify new biological characteristics of this parasite as well as of the host-parasite relationship.     

MUC5B Leads to Aggressive Behavior of Breast Cancer MCF7 Cells

The mucin MUC5B has a critical protective function in the normal lung, salivary glands, esophagus, and gallbladder, and has been reported to be aberrantly expressed in breast cancer, the second leading cause of cancer-related deaths among women worldwide. To understand better the implication of MUC5B in cancer pathogenesis, the luminal human breast cancer cell line MCF7 was transfected with a vector encoding a recombinant mini-mucin MUC5B and was then infected with a virus to deliver a short hairpin RNA to knock down the mini-mucin. The proliferative and invasive properties in Matrigel of MCF7 subclones and subpopulations were evaluated in vitro. A xenograft model was established by subcutaneous inoculation of MCF7 clones and subpopulations in SCID mice. Tumor growth was measured, and the tumors and metastases were assessed by histological and immunological analysis. The mini-mucin MUC5B promoted MCF7 cell proliferation and invasion in vitro. The xenograft experiments demonstrated that the mini-mucin promoted tumor growth and MCF7 cell dissemination. In conclusion, MUC5B expression is associated with aggressive behavior of MCF7 breast cancer cells. This study suggests that MUC5B may represent a good target for slowing tumor growth and metastasis.     

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (K_D?=?1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.     

Identification of new cancer biomarkers based on aberrant mucin glycoforms by in situ proximity ligation

Mucin glycoproteins are major secreted or membrane-bound molecules that, in cancer, show modifications in both the mucin proteins expression and in the O-glycosylation profile, generating some of the most relevant tumour markers in clinical use for decades. Thus far, the identification of these biomarkers has been based on the detection of either the protein or the O-glycan modifications. We therefore aimed to identify the combined mucin and O-glycan features, that is, specific glycoforms, in an attempt to increase specificity of these cancer biomarkers. Using in situ proximity ligation assays (PLA) based on existing monoclonal antibodies directed to MUC1, MUC2, MUC5AC and MUC6 mucins and to cancer-associated carbohydrate antigens Tn, Sialyl-Tn (STn), T, Sialyl-Le(a) (SLe(a)) and Sialyl-Le(x) (SLe(x)) we screened a series of 28 mucinous adenocarcinomas from different locations (stomach, ampulla of Vater, colon, lung, breast and ovary) to detect specific mucin glycoforms. We detected Tn/STn/SLe(a)/SLe(x)-MUC1 and STn/SLe(a)/SLe(x)-MUC2 glycoforms in ≥50% of the cases, with a variable distribution among organs. Some new glycoforms-T/SLe(a)-MUC2, STn/T/SLe(a) SLe(x)-MUC5AC and STn/T/SLe(a)/SLe(x)-MUC6-were identified for the first time in the present study in a variable percentage of cases from different organs. In conclusion, application of the PLA technique allowed sensitive detection of specific aberrant mucin glycoforms in cancer, increasing specificity to the use of antibodies either to the mucin protein backbone or to the O-glycan haptens alone.     

TGF-β1 increases invasiveness of SW1990 cells through Rac1/ROS/NF-κB/IL-6/MMP-2

Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II transmembrane proteins having a Golgi lumenal region that contains a catalytic domain with glycosyltransferase activity, and a C-terminal R-type (“ricin-like”) lectin domain. We investigated the effect of acetylation on catalytic activity of glycosyltransferase, and on fine carbohydrate-binding specificity of the R-type lectin domain of ppGalNAc-T2. Acetylation effect on ppGalNAc-T2 biological activity in vitro was studied using a purified human recombinant ppGalNAc-T2. Mass spectrometric analysis of acetylated ppGalNAc-T2 revealed seven acetylated amino acids (K103, S109, K111, K363, S373, K521, and S529); the first five are located in the catalytic domain. Specific glycosyltransferase activity of ppGalNAc-T2 was reduced 95% by acetylation. The last two amino acids, K521 and S529, are located in the lectin domain, and their acetylation results in alteration of the carbohydrate-binding ability of ppGalNAc-T2. Direct binding assays showed that acetylation of ppGalNAc-T2 enhances the recognition to αGalNAc residue of MUC1αGalNAc, while competitive assays showed that acetylation modifies the fine GalNAc-binding form of the lectin domain. Taken together, these findings clearly indicate that biological activity (catalytic capacity and glycan-binding ability) of ppGalNAc-T2 is regulated by acetylation.     

