Synthetic glycobiology

Cellular membranes comprise the physical barrier of a cell towards the environment performing a variety of functions, including communication with other cells or pathogens. A large portion of the cell surface constitutes of complex glycans, many with specific adhesion properties. To elucidate such interactions various assay techniques are utilised, a majority based on flat rigid surfaces such as ELISA and microarray glass slides. Spherical cell-size Giant Unilamellar Vesicles (GUVs) are increasingly used as cell models to study membrane interactions. The group has developed a fully synthetic method to produce fluorescently labelled cholesterylated glycopeptides for efficient incorporation into GUV membranes, forming an artificial glycocalyx. With this system the group has begun evaluating membrane receptor interactions with pathogens such as the Malaria infected red blood cells and intact viruses.

GalNac type O-glycosylation, contrary to N-glycosylation, is typically started in the Golgi apparatus. The polypeptide N-acetyl-Galactosamyl transferases (GALNTs) are initiating the pathway by generating the Tn glycan. GALNTs act on thousands of sites in cell surface and secreted proteins. GALNTs can be relocated from Golgi to the ER upon Src activation, a process called GALA. Dr Bard finds that GALA stimulates the glycosylation of about 20% of detectable glycosites. Roughly 200 proteins and 800 glycosites are strongly affected. Many of these proteins have been implicated in tumorigenesis. One of them is the metalloprotease MMP14, whose glycosylation strongly increases activity. GALA also affects the ER resident protein Calnexin, which is found to also be required for ECM degradation. In a mouse model of liver tumour, stimulating GALA leads to a strong acceleration of tumour progression, while inhibiting it blocks it. In sum, it is proposed that GALA is a glycosylation program that controls matrix degradation and tissue invasion and is required for tissue remodelling during tumour growth.

Glycans (also known as carbohydrates, saccharides or, simply, sugars) are among the most intriguing carriers of biological information in living systems. The structures of glycans not only convey the cells’ physiological state, but also regulate cellular communication and responses by engaging receptors on neighbouring cells and in the extracellular matrix. Despite their structural complexity, individual glycans rarely engage their protein partners with high affinity. Yet, glycans modulate biological processes with exquisite selectivity and specificity. To correctly evaluate glycan interactions and their biological consequences, one needs to look beyond individual glycan structures and consider the entirety of the cell-surface landscape. There, glycans are presented on protein scaffolds, or are linked directly to membrane lipids, forming a complex, hierarchically organised network with specialised functions, called the glycocalyx. Professor Godula's research program focuses on the development of nanoscale glycomaterials, which can mimic the various components of the glycocalyx, together with chemical methods for cell surface engineering to reveal how the presentation of glycans within the glycocalyx can influence their biological functions. In this presentation, Professor Godula will describe the recent efforts in this area, placing emphasis on the applications of glycomaterials to provide new insights into the mechanisms through which glycans mediate cellular differentiation and host-pathogen interactions.

Glycan-mediated interactions play critical roles in diverse biology processes. However, glycan-mediated interactions are often low-affinity and transient, impeding their characterisation by traditional biochemistry methods. Using synthetic sugar analogs, Professor Kohler's group has been able to incorporate a photoactivatable function group into glycoconjugates in living cells. Professor Kohler will present data demonstrating that both photocrosslinking GlcNAc and sialic acid can be incorporated into cell surface glycoconjugates in place of natural sugars. Cells metabolically labelled with photocrosslinking sugars can be subjected to UV irradiation, resulting in covalently crosslinking between glycoconjugates and neighbouring molecules. These crosslinked complexes can be characterised by standard biochemistry methods, including immunoblot and mass spectrometry-based proteomics analysis. Professor Kohler will also describe how the group used photocrosslinking sugar technology to determine that cholera toxin subunit B (CTB) recognises fucosylated glycoproteins on the surface of human colonic epithelial cells. The results of these experiments have led to the discovery that fucosylated molecules function in host cell intoxication, and to new fucose-based strategies that can competitively inhibit CTB binding to host cells and potentially block host cell intoxication. Metabolically incorporated photocrosslinking sugars can potentially be used to capture and define many molecular recognition events in which glycans play a critical role.

Dr Dwek will describe studies undertaken using lectins to identify proteins showing alterations in O-linked glycosylation in cancer. The strategies taken to identify proteins with altered glycosylation in cancer in both colorectal and breast cancer validated using tissue samples from patients and highlighting commonalities of glycosylation changes in both tumour types will be described. Serum tests for cancer are often based on glycoproteins but in the majority of tests the protein levels are monitored whilst glycosylation is ignored. A glycoproteomic approach was used to identify serum proteins with altered glycosylation in metastatic breast cancer. This was accompanied by development of a rapid screening method with a lectin-based recognition system. In this manner, serum biomarkers were identified – this will be described with respect to VE-cadherin – a biomarker for metastatic oestrogen receptor positive breast cancer. Glycosidases have been identified as mediators of cancer cell invasion and glycosidase inhibitors have emerged as potential compounds for prevention of cancer cell invasion. The pivotal role of the glycocalyx as a barrier, preventing cancer drug interaction at the cell surface, has led to development of a novel biosensor system for studying cell-protein interactions; this will be described alongside recent approaches for enhancing interaction between the drug trastuzumab (Herceptin) and HER2 in breast cancer. The importance of glycosylation in modulating the responsiveness of cancer cells to treatments as well novel approaches to enhancing cancer treatments will be discussed.

