Ulrika Westerlind

Glycopeptide-functionalized gold nanoparticles for antibody induction against the tumor associated mucin-1 glycoprotein.

We report the preparation of gold nanoparticle (AuNP)-based vaccine candidates against the tumor-associated form of the mucin-1 (MUC1) glycoprotein. Chimeric peptides, consisting of a glycopeptide sequence derived from MUC1 and the T-cell epitope P30 sequence were immobilized on PEGylated AuNPs and the ability to induce selective antibodies in vivo was investigated. After immunization, mice showed significant MHC-II mediated immune responses and their antisera recognized human MCF-7 breast cancer cells. Nanoparticles designed according to this report may become key players in the development of anticancer vaccines.

Synthetic antitumor vaccines containing MUC1 glycopeptides with two immunodominant domains-induction of a strong immune response against breast tumor tissues.

A shot in the arm for cancer treatment: two MUC1 tetanus toxoid vaccines were synthesized and induced a strong immune response in mice. The antibodies elicited by the vaccines show a high selectivity for the tumor cells in mammary carcinoma tissues and also distinguish between tumor tissues at different stages.

Assignment of Saccharide Identities through Analysis of Oxonium Ion Fragmentation Profiles in LC–MS/MS of Glycopeptides

Protein glycosylation plays critical roles in the regulation of diverse biological processes, and determination of glycan structure-function relationships is important to better understand these events. However, characterization of glycan and glycopeptide structural isomers remains challenging and often relies on biosynthetic pathways being conserved. In glycoproteomic analysis with liquid chromatography-tandem mass spectrometry (LC-MS/MS) using collision-induced dissociation (CID), saccharide oxonium ions containing N-acetylhexosamine (HexNAc) residues are prominent. Through analysis of beam-type CID spectra and ion trap CID spectra of synthetic and natively derived N- and O-glycopeptides, we found that the fragmentation patterns of oxonium ions characteristically differ between glycopeptides terminally substituted with GalNAcα1-O-, GlcNAcβ1-O-, Galβ3GalNAcα1-O-, Galβ4GlcNAcβ-O-, and Galβ3GlcNAcβ-O- structures. The difference in the oxonium ion fragmentation profiles of such glycopeptides may thus be used to distinguish among these glycan structures and could be of importance in LC-MS/MS-based glycoproteomic studies.

Preparation of Biomolecule Microstructures and Microarrays by Thiol–ene Photoimmobilization

A mild, fast and flexible method for photoimmobilization of biomolecules based on the light‐initiated thiol–ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 μm in size covering an area of up to 75×25 mm were created. Use of a confocal laser microscope with a UV laser as UV‐light source enabled further reduction of biotin feature size opening access to nanostructured biochips.

Arraying the post-translational glycoproteome (PTG).

Glycosylation is chemically the most complex post-translational modification of proteins and therefore understanding the structural and biological implications of post-translational glycosylation is a major challenge. The need for rapid and reliable investigations of protein-glycan interaction events and the substantial efforts required to synthesize glycans and glycopeptides with a variety of structures has called for the development of miniaturized analytical techniques. In the last decade, glycan and glycopeptide microarrays have enabled high-throughput analysis of diverse protein-glycan interactions. Evaluations of enzyme activities and substrate specificities, characterization of glycan binding proteins, mapping of antibody epitopes, detection of autoantibodies and serodiagnosis are typically conducted on microarrays. The most significant developments in synthesis, immobilization and applications of glycopeptide microarrays are covered in this review.

Distinctive MS/MS Fragmentation Pathways of Glycopeptide-Generated Oxonium Ions Provide Evidence of the Glycan Structure.

Post-translational glycosylation of proteins play key roles in cellular processes and the site-specific characterisation of glycan structures is critical to understanding these events. Given the challenges regarding identification of glycan isomers, glycoproteomic studies generally rely on the assumption of conserved biosynthetic pathways. However, in a recent study, we found characteristically different HexNAc oxonium ion fragmentation patterns that depend on glycan structure. Such patterns could be used to distinguish between glycopeptide structural isomers. To acquire a mechanistic insight, deuterium-labelled glycopeptides were prepared and analysed. We found that the HexNAc-derived m/z 126 and 144 oxonium ions, differing in mass by H2 O, had completely different structures and that high-mannose N-glycopeptides generated abundant Hex-derived oxonium ions. We describe the oxonium ion decomposition mechanisms and the relative abundance of oxonium ions as a function of collision energy for a number of well-defined glycan structures, which provide important information for future glycoproteomic studies.

