New analytical and synthetic tools for the study of protein-gycosaminoglycan interactions
View/ Open
word thesis.zip (38.43Mb)
Thomas2015.pdf (7.299Mb)
Date
30/06/2015Item status
Restricted AccessEmbargo end date
31/12/2100Author
Thomas, Sarah Jane
Metadata
Abstract
Glycosaminoglycans (GAGs) are linear polysaccharides found on most animal cell surfaces
and in extracellular matrices. Their key biological roles include cell signalling, cell-to-cell
recognition, bacterial and viral adhesion, and antibody production. They are composed of
disaccharide repeating units, containing uronic acid and amino sugar residues, and may be
highly heterogeneously sulfated. Protein-GAG complexes are thought to play an important
role in a number of aspects of cancer development, but are an under-studied area due to a lack
of enabling tools to facilitate their analysis as discussed in Chapter 1.
To obtain homogenous GAG structures, a range of conditions for the separation of GAG
oligosaccharides by Zwitterionic Hydrophilic Interaction Liquid Chromatography (ZIC-HILIC)
were developed using commercial chondroitin sulfate standards; these are discussed in
Chapter 2. Mixed-modal separation mechanisms were explored across a different buffer
compositions and elution programs to optimise the conditions to suit individual classes of
native and modified glycosaminoglycans. These methods were then applied to the separation
of chondroitin sulfate mixtures and heparin oligosaccharides. ZIC-HILIC may also be coupled
to Mass Spectrometry to produce an online analytical method for GAG mixtures.
A range of optimised conditions for the analysis of low molecular weight glycosaminoglycan
oligosaccharides by Electrospray Mass Spectrometry were developed using commercial
chondroitin sulfate disaccharide standards; as discussed in Chapter 3. These conditions were
then employed in the tandem mass spectrometry (MS/MS) of chondroitin sulfate to identify
diagnostic fragmentation patterns and this was applied in the characterisation of chondroitin
sulfate disaccharides derived from enzymatic cleavage. The position of ring substituents can
also be identified using this methodology, which is desirable in the analysis of chemically-labelled
GAG structures.
To allow for high resolution EPR and NMR studies of protein-GAG interactions, synthetic
procedures for the incorporation of paramagnetic centres into GAG oligosaccharides and
proteins were developed and these are discussed in Chapter 4. A propargyl-modified lysine
was synthesised for recombinant expression into myoglobin.
Copper-Catalysed Azide-Alkyne Cycloaddition (CuAAC) was employed in the spin labelling
of myoglobin, however traditional experimental conditions were found to reduce the TEMPO-based
spin labels used. Alternative conditions were developed using glutathione to stabilise
the copper (II) catalyst without reducing the spin label. Spin labelling of the modified lysine
was monitored by EPR.
Modification of the non-reducing end was explored for the spin labelling of GAG
oligosaccharides, to avoid the ring opening that typically occurs in reducing end labelling, and
make use of the unsaturated uronic acid that is derived when obtaining GAGs through
enzymatic depolymerisation. A hydrazide labelling of uronic acids was initially adopted,
however due to concerns regarding selectivity and availability of hydrazide spin labels, an
alternative Thiol-Ene Click (TEC) method was developed. TEC labelling of monosaccharides
and unnatural amino acids was found to proceed efficiently under aqueous conditions and was
monitored by NMR and HPLC. Preliminary studies in the TEC labelling of chondroitin
sulfate disaccharides proved promising, however further optimisation is required for this
method to be utilised in the study of protein-GAG interactions by NMR.