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.

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.

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.

Publications

10 25 50
 
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 Next generation LIF detector currently under evaluation for future development by UK industry.
First Year Of Impact 2018
Sector 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 09/2017 
End 08/2019
 
Description Seed Funding
Amount £2,600 (GBP)
Organisation Keele University 
Sector Academic/University
Country United Kingdom
Start 05/2016 
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 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
 
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