A Unified Strategy for the Synthesis of Mucin Cores 1–4 Saccharides and the Assembled Multivalent Glycopeptides

By displaying different O-glycans in a multivalent mode, mucin and mucin-like glycoproteins are involved in a plethora of protein binding events. The understanding of the roles of the glycans and the identification of potential glycan binding proteins are major challenges. To enable future binding studies of mucin glycan and glycopeptide probes, a method that gives flexible and efficient access to all common mucin core-glycosylated amino acids was developed. Based on a convergent synthesis strategy starting from a shared early stage intermediate by differentiation in the glycoside acceptor reactivity, a common disaccharide building block allows for the creation of extended glycosylated amino acids carrying the mucin type-2 cores 1-4 saccharides. Formation of a phenyl-sulfenyl-N-Troc (Troc=trichloroethoxycarbonyl) byproduct during N-iodosuccinimide-promoted thioglycoside couplings was further characterized and a new methodology for the removal of the Troc group is described. The obtained glycosylated 9-fluorenylmethoxycarbonyl (Fmoc)-protected amino acid building blocks are incorporated into peptides for multivalent glycan display.

Antibody Induction Directed against the Tumor‐Associated MUC4 Glycoprotein

Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti‐tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor‐associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor‐associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem‐repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4‐based vaccines induced very strong antigen‐specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies.

Synthesis of a glycopeptide vaccine conjugate for induction of antibodies recognizing O-mannosyl glycopeptides.

In spite of the clear importance of protein O‐mannosylation in brain glycobiology, tools are lacking for specific detection, enrichment, and identification of proteins containing these modifycations. We envisioned inducing antibodies that specifically recognize O‐mannose glycans on proteins and peptides. With this in mind, we prepared a glycopeptide vaccine construct containing the N‐acetyllactosamine‐extended mannose motif Galβ1‐4GlcNAcβ1‐2ManαThr, found as a common core structure on almost all mammalian O‐mannosyl glycoproteins identified. O‐mannose glycosylated amino acid building blocks and the corresponding glycopeptides were prepared by chemical synthesis and then conjugated to an immune carrier protein. After administration of the synthetic vaccine into rabbits, strong immune responses were obtained. Further evaluation by ELISA neutralization experiments and glycopeptide microarrays showed that the induced antibodies were highly specific to the glycopeptide antigen.

Protein O-Mannosylation in the Murine Brain: Occurrence of Mono-O-Mannosyl Glycans and Identification of New Substrates.

Protein O-mannosylation is a post-translational modification essential for correct development of mammals. In humans, deficient O-mannosylation results in severe congenital muscular dystrophies often associated with impaired brain and eye development. Although various O-mannosylated proteins have been identified in the recent years, the distribution of O-mannosyl glycans in the mammalian brain and target proteins are still not well defined. In the present study, rabbit monoclonal antibodies directed against the O-mannosylated peptide YAT(α1-Man)AV were generated. Detailed characterization of clone RKU-1-3-5 revealed that this monoclonal antibody recognizes O-linked mannose also in different peptide and protein contexts. Using this tool, we observed that mono-O-mannosyl glycans occur ubiquitously throughout the murine brain but are especially enriched at inhibitory GABAergic neurons and at the perineural nets. Using a mass spectrometry-based approach, we further identified glycoproteins from the murine brain that bear single O-mannose residues. Among the candidates identified are members of the cadherin and plexin superfamilies and the perineural net protein neurocan. In addition, we identified neurexin 3, a cell adhesion protein involved in synaptic plasticity, and inter-alpha-trypsin inhibitor 5, a protease inhibitor important in stabilizing the extracellular matrix, as new O-mannosylated glycoproteins.