Thymoquinone (TQ), a volatile oil component of black seed oil (derived from Nigella sativa), has been shown to have various biological effects including disease treatment and prevention. TQ is believed to share similar properties to the benzoquinones already in use as therapeutic drugs. Based on previous reports on the anti-inflammatory properties of black seed oil and TQ, it was originally hypothesized that TQ would inhibit lipopolysaccharide (LPS)-induced cellular sialidase activity in an anti-inflammatory manner. Sialidase activity was tested on live mouse bone marrow derived primary macrophage cells, BMC-2 macrophage cells, human embryonic kidney epithelial (HEK293) cells and human fibroblast cells using an assay that measures the cleavage of the sialidase specific fluorescent substrate 2’-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (4-MUNANA). The cleavage of 4-MUNANA causes the release of free 4-methylumbelliferone, which fluoresces at 450nm (blue) after excitation at 365nm. Unexpectedly, TQ induced sialidase activation in all three cell lines and wild type primary macrophage cells. TQ was unable to induce sialidase activity in primary macrophage cells isolated from Neu4 knockout mice suggesting that the TQ activates Neu4 sialidase enzyme. TQ-induced sialidase activity in these live cells was found to occur through intermediate GPCR-associated guanine nucleotide Gαi subunit and matrix metalloproteinase 9 (MMP9) by using specific inhibitors. In addition, TQ was found to induce sialidase activity in Toll-like receptor-deficient HEK293 cells. These latter data suggested that TQ may be activating GPCR Gαi and MMP9 signaling associated with Neu4 sialidase independent of TLRs. It is proposed that TQ-induced sialidase activity may activate Toll-like receptors in macrophage cells and the subsequent production of pro-inflammatory cytokines in the absence of LPS. Immunocytochemical staining of BMC-2 cells shows that TQ induced NFκB activation. NFκB activation was confirmed with electrophoretic mobility shift assay (EMSA) and western immunoblotting techniques. Cytokine arrays were used to test the pro-inflammatory cytokine response induced in mice by 5 hour treatment of TQ, compared to LPS. Mice treated with TQ exhibited an increase in IL-1β, IL-6 and TNF-α production, similar to LPS treatment. Taken together, the findings in these studies suggest that TQ is a novel ligand for Neu4 sialidase activation which consequently induces pro-inflammatory cytokine responses. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2009-04-21 17:38:10.413

No description available.

The IgG Fc domain mediates a range of antibody effector functions, including antibody dependent cell-mediated cytotoxicity (ADCC), complement activation, phagocytosis, and the recently emerged general anti-inflammatory effect of immunoglobulin therapy (IVIg). The conserved N-glycan attached to Fc N297 maintains the Fc structural integrity for the effector functions, while its glycoform is known to modulate the affinity for the Fc γ-receptors (FcγRs), complement, and the C-type lectin DC-SIGN. IgG Fc exhibits protein-directed glycosylation characterized by a series of biantennary complex type glycoforms, with a small population of sialylated species. The sialylated Fc has been proposed to bind DC-SIGN and initiate an anti-inflammatory signalling pathway. The restricted Fc glycan processing is partially attributed to the hydrophobic interaction between Fc glycan and the hydrophobic Fc protein backbone. Mutations within the hydrophobic Fc protein-glycan interface dramatically increases Fc glycan processing, while concomitantly decreases Fc affinity for the FcγRs. However, it is unclear whether this disrupted Fc-FcγR interaction was due to the increased terminal glycan processing, or the perturbed Fc protein-glycan interface. Here, the integrity of the Fc protein-glycan interface was demonstrated to be important in maintaining the productive Fc-FcγR interaction independently of glycoform. This glycoform-independent effect was exploited to generate novel inhibitory Fc variants. In addition, the interaction between sialylated IgG and the putative IVIg receptor DC-SIGN was re-evaluated. Analysis shows that IVIg binds DC-SIGN in a glycan-independent, Fab-mediated manner. Furthermore, the effect of IVIg sialylation on human antigen presenting cells was examined; evidence presented here indicate that IVIg deglycosylation, not desialylation, has an anti-inflammatory effect on human dendritic cells (DCs). These data suggest the need for a general re-evaluation of the current mechanistic model of anti-inflammatory IVIg.

