Advanced glycosaminoglycan sequencing
Lead Research Organisation:
Keele University
Department Name: Inst for Science and Tech in Medicine
Abstract
The interaction between proteins and cell-surface ligands underpin many biological processes such as cell growth, homeostasis, apoptosis and the ability of pathogens to invade host cells. One important family of cell surface ligands are the carbohydrate family of glycosaminoglycans (GAGs) found on the surface of almost all mammalian cells. Analysis of the sequence of the linear block-like arrangement of these GAGs is a significant technical challenge due to only miniscule quantities of pure material being (readily) available, poor chemistries and insensitive detection equipment.
This research aims to develop and test a new method for sequencing these GAG polysaccharides utilising recent fundamental improvements made by the applicant. These developments include the exploitation of the opposite end of the molecule than is conventionally used (non-reducing end), with a vastly improved labelling mechanism and an advanced detection system for the conventional end of the molecule (reducing end). This approach will provide a powerful and sensitive sequencing technique capable of employing both ends of the GAG saccharide for characterization.
The information gained from GAG sequencing will enable researchers to study previously elusive structures and processes that are biologically and medically significant.
This research aims to develop and test a new method for sequencing these GAG polysaccharides utilising recent fundamental improvements made by the applicant. These developments include the exploitation of the opposite end of the molecule than is conventionally used (non-reducing end), with a vastly improved labelling mechanism and an advanced detection system for the conventional end of the molecule (reducing end). This approach will provide a powerful and sensitive sequencing technique capable of employing both ends of the GAG saccharide for characterization.
The information gained from GAG sequencing will enable researchers to study previously elusive structures and processes that are biologically and medically significant.
Technical Summary
The sequence determination of the biologically important glycosaminoglycan (GAG) polysaccharides remains a significant technical challenge for several reasons. These include a difficulty in obtaining sufficient quanitties of purified GAGs from biological samples, problematic labelling chemistries, insensitive instrumentation and the reliance on rare, and thus expensive, exoglycosidase enzymes to elucidate sequence identity.
This project will develop, evaluate and validate a novel approach in which two fundamental and radical improvements are incorporated. These are to combine a recently developed approach for efficient tethering of GAG saccharides via their non-reducing ends together with a vastly improved fluorescent label (BODIPY) and state-of-the-art detection system (LIF), developed in collaboration with an Industrial partner (MI Engineering Ltd.). This permits a novel "dual-end" sequencing method to be developed, permitting access to sequence information from both ends of the molecule, and providing two complementary opportunities to obtain sequence information.
This improved sequencing strategy with vastly improved detection (at least 20,000 fold, accessing zeptomole levels for the first time) will provide a powerful sequencing tool for the high throughput analysis of biologically and medically important GAGs, and open up new opportunities to exploit GAGs in biomedical applications.
This project will develop, evaluate and validate a novel approach in which two fundamental and radical improvements are incorporated. These are to combine a recently developed approach for efficient tethering of GAG saccharides via their non-reducing ends together with a vastly improved fluorescent label (BODIPY) and state-of-the-art detection system (LIF), developed in collaboration with an Industrial partner (MI Engineering Ltd.). This permits a novel "dual-end" sequencing method to be developed, permitting access to sequence information from both ends of the molecule, and providing two complementary opportunities to obtain sequence information.
This improved sequencing strategy with vastly improved detection (at least 20,000 fold, accessing zeptomole levels for the first time) will provide a powerful sequencing tool for the high throughput analysis of biologically and medically important GAGs, and open up new opportunities to exploit GAGs in biomedical applications.
Planned Impact
The biologically and medically family of carbohydrates, the glycosaminoglycans (GAGs), are present on the cell surface of almost all mammalian cells and are implicated in many crucial biological mechanisms such as cell growth, division, homeostasis and pathogen invasion. However, analysis of their sequence is a significant technical challenge compounded by the difficulty in obtaining sufficient purified material from biological samples. Sequencing techniques to date rely on relatively large amounts of starting material and are generally difficult, labour intensive, time consuming and relatively insensitive.
The successful development of a rapid, sensitive and high throughput sequencing strategy will permit rapid progress to be made on investigations into the structure:function relationships of GAG saccharides and will ultimately contribute significantly to our understanding of GAG polysaccharides as major components of the glycome. This is a crucial facet of post-genome science, and will open up major new exploitation.
The successful development of a rapid, sensitive and high throughput sequencing strategy will permit rapid progress to be made on investigations into the structure:function relationships of GAG saccharides and will ultimately contribute significantly to our understanding of GAG polysaccharides as major components of the glycome. This is a crucial facet of post-genome science, and will open up major new exploitation.
