Of the many post-translational modifications organisms can undertake, glycosylation is the most
prevalent
and the most diverse. The research in this thesis focuses on the structural characterisation of
glycosylation in two classes of glycopolymer (lipopolysaccharide (LPS) and glycoprotein) in two
domains of life (bacteria and archaea). The common theme linking these subprojects is the
development and application of high sensitivity analytical techniques, primarily mass spectrometry
(MS), for studying prokaryotic glycosylation. Many prokaryotes produce glycan arrangements with
extraordinary variety in composition and structure. A further challenge is posed by additional
functionalities such as lipids whose characterisation is not always straightforward. Glycosylation
in prokaryotes has a variety of different biological functions, including their important roles in
the mediation of interactions between pathogens and hosts. Thus enhanced knowledge of bacterial
glycosylation may be of therapeutic value, whilst a better understanding of archaeal protein
glycosylation will provide further targets for industrial applications, as well as insight into
this post- translational modification across evolution and protein processing under extreme
conditions.
The first sub-project focused on the S-layer glycoprotein of the halophilic archeaon Haloferax
volcanii, which has been reported to be modified by both glycans and lipids. Glycoproteomic and
associated MS technologies were employed to characterise the N- and O-linked glycosylation and to
explore putative lipid modifications. Approximately 90% of the S-layer was mapped and N-glycans
were identified at all the mapped consensus sites, decorated with a pentasaccharide consisting of
two hexoses, two hexuronic acids and a methylated hexuronic acid. The O-glycans are homogeneously
identified as a disaccharide consisting of galactose and glucose. Unexpectedly it was found that
membrane-derived lipids were present in the S- layer samples despite extensive purification,
calling into question the predicted presence of covalently linked lipid. The H. volcanii
N-glycosylation is mediated by the products of the agl gene cluster and the functional
characterisation of members of the agl gene cluster was investigated by MS analysis of agl-mutant
strains of the S-layer.
Burkholderia pseudomallei is the causative agent of melioidosis, a serious and often fatal disease
in humans which is endemic in South-East Asia and other equatorial regions. Its LPS is vital for
serum resistance and the O-antigen repeat structures are of interest as vaccine targets. B.
pseudomallei is reported to produce several polysaccharides, amongst which the already
characterised ‘typical’ O-antigen of K96243 represents 97% of the strains. The serologically
distinct ‘atypical’ strain 576 produces a different LPS, whose characterisation is the subject of
this research project. MS strategies coupled with various hydrolytic and chemical derivatisation
methodologies were employed to define the composition and potential sequences of the O-antigen
repeat unit. These MS strategies were complemented by a novel NMR technique involving embedding of
the LPS into micelles. Taken together the MS and NMR data have revealed a highly unusual O-antigen
structure for atypical LPS which is remarkably different from the typical O-antigen.
The development of structural analysis tools in MS and NMR applicable to the illustrated types of
glycosylation in these prokaryotes will give a more consistent approach to sugar characterisation
and their modifications thus providing more informative results for pathogenicity and immunological
studies as well as
pathway comparisons.Open Acces