Mucolipidosis type IV (MLIV) is a rare, autosomal recessive, neurodegenerative, lysosomal storage disorder. MLIV is caused by mutations in a gene (MCOLN1) encoding a TRP channel family member known as Mucolipin 1 or TRPML1. TRPML1 is a lysosomal transmembrane protein that appears to be required for normal lysosomal pH regulation, recycling of molecules and membrane reorganisation including lysosomal biogenesis, fusion and exocytosis. The exact function of the channel is unknown but it is permeable to multiple ions including Ca²⁺, Na⁺ and K⁺, possibly also Fe²⁺ and Zn²⁺. How normal TRPML1 function regulates lysosomal processes is not clearly understood. Mutations in the MCOLN1 gene can lead to complete loss of TRPML1 function, partial loss of function or mislocalisation, all of which lead to lysosomal dysfunction, lysosomal lipid storage and ultimately neurodegeneration. The disease processes that lead to neurodegeneration are poorly understood and at present no therapy exists for MLIV. We have discovered that TRPML1 results in regulating lysosomal Ca²⁺ homeostasis that is the opposite of the Ca²⁺ dysregulation associated with Niemann-Pick type C disease (NPC). Our findings indicate that disrupted function of TRPML1 leads to enhanced Ca²⁺ release via the NAADP receptor, recently shown to be the lysosomal two-pore channel TPC2. This indicates that TRPML1 is not the NAADP receptor as suggested by others, indeed NAADP mediated Ca²⁺ release is enhanced with multiple NAADP induced lysosomal Ca²⁺ release events occurring in TRPML1 null cells compared to single releases in normal cells. This phenotype appears to be responsible for the cellular dysfunction associated with MLIV disease cells, enhanced lysosomal fusion, defective endocytosis and potentially even altered lysosomal pH. Several of these phenotypes are normalised by the NAADP receptor specific antagonist Ned-19. These findings illustrate that the NAADP receptor is central to MLIV disease pathology and may be a novel candidate for disease therapy.

In the post-genomic era, recognition of the importance of sugars is increasing in biological research. For the precise analysis of their functions, homogeneous materials are required. Chemical synthesis is a powerful tool for preparation of homogeneous oligosaccharides and glycoconjugates. Glycosidases are potent catalysts for this purpose because they realize high stereo- and regio- selectivities under conditions benign to biomolecules without repetitive protection/deprotection procedures. A glycosynthase is an aritificial enzyme which is derived from a glycosidase and is devised for glycosylation reaction. To suppress the mechanistically inherent oligomerization side reaction of this class of biocatalysts, a glycosidase with plastic substrate recognition was engineered to afford the first α-mannosynthase. This novel biocatalyst showed low occurrence of oligomerized products as designed and was applied to prepare a wide range of oligosaccharides. Glycosidases are also valuable tools for glycan engineering of glycoconjugates, which is a pivotal issue in the development of pharmaceutical agents, including immunoglobulin G (IgG)-based drugs. EndoS, an endo-β-N-acetylglucosaminidase from Streptococcus pyogenes, natively cleaves N-glycans on IgG specifically. When the latent glycosylation activity of this enzyme was applied, the N-glycan remodelling of full-length IgG was successfully achieved for the first time and a highly pure glycoform was obtained using the chemically synthesized oxazoline tetrasaccharide as glycosyl donor. This biocatalytic reaction allows development of a novel type of antibody-drug conjugates (ADCs) in which drug molecules are linked to N-glycans site-specifically. For this purpose, glycans with bioorthogonal reaction handles were synthesized and conjugated to IgG. A model reaction using a dye compound as reaction partner worked successfully and the synthetic method for this newly designed ADC was validated. Glycan trimming of glycoproteins expressed from Pichia pastoris was performed using exoglycosidases to derive homogeneous glycoform. Jack Bean α-mannosidase (JBM) trimmed native N-glycans down to the core trisaccharide structure but some of the glycoforms were discovered to be resistant to the JBM activity. Enzymatic analyses using exoglycosidases suggested that the JBM-resistant factor was likely to be β-mannoside. In summary, this work advanced application of modified glycosidases for preparation of oligosaccharides and also demonstrated biocatalytic utility of glycosidases to produce biologically relevant glycoconjugates with homogeneous glycoforms.

