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In the last decade, it was discovered that protein mucin-type O-glycosylation and O-GlcNAcylation modify Tyr residues besides the well explored Thr and Ser amino acids. Several glycoproteomic studies have identified α-GalNAc-O-Tyr modifications, and studies propose that β-GlcNAc-O-Tyr also exists as a new group of posttranslational modifications (PTMs). Specific bacterial toxins have further been identified to modify host GTPases with α-GlcNAc-O-Tyr to promote bacterial virulence. Despite being identified on numerous proteins, the biological roles, biosynthesis and expression of GalNAc- and GlcNAc-O-Tyr modifications are poorly understood. A major obstacle is the lack of tools to specifically detect and identify proteins containing these modifications. With this in mind, we prepared vaccine constructs and raised antibodies to enable selective detection of proteins carrying these new PTMs. The obtained polyclonal antibody sera were evaluated using ELISA and glycopeptide microarrays and were found to be highly selective for GlcNAc- and GalNAc-O-Tyr glycopeptides over the corresponding Ser- and Thr-modifications. For microarray analysis, synthetic GlcNAc- and GalNAc-O-Tyr Fmoc-amino acids were prepared and applied in Fmoc-SPPS to obtain an extensive O-glycopeptide library. After affinity purification, the antibodies were applied in western blot analysis and showed specific detection of α-GlcNAc-O-Tyr modified RhoA GTPase.

Mucin glycoproteins are essential components of the mucosal barrier, which protects the host from pathogens. Throughout evolution, bacteria have developed strategies to modulate and penetrate this barrier, and cause virulence by interacting with mucin O-glycans at the epithelial cell-surface. O-fucosylated glycan epitopes on mucins are key ligands of many bacterial lectins. Here, a chemoenzymatic synthesis strategy is described to prepare a library of fucosylated mucin core glycopeptides to enable studies of mucin-interacting and fucose-binding bacterial lectins. Glycan cores with biologically important Lewis and H-antigens were prepared decorating the peptide backbone at different sites and densities. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of glycan core and peptide backbone presentation of these antigens in binding interactions with the P. aeruginosa lectin LecB and the C. difficile toxin A.

MUC1 glycopeptides are attractive antigens for anti-cancer vaccine development. One potential drawback in using the native MUC1 glycopeptide for vaccine design is the instability of theO-glycosyl linkage between the glycan and the peptide backbone to glycosidase. To overcome this challenge, a MUC1 glycopeptide mimic has been synthesized with the galactose-galactosamine disaccharide linked with threonine (Thomsen-Friedenreich or Tf antigen) through an unnatural β-glycosyl bond. The resulting MUC1-β-Tf had a much-enhanced stability toward a glycosidase capable of cleaving the glycan from the corresponding MUC1 glycopeptide with the natural α-Tf linkage. The MUC1-β-Tf was subsequently conjugated with a powerful carrier bacteriophage Qβ. The conjugate induced high levels of IgG antibodies in clinically relevant human MUC1 transgenic mice, which cross-recognized not only the natural MUC1-α-Tf glycopeptide but also MUC1 expressing tumor cells, supporting the notion that a simple switch of the stereochemistry of the glycan/peptide linkage can be a strategy for anti-cancer vaccine epitope design for glycopeptides.

