Phage capsid nanoparticles with defined ligand arrangement block influenza virus entry

Multivalent interactions at biological interfaces occur frequently in nature and mediate recognition and interactions in essential physiological processes such as cell-to-cell adhesion. Multivalency is also a key principle that allows tight binding between pathogens and host cells during the initial stages of infection. One promising approach to prevent infection is the design of synthetic or semisynthetic multivalent binders that interfere with pathogen adhesion1,2,3,4. Here, we present a multivalent binder that is based on a spatially defined arrangement of ligands for the viral spike protein haemagglutinin of the influenza A virus. Complementary experimental and theoretical approaches demonstrate that bacteriophage capsids, which carry host cell haemagglutinin ligands in an arrangement matching the geometry of binding sites of the spike protein, can bind to viruses in a defined multivalent mode. These capsids cover the entire virus envelope, thus preventing its binding to the host cell as visualized by cryo-electron tomography. As a consequence, virus infection can be inhibited in vitro, ex vivo and in vivo. Such highly functionalized capsids present an alternative to strategies that target virus entry by spike-inhibiting antibodies5 and peptides6 or that address late steps of the viral replication cycle

Retrospective evaluation of whole exome and genome mutation calls in 746 cancer samples

Funder: NCI U24CA211006Abstract: The Cancer Genome Atlas (TCGA) and International Cancer Genome Consortium (ICGC) curated consensus somatic mutation calls using whole exome sequencing (WES) and whole genome sequencing (WGS), respectively. Here, as part of the ICGC/TCGA Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium, which aggregated whole genome sequencing data from 2,658 cancers across 38 tumour types, we compare WES and WGS side-by-side from 746 TCGA samples, finding that ~80% of mutations overlap in covered exonic regions. We estimate that low variant allele fraction (VAF < 15%) and clonal heterogeneity contribute up to 68% of private WGS mutations and 71% of private WES mutations. We observe that ~30% of private WGS mutations trace to mutations identified by a single variant caller in WES consensus efforts. WGS captures both ~50% more variation in exonic regions and un-observed mutations in loci with variable GC-content. Together, our analysis highlights technological divergences between two reproducible somatic variant detection efforts

Development and Evaluation of Tools to Explore Posttranslational HexNAc-Tyrosine and Mucin-Type O-Glycosylation

Glycosylation is the most abundant form of post-translational modification (PTM). Recently, O-glycosylation attracted much attention in the glycoproteomic field due to its association with various diseases, such as pathogenic infections and cancer. However, glycoproteomic analysis of O-linked glycosylation is highly challenging due its structural diversity and complexity. New and efficient methods need to be developed to obtain a better understanding of the biological functions of O-glycans. In the presented thesis, glycopeptide microarrays were used as tools to explore the role of mucin type O-glycosylation in cancer, bacterial adhesion processes and galectin recognition on a molecular level, and to get insights into a new group of tyrosine O-glycosylation. A better understanding of these carbohydrate-protein interactions on a molecular level could facilitate the development of glycomimetic inhibitors to fight bacterial infections or block glycan binding proteins involved in cancer progression, or improve the design of novel carbohydrate-based cancer vaccines. In the first part of this work, tools were developed to elucidate the role of a novel group of PTMs, where N-acetylhexosamine (HexNAc = α-GalNAc, α- or β-GlcNAc) was found to modify the hydroxyl group of tyrosine. Synthetic glycopeptides carrying this new modification, as well as glycopeptide microarray libraries were prepared to evaluate the abilities of plant lectins (carbohydrate-binding proteins) to detect HexNAc-O-Tyr modifications. These lectins are commonly used in glycoproteomic work flows to detect and enrich glycopeptides and -proteins. Additionally, HexNAc-O-Tyr-specific rabbit antibodies were raised and immunologically analyzed by enzyme-linked immunosorbent assays, western blot and microarray binding studies. In the second part of the presented thesis, synthetic mucin glycopeptide microarray libraries were prepared and employed to explore carbohydrate-protein interactions of galectins, bacterial lectins and tumor specific antibodies. Mucin glycoproteins are part of the mucus barrier that protects the host against invading pathogens. However, bacteria and viruses have co-evolved with the human host and have developed strategies to promote virulence, for example by adhering to glycans on the host cell-surface. To combat bacterial infections, their virulence and pathogenicity must be understood on a molecular level. In this work, mucin glycopeptides were enzymatically modified with different fucose motifs and used to determine the fine binding specificities of fucose-recognizing lectins LecB from Pseudomonas aeruginosa and the Clostridium difficile toxin A. Furthermore, a synthesis strategy was developed to generate simplified mucin core glycopeptides that could be used as scaffolds to enzymatically generate LacdiNAc modified glycopeptides. They could be used in microarray binding studies to evaluate the glycan binding preferences of various proteins, including the Helicobacter pylori lectin LabA and human galectins, which play roles in cancer development and progression. Aberrant glycosylation of mucin glycoproteins has been associated with various types of cancer. Tumor specific carbohydrate antigens on mucins represent attractive antigenic targets for the development of effective anti-cancer vaccines. In this work, antibodies induced by tumor-associated MUC1 glycopeptide-bacteriophage Qβ vaccine conjugates were immunologically analyzed using MUC1 glycopeptide microarray libraries