Tunicamycin nucleoside antibiotics were the first known to target the formation of peptidoglycan precursor lipid I in bacterial cell wall biosynthesis. They have also been used extensively as inhibitors of protein N-glycosylation in eukaryotes, blocking the biogenesis of early intermediate dolichyl-pyrophosphoryl-N-acetylglucosamine. Despite their unusual structures and useful biological properties, little is known about their biosynthesis. Elucidating the metabolic pathway of tunicamycins and gaining an understanding of the enzymes involved in key bond forming processes would not only be of great academic value in itself, it would also unlock a comprehensive toolbox of biosynthetic machinery for the production of tunicamycin analogues which have the potential to act as novel therapeutic antibiotics or as specific inhibitors of medicinally important NDP-dependent glycosyltransferases. I – Cloning the tunicamycin biosynthetic gene cluster. We report identification of the tunicamycin biosynthetic genes in Streptomyces chartreusis following genome sequencing and a chemically-guided strategy for in silico genome mining that allowed rapid identification and unification of an operon fractured across contigs. Heterologous expression established a likely minimal gene set necessary for antibiotic production, from which a detailed metabolic pathway for tunicamycin biosynthesis is proposed. II – Natural product isolation and degradation. We have developed efficient methods for the isolation of tunicamycins from liquid culture in preparative quantities. A subsequent relay synthesis furnished advanced biosynthetic intermediates for use as precursors in the production of tunicamycin analogues and as substrates for the in vitro characterisation of individual Tun enzymes. III – Functional characterisation of tun gene products. Individual tun gene products were over-expressed and purified from recombinant E. coli hosts, allowing in vitro functional studies to take place. An NMR assay of biosynthetic enzyme TunF showed it acted as a UDP-GlcNAc-4-epimerase. Putative glycosyltransferase TunD showed hydrolytic activity towards substrate UDP-GlcNAc but failed to accept to the expected natural acceptor substrate, providing unexpected insights into the ordering of biosynthetic events in the tunicamycin pathway. Initial studies into the over-expression of the putative sugar N-deacetylase TunE were also described. IV – Towards synthesis of tunicamycin fragments. Investigations into a novel synthesis of D-galactosamine – a structural motif within tunicamycin – led to the unexpected observation of inverted regioselectivity upon RhII-catalysed C-H insertion of a D-mannose-derived sulfamate. This was the first example of N-insertion at the beta- rather than gamma-C-H based on conformation alone and warranted further investigation. The X-ray structure of a key sulfamate precursor offered valuable insights as to the source of this unique selectivity.

