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

Lead Research Organisation: London Sch of Hygiene and Trop Medicine
Department Name: Infectious and Tropical Diseases

Abstract

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.

Technical Summary

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.

Planned Impact

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.
 
Description A large number of proteins in biological systems are modified with sugars, ie the proteins are glycosylated to form glycoproteins. Glycoproteins and the associated glycan structures are ubiquitous biomolecules involved in many biological processes ranging from immune recognition to cancer development. It has become clear that glycan structures have important, often underestimated biological functions. In contrast to protein and DNA, glycoproteins have escaped biotechnological applications so far. This is primarily due to the complexity of these essential biosynthetic pathways in mammailian cells. Comparison of well-characterised protein sequence database entries indicates that more than half of all proteins in nature will eventually be identified as glycoproteins. Glycoengineering-the synthesis of novel glyco-structures-is essential for studies to elucidate the crucial role of glycosylation in many aspects of biology. Glycoengineering is in its infancy, and with few suitable glycosylation pathways known in bacteria, producing recombinant glycoproteins in simple workhorse hosts such as E. coli has not been possible.

We have identified an N-linked general glycosylation pathway in bacteria from the epsilon proteobacteria that can add sugar structures to proteins to produce glycoproteins.

Thus an aim of this study was to characterise further general glycosylation systems among the epsilon proteobacteria . We identified and characterised three novel N-linked system from bacteria found in deep-sea vents found in some oceans. These appear to have different specificity and useful additions to the "Glyco-toolbox"
Exploitation Route We have identified an N-linked general glycosylation pathway in bacteria from the epsilon proteobacteria that can add sugar structures to proteins to produce glycoproteins. We have been able to clone this pathway into E. coli to produce recombinant glycoproteins. This discovery has had enormous advantages to tailor make production-line quality glycoproteins that are required in industry and medicine. Protein Glycan Coupling Technology (PGCT) as a method to produce recombinant glycoconjugates molecules in E. coli is now regularly used in academia and industry for multiple glycoengineering purposes. For example, in the vaccine industry used by GlycoVaxyn (recently purchased by GSK) and VaxAlta. The principles of PGCT have been used as a platform technology to initiate a Spin Out company ArcVax at the LSHTM.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description In this research program we were able to 1) efficiently express bacterial glycans such as O-antigens in E. coli and 2) transfer the O-antigen to a carrier protein in E. coli using novel oligosaccharyltransferase. This has helped to establish Protein Glycan Coupling Technology (PGCT) as a method to produce recombinant glycoconjugate vaccines in E. coli. This has potential in the vaccine industry used by GlycoVaxyn (recently purchased by GSK) and VaxAlta. The principles of PGCT have been used as a platform technology to initiate a Spin Out company ArcVax at the LSHTM.
First Year Of Impact 2010
Sector Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Description DSTL Glycoconjugate vaccine design
Amount £450,000 (GBP)
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom
Start 09/2011 
End 09/2014
 
Description DSTL Glycoconjugate vaccine design for Coxiella
Amount £600,000 (GBP)
Organisation Defence Science & Technology Laboratory (DSTL) 
Sector Public
Country United Kingdom
Start 08/2012 
End 08/2015
 
Description Developing a multivalent Streptococcus pneumoniae recombinant glycoconjugate vaccine for preventing meningitis'
Amount £220,000 (GBP)
Organisation Meningitis Now 
Sector Charity/Non Profit
Country United Kingdom
Start 04/2020 
End 04/2022
 
Description Developing and expanding the bacterial glycotoolbox for animal pathogens
Amount £200,000 (GBP)
Funding ID BB/M01925X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2015 
End 09/2017
 
Description Developing the E. coli GlycoCell
Amount £455,000 (GBP)
Funding ID BB/R008124/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 06/2018 
End 06/2021
 
Description Gates Grand Challenge
Amount $1,000,000 (USD)
Organisation Bill and Melinda Gates Foundation 
Sector Charity/Non Profit
Country United States
Start 03/2011 
End 03/2014
 
Description Glycoengineering of Veterinary Vaccines
Amount £5,300,000 (GBP)
Funding ID BB/N001591/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2016 
End 04/2021
 
Description The Future Vaccine Manufacturing Research Hub (Vax-Hub)
Amount £7,000,000 (GBP)
Funding ID EP/R013756/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 10/2018 
End 10/2022
 
Description Vaccine development for defence purposes 
Organisation Defence Science & Technology Laboratory (DSTL)
Country United Kingdom 
Sector Public 
PI Contribution New vaccine technology platform for making glycoconjugate vaccines against Burkholderia pseudomallei and Francisella tulerensis
Collaborator Contribution Technology know how, specific animal testing facilities
Impact Several vaccine candidates tested and some may go forward to vaccine trials
 
Description huvepharma 
Organisation Huvepharma
Country Bulgaria 
Sector Private 
PI Contribution Intellectual contribution, vaccine development and production.
Collaborator Contribution Intellectual contribution, vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Impact Intellectual contribution and vaccine development and production. Vaccine technology Vaccine upscaling and manufacture
Start Year 2020
 
Title Glycoconjugate Vaccines 
Description Novel method to construct combinations of glycoconjugate vaccines 
IP Reference WO4307P 
Protection Patent granted
Year Protection Granted
Licensed No
Impact Can cheaply produce novel glycoconjugate vaccines for humans and animals
 
Title Glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen 
Description New method for constructing glycoconjugate vaccine conferring protection against Francisella tularensis infections and a method to manufacture a glycoconjugate antigen 
IP Reference WO2018046955 
Protection Patent granted
Year Protection Granted 2015
Licensed Commercial In Confidence
Impact Method to make vaccines against Francisella tularensis for which there is no current human vaccine
 
Title MAGIC Mobile-element Assisted Glycoconjugate Insertion on Chromosome 
Description A method to improve glycosylation of proteins 
IP Reference US20150344928 
Protection Patent application published
Year Protection Granted 2018
Licensed No
Impact A method that could produce inexpensive glycoproteins
 
Title Oligosaccharyltransferase Polypeptide 
Description New enzyme to improve glycoengineering in E. coli 
IP Reference GB1704103.9 
Protection Patent application published
Year Protection Granted 2018
Licensed Commercial In Confidence
Impact Improve glycoengineering in the E. coli cell
 
Title Recombinant Protein Production 
Description The use of Protein Glycan Coupling Technology to produce low cost recombinant vaccines. In this example against Francisella tulerensis 
IP Reference GB1606036.0 
Protection Patent granted
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact The establishment of vaccine technology platform to engineer low cost recombinant glycoconjugate vaccines
 
Title Whole Cell Vaccines 
Description Development of Protein Glycan Coupling Technology for vaccines for veterinary purposes 
IP Reference GB1603958.8 
Protection Patent application published
Year Protection Granted 2016
Licensed Commercial In Confidence
Impact The establishment of vaccine technology platform to engineer low cost recombinant glycoconjugate vaccines
 
Description Broadcast interviews (Newsnight, Panorama, One Show) 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Public/other audiences
Results and Impact Interest in research at LSHTM

Better understanding of science
Year(s) Of Engagement Activity 2009,2010,2011,2012,2013,2014