Specificity of human anti-carbohydrate IgG antibodies as probed with polyacrylamide-based glycoconjugates

The TF, Tn, and SiaTn glycotopes are frequently expressed in cancer-associated mucins. Antibodies to these glycotopes were found in human serum. A set of polyacrylamide (PAA)--based glycoconjugates was applied to the direct and competitive enzyme-linked immunosorbent assays (ELISA) to characterize the specificity of serum IgG antibodies. The anti-TF, -Tn and -SiaTn IgG were affinity purified from serum of cancer patients and characterized using PAA-conjugates and free saccharides. The anti-TF and -Tn antibodies were shown to be specific. The anti-TF IgG bound both Galbeta1-3GalNAcalpha- and Galbeta1-3GalNAcbeta-PAA, the latter was three-four times more effective inhibitor of antibody binding. The anti-Tn IgG reacted only with GalNAcalpha-PAA. The anti-SiaTn IgG cross-reacted with Tn-PAA but SiaTn-PAA was five-six times more effective inhibitor in a competitive assay. The IC50 values for PAA-conjugates with the corresponding antibodies typically ranged from 2 to 5 x 10(-8) M. The antibodies display a low specificity to mucin-type glycoconjugates in comparison with PAA-conjugates as was shown for mucins isolated from human malignant tumor tissues, ovine submaxillary mucin (OSM) and asialo-OSM. The unusual IgG-antibody specificity to GalNAcbeta and GalNAcbeta1-3GalNAcbeta ligands was found in human serum.     

Mucin-induced apoptosis of monocyte-derived dendritic cells during maturation

Many tumors arising from epithelial tissues produce mucins, which readily come into contact with infiltrating cells in cancer tissues. MUC2 mucins were purified from the conditioned medium of a colorectal cancer cell line, LS180 cells. It is known that in cancer patients, the number of dendritic cells (DCs) is reduced and their function is impaired. Mature DCs were generated from human peripheral blood monocytes through successive treatments with GM-CSF and IL-4, and then with proinflammatory mediators. When monocytes were cultured in the presence of MUC2 mucins in addition to GM-CSF and IL-4 at an early stage of development, mature DCs expressing CD83 decreased and apoptotic cells increased in a dose-dependent manner. During the development of DCs, sialic acid-binding Ig-like lectin (Siglec)-3 was constantly expressed. We prepared recombinant soluble Siglec-3 corresponding to the ectodomain of Siglec-3 and confirmed the binding of soluble Siglec-3 to the MUC2 mucins, probably through alpha2,6-sialic acid-containing O-glycans including a sialyl Tn antigen, which is known to bind to Siglec-3. Apoptosis was partially inhibited by anti-Siglec-3 mAb or recombinant soluble Siglec-3. These results suggest that apoptosis was partially induced through the ligation of the MUC2 mucins with Siglec-3.     

Characterization of ppGalNAc-T18, a member of the vertebrate-specific Y subfamily of UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases

The first step of mucin-type O-glycosylation is catalyzed by members of the UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase (ppGalNAc-T; EC 2.4.1.41) family. Each member of this family has unique substrate specificity and expression profiles. In this report, we describe a new subfamily of ppGalNAc-Ts, designated the Y subfamily. The Y subfamily consists of four members, ppGalNAc-T8, -T9, -T17 and -T18, in which the conserved YDX(5)WGGENXE sequence in the Gal/GalNAc-T motif of ppGalNAc-Ts is mutated to LDX(5)YGGENXE. Phylogenetic analysis revealed that the Y subfamily members only exist in vertebrates. All four Y subfamily members lack in vitro GalNAc-transferase activity toward classical substrates possibly because of the UDP-GalNAc-binding pocket mutants. However, ppGalNAc-T18, the newly identified defining member, was localized in the endoplasmic reticulum rather than the Golgi apparatus in lung carcinoma cells. The knockdown of ppGalNAc-T18 altered cell morphology, proliferation potential and changed cell O-glycosylation. ppGalNAc-T18 can also modulate the in vitro GalNAc-transferase activity of ppGalNAc-T2 and -T10, suggesting that it may be a chaperone-like protein. These findings suggest that the new Y subfamily of ppGalNAc-Ts plays an important role in protein glycosylation; characterizing their functions will provide new insight into the role of ppGalNAc-Ts.     

Polypeptide N-acetylgalactosaminyltransferase 18 non-catalytically regulates the ER homeostasis and O-glycosylation

Mucin-type O-glycosylation plays important roles in various biological processes. It is initiated by a family of 20 conserved UDP-GalNAc: polypeptide N-acetylgalactosaminyltransferases (ppGalNAc-Ts). Unlike most ppGalNAc-Ts localized to the Golgi apparatus, ppGalNAc-T18 is predominantly distributed on the endoplasmic reticulum (ER) and exhibits no ppGalNAc-T catalytic activity in vitro. Herein, we found that ppGalNAc-T18 silencing in cells decreased O-glycosylation levels and activated ER stress leading to apoptosis. After treatment with chemical chaperone 4-phenylbutyric acid (PBA) or forced expression of ppGalNAc-T18 in the ppGalNAc-T18 knockdown cell, these defects could be significantly alleviated, suggesting that ppGalNAc-T18 is important for ER homeostasis and protein O-glycosylation. Furthermore, we found that ppGalNAc-T18 exerts its functions in O-glycosylation and ER stress via a non-catalytic mechanism. These results reveal a novel molecular role of ppGalNAc-Ts that the ER-localized ppGalNAc-T18 could regulate the O-glycosylation and ER homeostasis in a non-catalytic manner.     