Iminosugars are a class of small molecules defined by substitution of a sugar’s ring oxygen with nitrogen. Various chemical modifications of these basic structures (e.g. alkyl chain addition off of the ring nitrogen) have been developed during the last several decades. These molecules have been considered as therapeutics for a number of pathologies including viral infection, congenital disorders of glycosylation (of both glycoproteins and glycolipids), and diabetes. This thesis focuses on the application of a small subset of iminosugars, known as deoxynojirimycin derivatives, as therapeutics against dengue virus induced pathology. Dengue virus infection predominates in tropical climates, but autochthonous infection has recently emerged in areas of both southern Europe and the southern United States. With 390 million people infected annually, dengue is the most prevalent arthropod-borne viral infection worldwide, and the possibility of severe pathology including haemorrhage, shock, and/or death, necessitates development of effective antiviral therapies. Although the molecular mechanisms responsible for progression to severe dengue disease are not completely understood, there is considerable evidence for the role of both the innate and the adaptive immune responses in development of life-threatening complications. Excessive activation of the innate immune response, a phenomenon known as cytokine storm, has been hypothesised to explain development of symptoms related to vascular permeability, whereas the adaptive immune response has been implicated in severe disease through two hypotheses – the antibody dependent enhancement and original antigenic sin hypotheses. The evidence regarding each of these potential mechanisms of severe pathology is discussed throughout this thesis principally with respect to how iminosugar treatment could alter any detrimental effects of the immune response to dengue virus infection. The principal aim of this thesis is to consider the potential of deoxynojirimycin iminosugars as antiviral therapeutics in dengue infection with a focus on how these molecules exert their antiviral effects in primary human cells. I first consider the contributions of glycoprotein inhibition and glycolipid inhibition on production of infectious dengue virus. These experiments suggest that inhibition of glycoprotein folding is responsible for inhibition of infectious dengue virus production. I next consider the impact of treatment of a promising clinical candidate iminosugar, N9-methoxynonyl-deoxynojirimycin (MON-DNJ), on the primary human macrophage transcriptome. In uninfected macrophages as well as macrophages infected with dengue virus or treated with lipopolysaccharide to model bacterial sepsis, iminosugar treatment results in activation of the unfolded protein response and inhibition of several elements of the inflammatory response including signalling by the cytokines IFN-γ and TNF-α, and the inflammatory cascade mediated by NF-κB. Activation of the unfolded protein response as a result of treatment with MON-DNJ can be confirmed by analysis of phosphorylated (activated) NFE2L2, a transcription factor that functions principally to control oxidative stress in response to ER stress signals. Modulation of the inflammatory response of macrophages to dengue infection and bacterial sepsis is confirmed by analysis of secreted cytokines. As predicted by my transcriptomic experiments, levels of TNF-α and IFN-γ produced in response to dengue or lipopolysaccharide are reduced by treatment with MON-DNJ. Finally, I attempted to extend these observations to an animal model of dengue infection with a particular focus on TNF receptor and ligand superfamily members. Unfortunately, heterogeneity of cells types from tissue samples as well as limitations of the animal model complicate interpretation of these findings. Nevertheless, this thesis demonstrates that MON-DNJ is an effective dengue antiviral therapeutic and that this therapeutic activity may be related to both reduction of infectious virus as a consequence of inhibition of glycoprotein processing and as a result of changes to the host’s response to the pathogen. These results have been used in part to justify recently initiated clinical trials of MON-DNJ as a dengue antiviral therapy.