In this contribution Dr Tkac will describe an alternative to mass spectrometry-based analysis of glycans by the use of lectins for development of various types of bioanalytical devices. Application of lectins allows us to analyse glycans of the glycoproteins directly on an intact protein backbone. There is no need to release glycans from the proteins, which significantly simplifies assay procedure. The devices developed so far are based on electrochemical biosensors, fluorescent lectin microarrays and lectin-based ELLA (Enzyme-linked lectin assays) like platform of analysis. The most recent approach involves the use of magnetic nanoparticles in combination of ELLA for highly sensitive assay format requiring small sample consumption. The potential of lectin-based glycan analysis will be provided by the use of the bioanalytical devices for analysis of serum samples from people who have prostate cancer, breast cancer, rheumatoid arthritis and chronic lymphocytic leukaemia. The clinical potential of magnetic ELLA for diagnostics of prostate cancer will be discussed.

The last part of the presentation will show application of glycan biosensors with an immobilised Tn antigen to detect antibodies. Two different approaches were applied for immobilisation of a small glycan either directly to a modified surface (2D biosensor) or to a layer of human serum albumin formed on the surface (3D biosensor) and these two approaches are compared.

Professor Davletov will present the evidence showing that a chimera made of cholera and botulinum toxins gives long-lasting pain relief in rats without adverse effects and, in time, could replace opioid drugs as a safe and effective way of treating chronic pain. Cholera toxin is known to target GM1 gangliosides present on neurons and the cholera ganglioside binding subunit has been used widely as a strong neuronal tracer. The group deconstructed the botulinum (‘Botox’) molecule, which blocks neuromuscular junctions, and reassembled it with the ganglioside-binding part of the cholera toxin to make Cho-Bot – a compound which successfully targets pain neurons. Injected under the skin just once, Cho-Bot relieves chronic pain induced by a chemotherapeutic drug in a rodent model. It doesn’t affect muscles like the Botox used to reduce wrinkles but it does block nerve pain for up to four months without affecting normal pain responses. Thus Cho-Bot could avoid the adverse events of tolerance and addiction often associated with repeated opioid drug use. Chronic pain of ‘moderate to severe’ intensity is widespread affecting 7.8 million people in the UK and 19% of adult Europeans. Opioids like morphine are considered to be the gold standard for pain relief but there is little evidence that their long-term use is effective in treating chronic pain.

In summary, to prepare Cho-Bot the group developed a molecular Lego system which allows us to link the botulinum 'warhead' to a navigation molecule, in this case, the ganglioside binding cholera binding subunit, allowing the creation of widely desired long-lasting pain killer without the side effects of opioids.

Conditionally replicating (oncolytic) adenoviruses are promising tools for therapy of solid tumours but their wildtype tropism lacks effective recognition and binding to tumour cells. Genetic modification of the adenoviral fibers has proven difficult since frequently used tumour ligands such as scFvs or TCRs are usually incompatible with synthesis and assembly of adenoviral capsid proteins. In this study, Professor Gerardy-Schahn utilised endosialidase NF (endoNF), the trimeric tailspike protein of bacteriophage K1F, to replace the entire knob part of adenoviral fiber and to facilitate oncolytic adenovirus infection of tumours expressing the cell surface antigen polysialic acid (polySia). For this purpose, the group exchanged the fiber gene locus of the oncolytic adenovirus hTert-Ad by genes for fiber-endoNF fusion protein comprising the fiber capsid anchoring domain and an N-terminally truncated form of endoNF. Functional incorporation of chimeric fibers into the capsids of viral progeny was demonstrated by electron microscopic images of viral particles and detection of endoNF activity on isolated capsids. Chimeric viruses effectively infected cells in a polySia-dependent manner as shown by in an isogenic assay using HEK293 cells with or without polySia expression. Interestingly, the group found that the ability of endoNF to degrade polySia was essential for infection. Investigations on the oncolytic properties in comparison with the maternal virus hTertAd showed significantly improved oncolysis in a polysialic acid-expressing human neuroblastoma cell line and effective inhibition of tumour growth in subcutaneous neuroblastoma in nude mice. In summary, the group's engineered oncolytic adenoviruses with a genetically stable tropism towards polysia-expressing tumours represent promising tools for the treatment of clinically relevant cancers such as small cell lung cancer and neuroblastoma.