3,206 research outputs found
Mass Spectrometry in the Elucidation of the Glycoproteome of Bacterial Pathogens
Presently some three hundred post-translational modifications are known to occur in bacteria in vivo. Many of
these modifications play critical roles in the regulation of proteins and control key biological processes. One of the most
predominant modifications, N- and O-glycosylations are now known to be present in bacteria (and archaea) although they
were long believed to be limited to eukaryotes. In a number of human pathogens these glycans have been found attached
to the surfaces of pilin, flagellin and other surface and secreted proteins where it has been demonstrated that they play a
role in the virulence of these bacteria. Mass spectrometry characterization of these glycosylation events has been the enabling
key technology for these findings. This review will look at the use of mass spectrometry as a key technology for the
detection and mapping of these modifications within microorganisms, with particular reference to the human pathogens,
Campylobacter jejuni and Mycobacterium tuberculosis. The overall aim of this review will be to give a basic understanding
of the current ‘state-of-the-art’ of the key techniques, principles and technologies, including bioinformatics tools, involved
in the analysis of the glycosylation modifications
Structure analysis of biologically important prokaryotic glycopolymers
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
Recommended from our members
Studying Lactoferrin N-Glycosylation.
Lactoferrin is a multifunctional glycoprotein found in the milk of most mammals. In addition to its well-known role of binding iron, lactoferrin carries many important biological functions, including the promotion of cell proliferation and differentiation, and as an anti-bacterial, anti-viral, and anti-parasitic protein. These functions differ among lactoferrin homologs in mammals. Although considerable attention has been given to the many functions of lactoferrin, its primary nutritional contribution is presumed to be related to its iron-binding characteristics, whereas the role of glycosylation has been neglected. Given the critical role of glycan binding in many biological processes, the glycan moieties in lactoferrin are likely to contribute significantly to the biological roles of lactoferrin. Despite the high amino acid sequence homology in different lactoferrins (up to 99%), each exhibits a unique glycosylation pattern that may be responsible for heterogeneity of the biological properties of lactoferrins. An important task for the production of biotherapeutics and medical foods containing bioactive glycoproteins is the assessment of the contributions of individual glycans to the observed bioactivities. This review examines how the study of lactoferrin glycosylation patterns can increase our understanding of lactoferrin functionality
Algorithms for Glycan Structure Identification with Tandem Mass Spectrometry
Glycosylation is a frequently observed post-translational modification (PTM) of proteins. It has been estimated over half of eukaryotic proteins in nature are glycoproteins. Glycoprotein analysis plays a vital role in drug preparation. Thus, characterization of glycans that are linked to proteins has become necessary in glycoproteomics. Mass spectrometry has become an effective analytical technique for glycoproteomics analysis because of its high throughput and sensitivity. The large amount of spectral data collected in a mass spectrometry experiment makes manual interpretation impossible and requires effective computational approaches for automated analysis. Different algorithmic solutions have been proposed to address the challenges in glycoproteomics analysis based on mass spectrometry. However, new algorithms that can identify intact glycopeptides are still demanded to improve result accuracy.
In this research, a glycan is represented as a rooted unordered labelled tree and we focus on developing effective algorithms to determine glycan structures from tandem mass spectra. Interpreting the tandem mass spectra of glycopeptides with a de novo sequencing method is essential to identifying novel glycan structures. Thus, we mathematically formulated the glycan de novo sequencing problem and propose a heuristic algorithm for glycan de novo sequencing from HCD tandem mass spectra of glycopeptides.
Characterizing glycans from MS/MS with a de novo sequencing method requires high-quality mass spectra for accurate results. The database search method usually has the ability to obtain more reliable results since it has the assistance of glycan structural information. Thus, we propose a de novo sequencing assisted database search method, GlycoNovoDB, for mass spectra interpretation
- …