Peptides have attracted increasing attention as therapeutics in recent years, at least partially as a consequence of the widespread acceptance of protein therapeutics; but also as possible solutions to problems such as short half-life and delivery of molecules, and as therapeutics in their own right. The current work presents three projects that involve applications of peptides in a therapeutic environment. The first project studies the use of ER retaining peptides and CPPs (Cell penetrating peptides) in enhancing the effective concentration of DNJ (1-deoxynojirimycin), an α-glucosidase inhibitor, in cells. DNJ constructs with ER retaining peptides (6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL and 6-[N-(1-deoxynojirimycino)]-hexanoyl-KKAA) and CPPs (6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT and 6-[N-(1-deoxynojirimycino)]-hexanoyl-MAP) were synthesised and analysed for their inhibitory activity against α-glucosidase I and II in vitro. The constructs were then analysed in a cell-based assay to determine their inhibitory activity on α¬-glucosidase-mediated hydrolysis of N-linked oligosaccharides. FITC-labelled ER retaining peptides were also synthesised to determine the internalisation and trafficking of the constructs by FACS and IF-microscopy. While none of the DNJ-constructs showed higher cellular inhibition than NB-DNJ (N-butyl DNJ; Miglustat), the CPP construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-TAT showed comparable activity and the ER retaining construct 6-[N-(1-deoxynojirimycino)]-hexanoyl-KDEL showed a small but significant increase in activity following long-term administration. The second project focuses on beauveriolides, a cyclic depsipeptide family shown to have activity as ACAT inhibitors and thus a possible treatment for Alzheimer’s disease by the decrease in the production of Amyloid β (Aβ). A published total synthetic method was improved by the use of a cross-metathesis to reduce the total synthesis by 5 steps and increase its flexibility to allow the production of analogues. The synthesised beauveriolide III was used in attempts to develop an IF-FACS-based assay to measure the intracellular concentrations of Aβ. However, the location of γ-secretase in the used cell-line meant that levels of intracellular Aβ were not sufficient to track any decrease caused by ACAT inhibition. The third project involves the design of a cyclic peptide that could block the binding site for the influenza virus in the host cell. The cyclic peptide (cGSGRGYGRGWGVGA) was developed from a comparative study of four different sialic acid-binding proteins and synthesised by solution cyclisation of the linear peptide synthesised by traditional solid phase peptide synthesis (SPPS). An in silico study showed that the cyclic peptide allowed overlap with the binding site of Hemagglutinin. A 1H NMR titration determined the dissociation constant of the cyclic peptide to sialic acid. The KD corresponded to a low binding affinity, however the observed binding seemed to be specific and caused by a single bound conformation.

Pseudomonas aeruginosa (PA) est l’un des principaux germes impliqués dans les maladies nosocomiales et est aussi la principale cause de mortalité et morbidité des patients atteints de la mucoviscidose malgré l’utilisation massive d’antibiotiques. Dans la lutte contre PA, une alternative aux antibiotiques est l’inhibition de ses facteurs de virulence notamment ceux impliqués dans l’adhésion et la formation du biofilm via des interactions de type sucres/protéines. Ces protéines sont appelées lectines (PA-IL, PA-IIL, FliD). L’objectif de ce travail est la recherche de molécules inhibitrices (glycoclusters) de ces lectines impliquées dans la virulence de PA. Compte tenu du grand nombre de glycoclusters à tester et des faibles quantités de matériels biologiques disponibles, un outil de criblage innovant a été développé (glycoarray) à partir d’une lame de verre microstructurée et fonctionnalisée chimiquement afin d’immobiliser de manière organisée et ordonnée les glycoclusters. La méthode d’immobilisation choisie est la méthode d’immobilisation spécifique par hybridation de l’ADN appelée DDI : DNA Directed Immobilization. Sur ces glycoarrays, 3 méthodes indépendantes (lecture de fluorescence directe, IC50 et Kd) de mesure des interactions glycoclusters/lectines ont été mises au point et validées par une étude comparative donnant un classement similaire des glycoclusters pour leur affinité vis-à-vis des lectines Il faut noter que ces mesures faites sur glycoarrays ne consomment que quelques picomoles de glycoclusters comparées aux méthodes classiques (ITC, ELLA, RMN, …) qui nécessitent des micromoles de produits. A l’aide de ces glycoarrays, un criblage d’une bibliothèque d’une centaine de glycoclusters multivalents, de différentes topologies, charges et linkers a permis d’identifier deux structures montrant une très forte affinité