Organisations
- Keele University (Lead Research Organisation)
- Engineering and Physical Sciences Research Council (Co-funder)
- INTELLIHEP LTD (Collaboration)
- Queensland University of Technology (QUT) (Collaboration)
- San Raffaele Hospital (Collaboration)
- PUBLIC HEALTH ENGLAND (Collaboration)
- MI Engineering Ltd (Collaboration)
- Federal University of São Paulo (Collaboration)
- National Institute for Biological Standards and Control (NIBSC) (Collaboration)
- Liverpool John Moores University (Collaboration)
- Zucero Therapeutics (Collaboration)
- Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni (Collaboration)
- University of Queensland (Collaboration)
- Rosalind Franklin Institute (Collaboration)
- UNIVERSITY OF LIVERPOOL (Collaboration)
- M I Engineering Ltd (Project Partner)
People |
ORCID iD |
| Mark Skidmore (Principal Investigator) |
Publications
Boyle M
(2017)
Identification of Heparin Modifications and Polysaccharide Inhibitors of Plasmodium falciparum Merozoite Invasion That Have Potential for Novel Drug Development
in Antimicrobial Agents and Chemotherapy
Curtis A
(2015)
Heat Dissipation of Hybrid Iron Oxide-Gold Nanoparticles in an Agar Phantom
in Journal of Nanomedicine & Nanotechnology
Devlin A
(2019)
Tools for the Quality Control of Pharmaceutical Heparin.
in Medicina (Kaunas, Lithuania)
Devlin A
(2019)
Analysis of solid-state heparin samples by ATR-FTIR spectroscopy
Devlin AJ
(2023)
Analysis of Heparin Samples by Attenuated Total Reflectance Fourier-Transform Infrared Spectroscopy in the Solid State.
in ACS central science
Ghezzi S
(2017)
Heparin prevents Zika virus induced-cytopathic effects in human neural progenitor cells.
in Antiviral research
Holman J
(2018)
An Inexpensive, Pulsed, and Multiple Wavelength Bench-Top Light Source for Biological Spectroscopy
in Plasma
Hyatt JG
(2020)
Molecular Changes in Dengue Envelope Protein Domain III upon Interaction with Glycosaminoglycans.
in Pathogens (Basel, Switzerland)
Kareem N
(2018)
In vitro investigations on the effects of semi-synthetic, sulphated carbohydrates on the immune status of cultured common carp (Cyprinus carpio) leucocytes.
in Fish & shellfish immunology
| Description | 1) The limits of detection for Bodipy labelled GAG derived disaccharides has been established for conventional (Xe lamp-based) fluorescence and Laser Induced Fluorescence (LIF) using High Performance Anion Exchange Chromatography (HP-AEC). 2) Novel methodology has been developed to port the Bodipy (fluorescent-based) GAG disaccharide analysis into a reverse-phase format for HPLC. RP-HPLC is more commonplace in research laboratories, cheaper and does not require corrosive solvents in contrast to HP-AEC. 3) Methodology for the aqueous Bodipy labeling of GAG-derived oligosaccharides has been established for the first time. |
| Exploitation Route | 1) The use of Laser Induced Fluorescence for GAG disaccharide analysis will facilitate structure-function studies on sub-femtomolar concentrations of GAG; previously not possible using conventional, bulb based detectors. This technological advance opens up the possibility of GAG analysis on extremely scarce samples, e.g. those obtained via microdisection. 2) The development of a reverse-phase HPLC method for Bodipy-GAG disaccharide analysis will enhance significantly the availability and future use of this analysis technique. The current technique (HP-AEX) requires specialist equipment, whereas RP-HPLC instrumentation is widely utilised in most research laboratory environments. 3) Fluorescent labelling in aqueous conditions permits high sensitivity structural analysis of GAG-derived oligosaccharides previously not possible with the Bodipy fluorophore. This was due to solubility issues with conventional strategies and the solvent of choice, DMSO. |
| Sectors | Agriculture Food and Drink Healthcare Pharmaceuticals and Medical Biotechnology |
| Description | Research into commercialisation of detector currently underway. |
| First Year Of Impact | 2021 |
| Sector | Electronics,Manufacturing, including Industrial Biotechology |
| Impact Types | Economic |
| Description | Laser Induced Fluorescence Benchmarking |
| Amount | £15,000 (GBP) |
| Organisation | MI Engineering Ltd |
| Sector | Private |
| Country | United Kingdom |
| Start | 08/2017 |
| End | 08/2019 |
| Description | Seed Funding |
| Amount | £2,600 (GBP) |
| Organisation | Keele University |
| Sector | Academic/University |
| Country | United Kingdom |
| Start | 04/2016 |
| End | 06/2016 |
| Title | Aqueous Bodipy labelling of oligosaccharides for structural analysis |
| Description | Methodology has been developed to allow for the first time the Bodipy labelling of GAG derived oligosaccharides prior to subsequent structural analysis. Previously, only small saccharides could be labelled owing to the significant charge of the molecules and their limited solubility in DMSO. |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2017 |
| Provided To Others? | Yes |
