A plethora of N-glycosylation pathways from the epsilon Proteobacteria - a resource for glycoprospecting and toolbox for glycoengineering

Glycoproteins (proteins that are modified with sugar structures) are ubiquitous biomolecules involved in most basic biological phenomena in complex living organisms such as humans, ranging from immune recognition to cancer development. They often have underestimated biological functions and in contrast to proteins and nucleic acids, glycans have escaped the cloning revolution. The experimentally tractable model bacterium Escherichia coli is often used as a 'cellular factory' to produce practically inexhaustible amounts of purified proteins for various uses. However, until recently it has not been possible to generate glycoproteins in this bacterium as these simple organisms do not make glycoproteins of this type. This how now changed. We recently identified and characterised a cluster of pgl genes which is responsible for the synthesis of glycoproteins in the simple gut bacterium Campylobacter jejuni. This is the first bacterium known to glycosylated their proteins in this way. Furthermore we have been able to transfer the segment of C. jejuni DNA containing the pgl genes into E. coli to produce recombinant glycoproteins, thus opening up the field of glycoengineering. The key enzyme in the C. jejuni pathway that couples proteins to sugars is the transferase protein termed CjPglB. Although CjPglB can transfer many sugar structures unfortunately there are many important glycostructures that it cannot. Recently we have identified dozens more bacteria that are related to C. jejuni that have different PglB sugar transferase enzymes. Indeed some of the bacteria have more than one PglB, the first time that this has been observed in bacteria, suggesting that they may have subtly different abilities to transfer different sugars and hence be invaluable for glycoengineering. In this proposal we wish to fully characterize the plethora of new PglB enzymes and their associated pathways to expand the range of genetic tools that could be used for glycoengineering. The proposal will also help answer fundamental questions as to why some bacteria require more than one PglB and the evolutionary origin of these unusual systems in bacteria. The program of work will benefit scientists interested in basic research and also in applied research particularly in the burgeoning glycobiotechnology industry.

We along with collaborators have characterized the first proven Bacterial N-linked glycosylation system in Campylobacter jejuni and subsequently transferred this system to E. coli to produce for the first time recombinant glycoproteins. The key enzyme in this process, encoded by the C. jejuni pglB gene, is the oligosaccharyltransferase that couples glycan to protein. Recently we have identified many more PglB-based bacterial glycosylation systems in species from the delta/epsilon Proteobacteria. These contain pglB genes associated with diverse sets of glycosyltransferases suggesting structural diversity in N-linked glycans. Some species encode two distinct PglB enzymes (a first for bacterial species) and may have dual glycosylation systems. This not only expands the range of PglB enzymes to investigate, but also provides novel glycostructures encoded by glycosyltransferases and associated enzymes within the variant Pgl pathways. We will (i) exploit a fluorescent peptide based assay for oligosaccharyltransferase activity to obtain PglB derived N-linked glycopeptides from a wide range of species for both structural analysis and to investigate the role of individual enzymes in glycan biosynthesis, (ii) explore the role of dual PglB glycosylation systems, (iii) identify novel glycoproteins in selected Helicobacter and Campylobacter species and (iv) further develop assays to screen the range of glycan specificity of diverse PglBs from the epsilon Proteobacteria. These studies will further our understanding of the evolution of N-linked glycosylation and act as tractable systems to understand more complex Eukaryotic N-linked glycosylation systems. Characterisation of novel oligosaccharyltransferases, associated glycosyltransferases and glycan structures in the Pgl pathways will expand our knowledge of these important enzymes and structures. Overall this will significantly contribute to the toolbox for glycoengineering with concomitant applications in glycobiotechnology.

The impact of the proposed basic research is likely to be considerable in terms of glycoengineering and glycobiotechnology. For example, the most successful vaccines such as those that protect against the deadly bacteria Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae are glycoconjugate vaccines. However, using current technology it is difficult to produce and manufacture such vaccines requiring purification of the glycan from the native pathogen and chemical coupling to a protein carrier. Our pioneering studies on the PglBs and the demonstration that they can be used to produce recombinant glycans, promises to resolve these technological problems. Given that vaccination is a core public health measure in reducing the infectious disease burden, the proposed studies could make a highly significant impact for the health and well-being of both animals and humans. A longer-term impact would be to develop the Pgl-based technology for the modification of human proteins used as therapeutics in the Pharmaceutical industry. Although there are technological hurdles to overcome before this is a reality, the proposed research to understand the basic function of dual N-linked bacterial glycosylation systems will contribute to this long-term goal. The characterization of glycan biosynthetic pathways using the innovative fluorescent-labelled peptide system will impact synthetic biology approaches providing cassettes of genes that encode known glycan structures that could have diverse functions. We propose to disseminate our studies through publication in international peer-reviewed journals (where the applicants have a strong record) and by poster and oral presentations at major national and international meetings. The applicants have a track record of communicating the results of their research to the public and the mass media. Where possible these links will be used to promote this research. The potential impact of the research will also be realised though our respective technology transfer offices, material transfer agreements and patents. We will continue to collaborate with Glycovaxyn, a 40+SME dedicated to the design and manufacture of glycoconjugate vaccines using PglB-based technology. Glycovaxyn and other potential biotechnology companies with an interest in glycoengineering and/or vaccine development would provide the route to realize the potential of the proposed work subject to appropriate licensing agreements. Currently, we have an international lead in the study and exploitation of bacterial N-linked protein glycosylation systems and it is imperative that we continue these basic studies coupled with glycotool development to maintain this lead.