Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal
cell surfaces and within extracellular matrices. GAGs usually occur as parts of
proteoglycans and often accomplish their biological functions through their interactions
with proteins. GAG oligosaccharides for this work were produced via enzymatic digest
of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide
species obtained from this digest were characterised using 1H and 13C NMR
spectroscopy.
Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement
system, a major component of human innate immunity. Acting as a cofactor to
factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells
from auto immune attack. This study focused on the interaction of factor H with GAGs,
which are thought to be among the markers allowing factor H to distinguish between self
and non self surfaces. Binding studies of two heparin-binding sites in fH are presented.
These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8
and fH~19-20 were produced recombinantly in various isotope forms. The techniques
used to study the protein-GAG interactions in this work encompass NMR spectroscopy,
mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking.
Several genetic studies suggest that a common polymorphism in the heparin-binding
module fH~7, Y402H, plays a role in the development of age-related macular degeneration
(AMD). The work presented here included preparation and backbone resonance
assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments.
Further NMR experiments were employed to investigate the role of the lysine
and arginine sidechains of fH~7 in GAG binding. These studies were combined with
the preparation and characterisation of a covalently cross linked GAG-protein complex
using NMR and mass spectrometry.
A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical
haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation
between the disease and the heparin binding properties of the aHUS mutants was observed.
The mutant proteins were also characterised with respect to their ability to
compete with full-length fH in a physiological complement assay. Simultaneous binding
of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using
NMR.
NMR experiments were also conducted with NK1, which comprises the two N-terminal
heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin
as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin,
HBD2, were performed to assess the role of GAGs in HBD2 self-association