| Impact | Further cemented collaborative links with the University of Liverpool and Intellihep Limited to utilise this methodology. |
| Title | Bodipy RP-HPLC |
| Description | Reverse phase fluorescent (Bodipy labelled) disaccharide analysis of GAGs. |
| Type Of Material | Biological samples |
| Year Produced | 2016 |
| Provided To Others? | Yes |
| Impact | Development of a new collaboration with Dr Andrew Powell (Liverpool John Moores University) to exploit new methodologies. |
| Title | FTIR-ATR Chemometric analysis of GAGs |
| Description | The use of FTIR-ATR based chemometrics in the structure:function studies of GAGs |
| Type Of Material | Technology assay or reagent |
| Year Produced | 2018 |
| Provided To Others? | No |
| Impact | Method currently in the process of being benchmarked through collaborations with The Ronzoni Institute and NIBSC. |
| Description | Intellihep |
| Organisation | Intellihep Ltd |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Use of modified heparin libraries and the sequence determination thereof. |
| Collaborator Contribution | Provision of chemically modified heparin libraries for structural charachterisation |
| Impact | Outputs are in progress |
| Start Year | 2016 |
| Description | Liverpool John Moores University - Dr Powell |
| Organisation | Liverpool John Moores University |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Knowledge transfer for new methodology. |
| Collaborator Contribution | Independent method validation. |
| Impact | None to date (papers in preparation). |
| Start Year | 2015 |
| Description | MI Engineering Ltd |
| Organisation | MI Engineering Ltd |
| Country | United Kingdom |
| Sector | Private |
| PI Contribution | Ongoing benchmarking & development of next-generation LIF based detector. |
| Collaborator Contribution | Donation of prototype next-generation LIF based detector. Use of manufacturing facilities & associates staff. |
| Impact | Multidisciplinary collaboration - Engineering, Biophysics, Optics and Electronics. Knowledge transferred from the industrial collaborator to the academic partner has led to research output in the form of one publication to-date (Curtis, A. et al. Heat dissipation of hybrid iron oxide-gold nanoparticles in an agar phantom. (2015) Journal of Nanomedicine & Nanotechnology. DOI 10.4172/2157-7439.1000335). |
| Start Year | 2015 |
| Description | NIBSC |
| Organisation | National Institute for Biological Standards and Control (NIBSC) |
| Country | United Kingdom |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Provided API for heparin and analysis of carbohydrates as used in COVID studies. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 https://www.biorxiv.org/content/10.1101/2020.02.29.971093v2.full https://www.biorxiv.org/content/10.1101/2020.04.28.066761v2.full https://www.biorxiv.org/content/10.1101/2020.04.29.068486v1.full https://www.biorxiv.org/content/10.1101/2020.04.29.068767v1.full |
| Start Year | 2020 |
| Description | Public Health England |
| Organisation | Public Health England |
| Department | Public Health England Porton Down |
| Country | United Kingdom |
| Sector | Public |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Live SARS-CoV-2 viral invasion assays were conducted on carbohydrates provided. |
| Impact | https://bpspubs.onlinelibrary.wiley.com/doi/10.1111/bph.15304 |
| Start Year | 2020 |
| Description | Queensland University of Technology |
| Organisation | Queensland University of Technology (QUT) |
| Country | Australia |
| Sector | Academic/University |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Carried out COVID-19 related modelling studies. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 |
| Start Year | 2020 |
| Description | Ronzoni |
| Organisation | Istituto di Ricerche Chimiche e Biochimiche G. Ronzoni |
| Country | Italy |
| Sector | Academic/University |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Use of advanced carbohydrate NMR facilities. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 https://www.biorxiv.org/content/10.1101/2020.02.29.971093v2.full https://www.biorxiv.org/content/10.1101/2020.04.28.066761v2.full https://www.biorxiv.org/content/10.1101/2020.04.29.068486v1.full https://www.biorxiv.org/content/10.1101/2020.04.29.068767v1.full https://www.nrronline.org/article.asp?issn=1673-5374;year=2020;volume=15;issue=8;spage=1546;epage=1553;aulast=Mycroft%2DWest https://www.biorxiv.org/content/10.1101/744532v1 https://www.mdpi.com/1660-3397/17/5/293 https://www.biorxiv.org/content/10.1101/538074v2 |
| Start Year | 2020 |
| Description | Rosalind Franklin Institute |
| Organisation | Rosalind Franklin Institute |
| Country | United Kingdom |
| Sector | Charity/Non Profit |
| PI Contribution | Provision of defined carbohydrates and characterisation of glycosaminoglycans. |
| Collaborator Contribution | Provision of proteins and structure:function studies. |
| Impact | Multidisciplinary : Carbohydrate chemistry, biochemistry, structural biology, cell biology, biophysics and protein science. |