Mucin-1 (MUC1) is a highly attractive antigenic target for anticancer vaccines. Naturally existing MUC1 can contain multiple types of O-linked glycans, including the Thomsen–Friedenreich (Tf) antigen and the Sialyl Thomsen-nouveau (STn) antigen. In order to target these antigens as potential anticancer vaccines, MUC1 glycopeptides SAPDT*RPAP (T* is the glycosylation site) bearing the Tf and the STn antigen, respectively, have been synthesized. The bacteriophage Qβ carrier is a powerful carrier for antigen delivery. The conjugates of MUC1-Tf and -STn glycopeptides with Qβ were utilized to immunize immune-tolerant human MUC1 transgenic (MUC1.Tg) mice, which elicited superior levels of anti-MUC1 IgG antibodies with titers reaching over 2 million units. The IgG antibodies recognized a wide range of MUC1 glycopeptides bearing diverse glycans. Antibodies induced by Qβ-MUC1-Tf showed strongest binding, with MUC1-expressing melanoma B16-MUC1 cells, and effectively killed these cells in vitro. Vaccination with Qβ-MUC1-Tf first followed by tumor challenge in a lung metastasis model showed significant reductions of the number of tumor foci in the lungs of immunized mice as compared to those in control mice. This was the first time that a MUC1-Tf-based vaccine has shown in vivo efficacy in a tumor model. As such, Qβ-MUC1 glycopeptide conjugates have great potential as anticancer vaccines.

Distinct structural changes of the α2,3/α2,6-sialic acid glycosidic linkages on glycoproteins are of importance in cancer biology, inflammatory diseases, and virus tropism. Current glycoproteomic methodologies are, however, not amenable toward high-throughput characterization of sialic acid isomers. To enable such assignments, a mass spectrometry method utilizing synthetic model glycopeptides for the analysis of oxonium ion intensity ratios was developed. This method was successfully applied in large-scale glycoproteomics, thus allowing the site-specific structural characterization of sialic acid isomers.

Human mucin-1 (MUC1) is a highly attractive antigen for the development of anticancer vaccines. However, in human clinical trials of multiple MUC1 based vaccines, despite the generation of anti-MUC1 antibodies, the antibodies often failed to exhibit much binding to tumor presumably due to the challenges in inducing protective immune responses in the immunotolerant environment. To design effective MUC1 based vaccines functioning in immunotolerant hosts, vaccine constructs were first synthesized by covalently linking the powerful bacteriophage Qβ carrier with MUC1 glycopeptides containing 20-22 amino acid residues covering one full length of the tandem repeat region of MUC1. However, IgG antibodies elicited by these first generation constructs in tolerant human MUC1 transgenic (Tg) mice did not bind tumor cells strongly. To overcome this, a peptide array has been synthesized. By profiling binding selectivities of antibodies, the long MUC1 glycopeptide was found to contain immunodominant but nonprotective epitopes. Critical insights were obtained into the identity of the key protective epitope. Redesign of the vaccine focusing on the protective epitope led to a new Qβ-MUC1 construct, which was capable of inducing higher levels of anti-MUC1 IgG antibodies in MUC1.Tg mice to react strongly with and kill a wide range of tumor cells compared to the construct containing the gold standard protein carrier, i.e., keyhole limpet hemocyanin. Vaccination with this new Qβ-MUC1 conjugate led to significant protection of MUC1.Tg mice in both metastatic and solid tumor models. The antibodies exhibited remarkable selectivities toward human breast cancer tissues, suggesting its high translational potential.

Mucin glycoproteins are essential components of the mucosal protective barrier, which constantly senses and clears the host from pathogens. Throughout evolution, bacteria and virus have developed strategies to modulate and penetrate the mucosal barrier and cause virulence by interacting with the glycans of membrane-bound mucins at the epithelial cell-surface. These interactions may promote bacteria cell-adhesion, biofilm formation, protein toxin delivery, or cause an inflammatory environment. O-fucosylated glycan epitopes are commonly found on mucin glycoproteins, and are key ligands of many bacterial and viral lectins (glycan binding proteins). Herein we describe a chemoenzymatic synthesis strategy to efficiently prepare an extensive library of fucosylated mucin core tandem repeats glycopeptides to elucidate the fine fucose-binding specificities of the Pseudomonas aeruginosa lectin LecB and the Clostridium difficile toxin A. Therefore, glycan core structures were decorated with terminal Lewis and H-antigens, which play critical roles in infection biology. The fucosylated mucin glycopeptides were applied in microarray binding studies to explore the importance of the glycan and peptide backbone presentation of these terminal antigens in binding interactions with the two bacterial lectins.