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

Type III Secretion Effectors with Arginine N-Glycosyltransferase Activity

Type III secretion systems are used by many Gram-negative bacterial pathogens to inject proteins, known as effectors, into the cytosol of host cells. These virulence factors interfere with a diverse array of host signal transduction pathways and cellular processes. Many effectors have catalytic activities to promote post-translational modifications of host proteins. This review focuses on a family of effectors with glycosyltransferase activity that catalyze addition of N-acetyl-d-glucosamine to specific arginine residues in target proteins, leading to reduced NF-κB pathway activation and impaired host cell death. This family includes NleB from Citrobacter rodentium, NleB1 and NleB2 from enteropathogenic and enterohemorrhagic Escherichia coli, and SseK1, SseK2, and SseK3 from Salmonella enterica. First, we place these effectors in the general framework of the glycosyltransferase superfamily and in the particular context of the role of glycosylation in bacterial pathogenesis. Then, we provide detailed information about currently known members of this family, their role in virulence, and their targetsSpanish Ministerio de Economía, Industria y Competitividad , Agencia Estatal de Investigación, and the European Regional Development Fund, grant number SAF2016‐75365‐REuropean Union’s Horizon 2020 e Marie Skłodowska‐Curie grant agreement No 84262

Chemical synthesis of charbohydrates and glycopeptides for biological application

This thesis describes chemical synthesis of carbohydrates and glycopeptides useful in studies of biologically interesting systems. The following topics are addressed: [Papers I-IV and supporting information]: [I] The non-reducing end di- and trisaccharide structures of an erythrocyte glycolipid responsible for the rare NOR polyagglutination were chemically synthesized. The syntheses were based on a α-D-Galp-(1→4)-β-D-GalpNAc-(1→) structural element found only recently in Nature, and derivatives thereof have not been synthesized before. Both the synthesized oligosaccharides specifically inhibited human anti-NOR antibodies, with the trisaccharide being 300 times more active than the disaccharide. [II] Derivatives of lactose with the galactose ring substituents replaced by 2´, 3´, 4´ and 6´ deoxy and 3´ acylamino functions were prepared. The lactosyl derivatives were tested as acceptors for the Neisseria menigitidis N-acetylglucosaminyltransferase catalyzed β-(1→3) glycosylation reaction, using UDP-GlcNAc as donor. The 6´-deoxy compound showed nearly a threefold increase in activity compared with the reference substance phenyl β-lactoside, whereas the 2´ and 4´-deoxy derivatives were less active. The 3´-deoxy and 3´-acylamino derivatives will be used in studies of the inhibitory capacity. [III] In order to develop the non-viral Bioplex vector system for non-viral gene delivery to hepatocytes, biotinylated ligands were synthesised to study the structure-function relationship of specific binding and uptake to the asialoglycoprotein receptor ASGPr. Cluster glycosides containing two, three and six β-D-GalpNAc residues were synthesized and tested for binding and uptake to liver cells. The derivative displaying six GalNAc units showed the highest uptake efficacy. However, the number of GalNAc units above three seems only to have a minor contribution to the overall affinity, while using longer spacer between the GalNAc ligands markedly influenced the uptake efficacy. [IV] An analgetically active glycopeptide from the cone snail Conus geographus, contulakin G, has recently been analyzed and synthesized. Contulakin-G has been found to be a neurotensin agonist and have entered pre-clinical trials for short-term management of post-operative pain. The glycan part of contulakin-G has been found to be important for the biological in vivo activity. In order to further investigate the importance of the glycan part, three analogues of contulakin-G have been synthesized, were the α/β conformation of the anomeric centers as well as the glycosidic bond pattern of the disaccharide have been altered. In addition to the contulakin G analogues, a heavily posttranslational modified glycopeptide from Conus textile, tx5a, has been synthesized

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

Glycosylation of Amino Acids and Efficient Synthesis of Glycosphingolipids

Glycoscience is an emerging field of science that focuses on the study of the structure, biosynthesis, biology, and evolution of saccharides (sugars). It covers a broad range of subjects including microwave-assisted synthesis as well as sphingolipid synthesis. In this field, knowledge is limited due to the complexity of carbohydrates and their derivatives. Therefore, it is all the more important that synthesis of these complex molecules occurs in order to fully understand their biological significance. The following report summarizes two aspects of glycoscience and discusses their biological applications

Oligosaccharyltransferase is highly specific for the hydroxy amino acid in Asn-Xaa-Thr/Ser