| Start Year | 2021 |
| Description | San Raffaele Hospital |
| Organisation | San Raffaele Hospital |
| Department | Department of Biological and Technological Research (DIBIT) |
| Country | Italy |
| Sector | Academic/University |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Live SARS-CoV-2 viral invasion assays were conducted on carbohydrates provided. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 https://www.biorxiv.org/content/10.1101/2020.02.29.971093v2.full https://www.biorxiv.org/content/10.1101/2020.04.28.066761v2.full https://www.biorxiv.org/content/10.1101/2020.04.29.068486v1.full https://www.biorxiv.org/content/10.1101/2020.04.29.068767v1.full |
| Start Year | 2020 |
| Description | The University of Queensland |
| Organisation | University of Queensland |
| Country | Australia |
| Sector | Academic/University |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Modelling studies of GAG to SARS-CoV-2 Spike protein. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 |
| Start Year | 2020 |
| Description | UNIFESP |
| Organisation | Federal University of São Paulo |
| Country | Brazil |
| Sector | Academic/University |
| PI Contribution | Led international consortium describing how GAGs (including heparin) interact with the SARS-CoV-2 Spike protein and block viral invasion. Provided carbohydrate samples for screening against SARS-CoV-2. This has led to further research studies involving industry and clinical trials worldwide. |
| Collaborator Contribution | Provided carbohydrate standards alongside protein expression, CD and NMR facilities. |
| Impact | https://www.thieme-connect.de/products/ejournals/abstract/10.1055/s-0040-1721319 https://www.biorxiv.org/content/10.1101/2020.02.29.971093v2.full https://www.biorxiv.org/content/10.1101/2020.04.28.066761v2.full https://www.biorxiv.org/content/10.1101/2020.04.29.068486v1.full https://www.biorxiv.org/content/10.1101/2020.04.29.068767v1.full |
| Start Year | 2020 |
| Description | University of Liverpool - Yates |
| Organisation | University of Liverpool |
| Department | Institute of Integrative Biology |
| Country | United Kingdom |
| Sector | Academic/University |
| PI Contribution | Knowledge transfer of state-of-the-art GAG detection methodologies |
| Collaborator Contribution | Fully characterized modified GAG compounds |
| Impact | Publication of 2 research papers. Multi-disciplinary: Carbohydrate chemistry Biophysical analysis Glycobiology |
| Start Year | 2015 |
| Description | Zucero Therapeutics |
| Organisation | Zucero Therapeutics |
| Country | Australia |
| Sector | Private |
| PI Contribution | Conducted COVID related assays in comparison to GAG libraries of defined characteristics. |
| Collaborator Contribution | Provided Pixatimod (formerly PG545) carbohydrate for screening against COVID19. Pxiatimod is a first-in-class innate immunomodulatory small molecule, which has been tested in Phase I clinical trials. |
| Impact | https://www.biorxiv.org/content/10.1101/2020.06.24.169334v3.full |
| Start Year | 2020 |
| Title | GAG analogues |
| Description | Library of semi-synthetic GAG analogues for selective intervention in GAG modulated/regulated disease states. |
| Type | Therapeutic Intervention - Drug |
| Current Stage Of Development | Initial development |
| Year Development Stage Completed | 2017 |
| Development Status | Under active development/distribution |
| Impact | Library of GAG analogues as been outsourced to relevant research groups and preliminary data of efficacy obtained for Zika, HIV and Influenza viruses, parasitic infections and BACE inhibition in Alzheimer;s disease. |
| Description | Sociedade Brasileira para o Progresso da Ciência - Outreach activity |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Public/other audiences |
| Results and Impact | Raised awareness and profile of GAGs, the need to understand sequence, which underpins elucidating structure:function relationships and how this cna be used to the advantage of society, regarding therapeutics interventions. |
| Year(s) Of Engagement Activity | 2017 |
| URL | http://ra.sbpcnet.org.br/belohorizonte/ |
| Description | UNIFESP |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Postgraduate students |
| Results and Impact | Research presentation at UNIFESP - Universidade Federal de São Paulo. Audience included academics, post-graduates, under-graduates, clinicians and policy makers. |
| Year(s) Of Engagement Activity | 2016 |
| Description | Workshop at UNESP Institute of Biotechnology (IBTEC), Botucatu, Brazil |
| Form Of Engagement Activity | Participation in an activity, workshop or similar |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Industry/Business |
| Results and Impact | Workshop on International Scientific Cooperation between UNESP Institute of Biotechnology (IBTEC), Botucatu, Brazil and Keele University. New collaborations are in initial stages. |
| Year(s) Of Engagement Activity | 2015 |