vis-à-vis des lectines de PA. Ces glycoclusters sont actuellement en test in vitro et in vivo. Ces études d’interactions sur DDI-glycoarray ont été étendues à d’autres agents pathogènes tels que les bactéries Burkholderia ambifaria, Viscum album ou contre le virus de la grippe. Dans le futur, pour mieux appréhender les mécanismes d’interactions sucres/protéines, il serait intéressant de pouvoir suivre en temps réel ces interactions en utilisant des systèmes de détection sans marquage tel que, par exemple, la résonance plasmonique de surface. Aussi, le dernier chapitre donne les prémices d’une adaptation de la méthode DDI sur glycoarray sur surface d’or. / Pseudomonas aeruginosa (PA) is one of the predominant bacterium encountered in nosocomial infections. PA infections often lead to chronic inflammation and eventually to death despite aggressive antibiotic therapy. A promising approach is to inhibit the virulence factors of PA such as PA-IL, PA-IIL, FliD (lectins). Therefore, there is a great interest for studying carbohydrate/lectin interactions in order to design new treatments. The goal of this work is the research for inhibitory molecules (glycoclusters ) of these lectins involved in the virulence of PA. An innovative screening tool for studying carbohydrate/lectin interactions has been developed (glycoarray). Glycoarray are microstructured glass-slides, chemically functionalized in order to immobilize, organized and orderly, glycoclusters at the surface. The immobilization method is the specific immobilization method based on DNA hybridization called DDI (DNA Directed Immobilization). This miniaturized analytical biosystem allows multiplex test performed in one single microwell. Moreover, three independent methods of affinity measurement (direct fluorescence read-out, IC50 and Kd) have been developed and validated by a comparative study giving a similar ranking of glycoclusters for their affinity towards PA-IL. These measurements on glycoarrays consume only a few picomoles glycoclusters compared to conventional methods (ITC, ELLA...) that require micromoles of products. Using these glycoarrays, the screening of a library of hundreds of glycoclusters presenting different topologies, multivalencies, charges and linkers led to the identification of two structures showing a very strong affinity for PA lectins. These glycoclusters are currently in vitro assay and in vivo. These interaction studies on DDI-glycoarray were extended to other pathogens such as Burkholderia ambifaria bacteria, Viscum album or against the influenza virus. In the future, to better understand the mechanisms of sugar / protein interactions, it would be interesting to monitor in real time the interactions using label-free detection systems such as, for example, the surface plasmon resonance (SPR). Also, the last chapter gives the beginnings of an adaptation of the method of DDI glycoarray on gold surface

The bacteria Streptococcus pyogenes produces a multitude of proteins which interact with and alter the functions of the host immune system. Two such proteins, Endoglycosidase S (EndoS) and Immunoglobulin G-degrading enzyme from S. pyogenes (IdeS) are able to specifically alter the effector functions of immunoglobulin G (IgG). EndoS is a glycoside hydrolase which removes the conserved <i>N</i>-linked glycan from IgG Fc whereas IdeS is a cysteine protease that cleaves the exible protein hinge of IgG. The activity of both proteins results in the reduced ability of IgG to elicit immune responses through Fc receptor binding and complement activation. Amongst other applications, both EndoS and IdeS are actively being explored as new therapeutics for IgG-mediated autoimmune diseases. Given the therapeutic potential of EndoS and IdeS, experiments were designed to investigate the structural and functional characteristics of these enzymes in an effort to understand their specficity for and activity against IgG. Here, bioinformatic and biophysical characterisation of EndoS identified subdomains outside of the catalytic domain which contribute to glycoside hydrolase activity. The substrate specificity of EndoS was also explored and showed that EndoS hydrolyses a broad range of glycans from the IgG scaffold. EndoS was also shown to have activity against alternative glycoprotein substrates, however, this non-specific activity was negligible in the context of whole serum. The effect of EndoS-mediated deglycosylation on the structure of the IgG Fc domain was explored using both X-ray crystallography and small-angle X-ray scattering. Small angle X-ray scattering was also used to characterise both EndoS and IdeS in complex with IgG Fc. Solution-state models of each complex were produced providing preliminary data towards how these enzymes interact with IgG. Overall, the results presented here contribute to our understanding of these enzymes which is of importance as they go forward into clinical applications.