Periodic Reporting for period 2 - synBIOcarb (Synthetic biology of carbohydrate-binding proteins: engineering protein-carbohydrate interactions for diagnostics and cell targeting)
Berichtszeitraum: 2020-10-01 bis 2023-03-31
Every living cell is covered in a coating of complex carbohydrates and so anything that approaches the cell membrane must descend through this forest of carbohydrates. The interaction of these carbohydrates with enzymes and proteins is an essential part of healthy cell activity but at the same time many parasites, viruses, bacteria and their toxins exploit these mechanisms to colonise tissues and enter cells. Understanding such processes can lead to new methods for diagnosis and treatment of infectious diseases. In addition the carbohydrate coating of cells changes when they become cancerous so that many of their carbohydrate structures become over-expressed, or uniquely expressed making them useful cancer biomarkers that can be exploited for diagnosis, or targeted immunotherapies/drug delivery.
Our growing understanding of glycoscience, and the advent of chemical and synthetic biology methodologies, presents an opportunity to redesign and synthesise these biological components for diverse analytical, diagnostic and targeted therapeutic applications. This is the field of Synthetic Glycobiology.
synBIOcarb brings together a diverse team of chemists, structural biologists, biophysicists, cell biologists and protein engineers who are pioneering the development of Synthetic Glycobiology, and four SMEs that are leading industrial innovation in glycoscience and protein engineering.
During the project we aimed to:
1. further our fundamental understanding of structure-activity relationships for protein-carbohydrate interactions. In particular, how changes in the architecture of lectins affects their binding selectivity and ability to interact with, bend and fuse membranes together.
2. expand our toolbox of Synthetic Glycobiology building blocks that can be used to functionalise complex surfaces including supported bilayers, GUVs, living cells. This work includes chemical and enzymatic synthesis of new glycosylated lipidated peptides and use of bioorthogonal coupling to attach lectins to surfaces in defined orientations.
3. move Synthetic Glycobiology towards practical applications in diagnostic and analytical devices through developing methods to detect tumour associated cancer antigens and specific glycans important in the quality control of pharmaceutical glycoproteins.
4. move Synthetic Glycobiology towards practical applications in cell targeting and drug delivery, through investigation of how lectins attached to polymers, other lectins, or antibody fragments/mimetics, either through bioorthogonal coupling or genetic fusion, can be used to target cancer cells for drug delivery or immunotherapies.
Our growing understanding of glycoscience, and the advent of chemical and synthetic biology methodologies, presents an opportunity to redesign and synthesise these biological components for diverse analytical, diagnostic and targeted therapeutic applications. This is the field of Synthetic Glycobiology.
synBIOcarb brings together a diverse team of chemists, structural biologists, biophysicists, cell biologists and protein engineers who are pioneering the development of Synthetic Glycobiology, and four SMEs that are leading industrial innovation in glycoscience and protein engineering.
During the project we aimed to:
1. further our fundamental understanding of structure-activity relationships for protein-carbohydrate interactions. In particular, how changes in the architecture of lectins affects their binding selectivity and ability to interact with, bend and fuse membranes together.
2. expand our toolbox of Synthetic Glycobiology building blocks that can be used to functionalise complex surfaces including supported bilayers, GUVs, living cells. This work includes chemical and enzymatic synthesis of new glycosylated lipidated peptides and use of bioorthogonal coupling to attach lectins to surfaces in defined orientations.
3. move Synthetic Glycobiology towards practical applications in diagnostic and analytical devices through developing methods to detect tumour associated cancer antigens and specific glycans important in the quality control of pharmaceutical glycoproteins.
4. move Synthetic Glycobiology towards practical applications in cell targeting and drug delivery, through investigation of how lectins attached to polymers, other lectins, or antibody fragments/mimetics, either through bioorthogonal coupling or genetic fusion, can be used to target cancer cells for drug delivery or immunotherapies.
During the synBIOcarb training network, our team has made significant progress in the development of synthetic glycobiology which has led to over 25 peer-reviewed papers published to date.
We have produced novel lectins for biomedically-relevant target glycans and incorporated them into re-engineered proteins with novel properties. We have demonstrated that “superlectins” based on multimeric complexes of bacterial toxins can mediate membrane fusion. We have developed improved methods for producing such proteins on scale that has facilitated the creation of a family of such superlectins that help us to understand the structure-activity relationships for their fusogenic behaviour.
We have developed novel chimeric “Janus lectins” that can be used to crosslink homogalacturonan and fucosylated xylo-glucans to create an artificial plant cell wall on a lipid bilayer. Another Janus lectin specific for fucose and α-galactose has been shown to form proto-tissue-like structures by crosslinking glycosylated giant unilamellar vesicles, and can recognize and bind to human lung epithelial cancer cells.
We have also created “lectibodies”: bispecific constructs comprising a lectin and an antibody fragment. These lectibodies target the tumor-related glycosphingolipid Gb3, and effectively recruit cytotoxic T cells to induce cancer cell death, showcasing their potential in cancer immunotherapy.