Novel Approaches To Design Glycan-Based Antibacterial Inhibitors

The interactions between bacterial lectins and carbohydrates on the host cell surface can mediate bacterial adhesion, invasion, and immune evasion. Multivalency plays a key role in these binding events. However, additional molecular mechanisms greatly impact multivalent binding recognition. To develop specific and effective bacterial inhibitors, a deeper understanding of the complex underlying mechanisms of bacterial adhesion processes is necessary. By interfering with bacterial adhesion, synthetic multivalent glycoconjugates do not only have the potential to improve or replace antibiotic treatments, but also represent useful tools to study carbohydrate-pathogen interactions. In this review, we highlight a few recent advances in the synthesis and application of synthetic glycan-based scaffolds to uncover the nature of glycan-bacteria interactions and to design efficient bacterial inhibitors

Glycopeptides and -Mimetics to Detect, Monitor and Inhibit Bacterial and Viral Infections : Recent Advances and Perspectives

The initial contact of pathogens with host cells is usually mediated by their adhesion to glycan structures present on the cell surface in order to enable infection. Furthermore, glycans play important roles in the modulation of the host immune responses to infection. Understanding the carbohydrate-pathogen interactions are of importance for the development of novel and efficient strategies to either prevent, or interfere with pathogenic infection. Synthetic glycopeptides and mimetics thereof are capable of imitating the multivalent display of carbohydrates at the cell surface, which have become an important objective of research over the last decade. Glycopeptide based constructs may function as vaccines or anti-adhesive agents that interfere with the ability of pathogens to adhere to the host cell glycans and thus possess the potential to improve or replace treatments that suffer from resistance. Additionally, synthetic glycopeptides are used as tools for epitope mapping of antibodies directed against structures present on various pathogens and have become important to improve serodiagnostic methods and to develop novel epitope-based vaccines. This review will provide an overview of the most recent advances in the synthesis and application of glycopeptides and glycopeptide mimetics exhibiting a peptide-like backbone in glycobiology

Antibodies directed against GalNAc- and GlcNAc-O-Tyrosine posttranslational modifications – a new tool for glycoproteomic detection

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 top 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

Antibodies directed against GalNAc- and GlcNAc-O-Tyrosine posttranslational modifications – a new tool for glycoproteomic detection

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.Originally included in thesis in manuscript form. </p

Photochemical Degradation of Various Bridge-Substituted Fluorene-Based Materials

Photochemical degradation is an important issue to be overcome in advancing the lifetime of fluorene-containing conjugated polymers. In order to optimize the inertness of the materials, a quantitative measure for the efficiency of degradation is needed. Here, we introduce a method to measure a relative quantum yield of the photochemical degradation by monitoring the kinetics of the process by means of UV/vis spectroscopy and liquid chromatography (LC) techniques. This method is employed to a set of differently substituted 2,7-diphenylfluorenes, serving as model compounds for polyfluorene materials. Our measurements show that the quantum yield changes by orders of magnitude upon varying the bridge substituents and that altered kinetics indicate changing degradation mechanisms

Sialyl-tn antigen-imprinted dual fluorescent core-shell nanoparticles for ratiometric Sialyl-Tn antigen detection and dual-color labeling of cancer cells

Sialyl-Tn (STn or sialyl-Thomsen-nouveau) is a carbohydrate antigen expressed by more than 80% of human carcinomas. We here report a strategy for ratiometric STn detection and dual-color cancer cell labeling, particularly, by molecularly imprinted polymers (MIPs). Imprinting was based on spectroscopic studies of a urea-containing green-fluorescent monomer 1 and STn-Thr-Na (sodium salt of Neu5Acα2-6GalNAcα-O-Thr). A few-nanometer-thin green-fluorescent polymer shell, in which STn-Thr-Na was imprinted with 1, other comonomers, and a cross-linker, was synthesized from the surface of red-emissive carbon nanodot (R-CND)-doped silica nanoparticles, resulting in dual fluorescent STn-MIPs. Dual-color labeling of cancer cells was achieved since both red and green emissions were detected in two separate channels of the microscope and an improved accuracy was obtained in comparison with single-signal MIPs. The flow cytometric cell analysis showed that the binding of STn-MIPs was significantly higher (p &lt; 0.001) than that of non-imprinted polymer (NIP) control particles within the same cell line, allowing to distinguish populations. Based on the modularity of the luminescent core-fluorescent MIP shell architecture, the concept can be transferred in a straightforward manner to other target analytes

Fucose binding motifs on mucin core glycopeptides impact bacterial lectin recognition

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