AbstractPig liver oligosaccharyltransferase (OST), which is involved in the en bloc transfer of the Dol-PP-linked GlcNAc2-Man9-Glc3 precursor on to asparagine residues in the Asn-Xaa-Thr/Ser sequence, is highly stereospecific for the conformation of the 3-carbon atom in the hydroxy amino acid. Moreover, substitution of the hydroxy group by either SH as in cysteine, or NH2 as in β,γ-diamino-butanoic acid as reported previously [Bause, E. et al., Biochem. J. 312 (1995) 979–985], followed by the determination of the pH optimum for enzymatic activity, indicates that neither a negative nor a positive charge in the hydroxy amino acid position is tolerated by the enzyme. Binding of the threonine β-methyl group by OST is also specific, with serine, L-threo-β-hydroxynorvaline and L-β-hydroxynorleucine containing tripeptides all bound much less efficiently than the threonine peptide itself. The data are interpreted in terms of a highly stereospecific hydrophobic binding pocket for the threonine CH3-CH(OH) group

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses

Mucin-1(MUC1)glycopeptidesareexceptionalcandidatesforpotentialcancervaccines.However,theirautoantigenicnatureoftenresultsinaweakimmuneresponse.Toovercomethisdrawback,wecarefullyengineeredsyntheticantigenswithprecisechemicalmodifications.Tobeeffectiveandstimulateananti-MUC1response,artificialantigensmustmimictheconforma-tionaldynamicsofnaturalantigensinsolutionandhaveanequivalentorhigherbindingaffinitytoanti-MUC1antibodiesthantheirnaturalcounterparts.Asa proofofconcept,wehavedevelopeda glycopeptidethatcontainsnoncanonicalaminoacid(2S,3R)-3-hydroxynorvaline.Theunnaturalantigenfulfillsthesetwopropertiesandeffectivelymimicsthethreonine-derivedantigen.Ontheonehand,conformationalanalysisinwatershowsthatthissurrogateexploresalandscapesimilartothatofthenaturalvariant.Ontheotherhand,thepresenceofanadditionalmethylenegroupinthesidechainofthisanalogcomparedtothethreonineresidueenhancesa CH/interactionintheantigen/antibodycomplex.Despiteanenthalpyentropybalance,thissyntheticglycopeptidehasabindingaffinityslightlyhigherthanthatofitsnaturalcounterpart.Whenconjugatedwithgoldnanoparticles,thevaccinecandidatestimulatestheformationofspecificanti-MUC1IgGantibodiesinmiceandshowsefficacycomparabletothatofthenaturalderivative.Theantibodiesalsoexhibitcross-reactivitytoselectivelytarget,forexample,humanbreastcancercells.Thisinvestigationreliedonnumerousanalytical(e.g.,NMRspectroscopyandX-raycrystallography)andbiophysicaltechniquesandmoleculardynamicssimulationstocharacterizetheantigenantibodyinteractions.Thisworkflowstreamlinesthesyntheticprocess,savestime,andreducestheneedforextensive,animal-intensiveimmunizationprocedures.Theseadvancesunderscorethepromiseofstructure-basedrationaldesignintheadvanceofcance

Structure-Guided Approach for the Development of MUC1-Glycopeptide-Based Cancer Vaccines with Predictable Responses

Mucin-1 (MUC1) glycopeptides are exceptional candidates for potential cancer vaccines. However, their autoantigenic nature often results in a weak immune response. To overcome this drawback, we carefully engineered synthetic antigens with precise chemical modifications. To be effective and stimulate an anti-MUC1 response, artificial antigens must mimic the conformational dynamics of natural antigens in solution and have an equivalent or higher binding affinity to anti-MUC1 antibodies than their natural counterparts. As a proof of concept, we have developed a glycopeptide that contains noncanonical amino acid (2S,3R)-3-hydroxynorvaline. The unnatural antigen fulfills these two properties and effectively mimics the threonine-derived antigen. On the one hand, conformational analysis in water shows that this surrogate explores a landscape similar to that of the natural variant. On the other hand, the presence of an additional methylene group in the side chain of this analog compared to the threonine residue enhances a CH/π interaction in the antigen/antibody complex. Despite an enthalpy–entropy balance, this synthetic glycopeptide has a binding affinity slightly higher than that of its natural counterpart. When conjugated with gold nanoparticles, the vaccine candidate stimulates the formation of specific anti-MUC1 IgG antibodies in mice and shows efficacy comparable to that of the natural derivative. The antibodies also exhibit cross-reactivity to selectively target, for example, human breast cancer cells. This investigation relied on numerous analytical (e.g., NMR spectroscopy and X-ray crystallography) and biophysical techniques and molecular dynamics simulations to characterize the antigen–antibody interactions. This workflow streamlines the synthetic process, saves time, and reduces the need for extensive, animal-intensive immunization procedures. These advances underscore the promise of structure-based rational design in the advance of cancer vaccine development