We have successfully incorporated the unnatural amino acids azidolysine into bacterial toxins and antibody fragments to allow bioorthogonal functionalisation of the proteins. We have developed industrial manufacturing methods for the modified proteins using a growth-decoupled E. coli expression strain. A clickable Shiga Toxin B-subunit has allowed formation of lectin-drug conjugates that can selectively target Gb3-expressing tumor cells. A ‘ready-to-click’ T-cell receptor-specific scFv antibody showed effective binding to T cells at nanomolar concentrations and has potential for making controlled anti-T cell antibody-drug conjugates and imaging immune T cell migration.
We developed methods for classification and prediction of beta-propeller lectins, and used them to discover a novel lectin, SaroL-1, in choanoflagellates. This remarkable protein has a lectin domain that can bind to the glycosphingolipid Gb3 that is upregulated in some cancers, and a beta-sheet domain that can assemble into a beta-barrel that can punch a pore through the cell membrane.
We have used glycosyltransferase enzymes to make biologically important glycans to be incorporated into protocell systems. We have demonstrated the potential of soluble glycosyltransferases to modify glycolipids in synthetic membrane systems, opening avenues for engineering glycolipids for various biomedical applications.
We have developed improved methods for glycomics analysis relevant to cancer diagnosis. The application of MXene as a novel cartridge for N-glycan enrichment exhibits superior enrichment of sialylated and bisecting N-glycans compared to conventional HILIC columns. MXene-based cartridges have strong potential for glycomic analysis from human samples.
In addition we have organised five international training events equipping our early stage researchers with scientific knowledge in protein and glycan preparation, structural glycoscience. Protocell studies and glycomic analysis, alongside complementary skills training in ethics, presentation skills and outreach video production.
We have produced novel lectins for biomedically-relevant target glycans and incorporated them into re-engineered proteins with novel properties. We have demonstrated that “superlectins” based on multimeric complexes of bacterial toxins can mediate membrane fusion. We have developed improved methods for producing such proteins on scale that has facilitated the creation of a family of such superlectins that help us to understand the structure-activity relationships for their fusogenic behaviour.
We have developed novel chimeric “Janus lectins” that can be used to crosslink homogalacturonan and fucosylated xylo-glucans to create an artificial plant cell wall on a lipid bilayer. Another Janus lectin specific for fucose and α-galactose has been shown to form proto-tissue-like structures by crosslinking glycosylated giant unilamellar vesicles, and can recognize and bind to human lung epithelial cancer cells.
We have also created “lectibodies”: bispecific constructs comprising a lectin and an antibody fragment. These lectibodies target the tumor-related glycosphingolipid Gb3, and effectively recruit cytotoxic T cells to induce cancer cell death, showcasing their potential in cancer immunotherapy.
We have successfully incorporated the unnatural amino acids azidolysine into bacterial toxins and antibody fragments to allow bioorthogonal functionalisation of the proteins. We have developed industrial manufacturing methods for the modified proteins using a growth-decoupled E. coli expression strain. A clickable Shiga Toxin B-subunit has allowed formation of lectin-drug conjugates that can selectively target Gb3-expressing tumor cells. A ‘ready-to-click’ T-cell receptor-specific scFv antibody showed effective binding to T cells at nanomolar concentrations and has potential for making controlled anti-T cell antibody-drug conjugates and imaging immune T cell migration.
We developed methods for classification and prediction of beta-propeller lectins, and used them to discover a novel lectin, SaroL-1, in choanoflagellates. This remarkable protein has a lectin domain that can bind to the glycosphingolipid Gb3 that is upregulated in some cancers, and a beta-sheet domain that can assemble into a beta-barrel that can punch a pore through the cell membrane.
We have used glycosyltransferase enzymes to make biologically important glycans to be incorporated into protocell systems. We have demonstrated the potential of soluble glycosyltransferases to modify glycolipids in synthetic membrane systems, opening avenues for engineering glycolipids for various biomedical applications.
We have developed improved methods for glycomics analysis relevant to cancer diagnosis. The application of MXene as a novel cartridge for N-glycan enrichment exhibits superior enrichment of sialylated and bisecting N-glycans compared to conventional HILIC columns. MXene-based cartridges have strong potential for glycomic analysis from human samples.
In addition we have organised five international training events equipping our early stage researchers with scientific knowledge in protein and glycan preparation, structural glycoscience. Protocell studies and glycomic analysis, alongside complementary skills training in ethics, presentation skills and outreach video production.
During the project we have discovered previously unknown lectins such as SaroL-1, which presents opportunities for cancer therapy based on carbohydrate-dependent membrane pore-formation. We have combined lectin domains into novel multimeric architectures opening routes to constructing artificial tissues and cell walls. We have gained new understanding in how reengineered lectins can achieve membrane disruption which could allow the development of drug delivery systems that can target specific tissues. We have created novel hybrid structures including lectibodies that have potential in cancer immunotherapy. We have developed industrially relevant expression systems for incorporating non-canonical amino acids into proteins to make bacterial toxins and single chain fragment variable antibodies that can be used to develop the next generation of targeted drug delivery systems or immunotherapies. We have created new methods for functionalising surfaces with glycans that could be used in diagnostic devices to detect bacterial toxins. We have developed novel methods for glycomics analysis that have potential to improve glycomics-based cancer diagnostic methods.