Mucin-type O-glycosylation and its potential use in drug and vaccine development

Mucin-type O-glycans are found on mucins as well as many other glycoproteins. The initiation step in synthesis is catalyzed by a large family of polypeptide GalNAc-transferases attaching the first carbohydrate residue, GalNAc, to selected serine and threonine residues in proteins. During the last decade an increasing number of GalNAc-transferase isoforms have been cloned and their substrate-specificities partly characterized. These differences in substrate specificities have been exploited for in vitro site-directed O-glycosylation. In GlycoPEGylation, polyehylene glycol (PEG) is transferred to recombinant therapeutics to specific acceptor sites directed by GalNAc-transferases. GalNAc-transferases have also been used to control density of glycosylation in the development of glycopeptide-based cancer vaccines. The membrane-associated mucin-1 (MUC1) has long been considered a target for immunotherapeutic and immunodiagnostic measures, since it is highly overexpressed and aberrantly O-glycosylated in most adenocarcinomas, including breast, ovarian, and pancreatic cancers. By using vaccines mimicking the glycosylation pattern of cancer-cells, it is possible to overcome tolerance in transgenic animals expressing the human MUC1 protein as a self-antigen providing important clues for an improved MUC1 vaccine design. The present review will highlight some of the potential applications of site-directed O-glycosylation.     

Comparison of antigen constructs and carrier molecules for augmenting the immunogenicity of the monosaccharide epithelial cancer antigen Tn

We have demonstrated previously that the optimal method for inducing an antibody response against defined cancer antigens is covalent conjugation of the antigen to keyhole limpet hemocyanin (KLH) and use of the potent saponin adjuvant QS-21. Single molecules of glycolipids (tetrasaccharides, pentasaccharides, or hexasaccharides) and MUC1 peptides (containing between one and five MUC1 tandem repeats) conjugated to KLH have proven sufficient for antibody recognition and vaccine construction. However, cancer specificity of monoclonal antibodies against the monosaccharide Tn and disaccharide sTn comes largely from recognition of clusters (c) of these molecules on the cell surface. Tn consists of a monosaccharide (GalNAc) O-linked to serine or threonine on epithelial cancer mucins which are uniquely rich in serines and threonines. We test here several Tn constructs: Tn monosaccharide, Tn(c) prepared on a triple threonine backbone, and Tn prepared on a partially or fully glycosylated MUC1 backbone. We determine that Tn(c) is more effective than Tn, and conjugation to KLH is more effective than conjugation to BSA or polystyrene beads for inducing ELISA reactivity against Tn, and FACS reactivity against Tn-positive tumor cells. Surprisingly, MUC1 glycosylated with Tn at three or five sites per 20 amino acid MUC1 tandem repeat and conjugated to KLH, induced the strongest antibody response against Tn and tumor cells expressing Tn, and had the additional advantage of inducing antibodies against MUC1.     

Antibodies against tumor cell glycolipids and proteins, but not mucins, mediate complement-dependent cytotoxicity

One of several effector mechanisms thought to contribute to Ab efficacy against cancer is complement-dependent cytotoxicity (CDC). Serological analysis of a series of clinical trials conducted over a 10-year period suggested that six vaccines containing different glycolipids induced Abs mediating CDC whereas four vaccines containing carbohydrate or peptide epitopes carried almost exclusively by mucin molecules induced Abs that did not mediate CDC. To explore this further, we have now compared cell surface reactivity using flow cytometry assays (FACS), complement-fixing ability, and CDC activity of a panel of mAbs and immune sera from these trials on the same two tumor cell lines. Abs against glycolipids GM2, globo H and Lewis Y, protein KSA (epithelial cell adhesion molecule, also known as EpCAM) and mucin Ags Tn, sialylated Tn, Thomsen Friedenreich (TF), and MUC1 all reacted comparably by FACS with tumor cells expressing these Ags. Compared with the strong complement binding and CDC with Abs against glycolipids and KSA, complement binding was diminished with Abs against mucin Ags and no CDC was detected. A major difference between these two groups of Ags is proximity to the cell membrane. Glycolipids and globular glycoproteins extend less than 100 A from the cell membrane while mucins extend up to 5000 A. Although complement activation at sites remote from the cell membrane has long been known as a mechanism for resistance from complement lysis in bacteria, it is identified here for the first time as a factor which may contribute to resistance from CDC against cancer cells.