Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant genetic disorder that results from a mutation of one of two key signaling receptors for the transforming growth factor beta (TGFβ) superfamily: endoglin and activin receptor-like kinase 1 (ALK1). These mutations result in development of HHT Type 1 and HHT Type 2, respectively. Patients suffering from HHT experience spontaneous blood vessel growth that can lead to telangiectasia, arteriovenous malformation (AVM) development, and other related health problems. ALK1 is a serine/threonine kinase receptor found on the cell membrane of endothelial cells. ALK1 and its co-receptor endoglin, are activated by binding to the circulating BMP9 ligand. The ALK1-endoglin-BMP9 complex will then regulate endothelial proliferation by activating the SMAD pathway by phosphorylation. Mutations in the ACVRL1 gene can form a modified ALK1 protein that has a high potential to inhibit this function, causing the hyperproliferation of endothelial cells and the development of AVMs, and ultimately HHT Type 2. It is believed, however unproven, that ALK1 is heavily glycosylated in the extracellular domain. My thesis research was aimed at studying the glycosylation of ALK1 and at exploring the relevance of this glycosylation to the development of HHT. The glycosylation of ALK1 was investigated by using: (i) a computational prediction approach (NetNGlyc 1.0 bioinformatics server), (ii) a glycosylation inhibiting drug (tunicamycin), (iii) an in vitro enzymatic approach of glycosylation breakdown, and (iv) site-directed mutagenesis to identify the ASP residue glycosylated on ALK1. The bioinformatics software NetNGlyc predicted a N-linked glycosylation site on an asparagine (ASN) residue located at position 98 in the extracellular domain of ALK1. I further found that, based on western blot analysis, ALK1 proteins shifted to a lighter molecular weight (5-8 kDa) when treated with tunicamycin, as well as endo H and PNGase F enzymes, which represent two glycosidases able to remove N-linked oligosaccharides on proteins. Western blot analysis also revealed an identical shift in protein size (5-8 kDa) when comparing wild type ALK1 to an asparagine98-to-alanine (N98A) mutant ALK1 construct. The 5-8 kDa shift observed in the drug and enzymatic experiments indicate the removal of a bulky oligosaccharide from the wild type ALK1 protein. This 5-8 kDa shift observed in the mutagenesis experiment indicated that the same oligosaccharide addition could not occur on ALK1 when ASP98 was missing. Thus proving that the asparagine at the 98th position of ALK1 is involved in N-linked glycosylation. These important findings on ALK1 modification offer a greater understanding of the mechanisms behind ALK1 regulation and function, especially its role in controlling angiogenesis. Furthermore, this data provides grounds for further research into the importance of ALK1 glycosylation in the pathogenesis of HHT, as well as the investigation into new treatment regiments.

The receptor for advanced glycation end products (RAGE) is a member of the immunoglobulin superfamily. The result of RAGE-ligand interactions augments the proinflammatory mechanisms acting in chronic inflammatory diseases. RAGE recognises a wide range of ligands that have no apparent structural similarities. It is unclear what controls this promiscuity of RAGE. The extracellular domain of RAGE has two potential glycosylation sites. It is speculated that N-linked glycosylation may have significant impact on ligand recognition, especially of S100 calcium binding protein ligands. Two objectives of this thesis were to establish whether S100A9 acts as a ligand for RAGE and to investigate whether glycosylation of RAGE has any influence on ligand recognition. These were achieved by generating two forms of RAGE. HEK 293 cells were transfected to express full-length, membrane-bound RAGE or a secreted form comprising the extracellular domain of RAGE. Site-directed mutagenesis of RAGE showed that asparagine at position 25 is the pre-dominant N-linked glycosylation site. The carbohydrate added to asparagine 25 was further modified to a non-sialylated carboxylated N-linked glycan, specifically recognised by monoclonal antibody GB 3.1. Binding studies showed that different RAGE ligands have individual requirements for glycosylation of the receptor. Binding of AGE-modified AGE-BSA or of S100B to RAGE occured independent of N-linked glycosylation of the receptor. RAGE also binds the S100 protein, MRP-14 (S100A9). In contrast to AGE-BSA or S100B, the non-sialylated carboxylated N-glycan expressed on RAGE is crucial for binding to MRP-14. However, RAGE produced in tunicamycin containing medium and thus lacking N-linked glycosylation, shows strong binding to MRP-14. It was concluded that two forms of binding are involved: the first mechanism relies on the non-sialylated carboxylated N-glycan attached to RAGE and acts in a "tethering" fashion. The second mechanism involves a conformational change of RAGE, which results in exposure of a binding site(s) and a more conventional receptor-ligand interaction. Another objective for this thesis is to study the expression of RAGE and its alternatively spliced variants. PCR analysis has revealed several variants of RAGE that result from alternative splicing mechanisms. The variant proteins are soluble due to a lack of membrane localising sequence. PCR results confirmed the presence of transcripts encoding for spliced variants of RAGE in several tumour cell lines. Among these were transcripts that should encode a soluble form of sRAGE 2. Furthermore, it was shown that sRAGE 2 transcript can be present in forms that contain the ligand-binding V-domain of RAGE or that are N-truncated and lack the V-domain. This is the first report of a soluble, N-truncated sRAGE 2 variant. The results in this thesis add to our knowledge of RAGE biology. MRP-14 (S100A9) is identified as a new ligand. The control of MRP-14/RAGE interaction relies on N-linked glycosylation of the receptor and further modification of the carbohydrate. "Tethering" or stronger receptor-ligand interactions are suggested as mechanisms for controlling RAGE recognition of multiple ligands. Soluble RAGE variants that lack or contain V-domain binding regions, and hence sites for glycosylation were produced. These have the capacity to compete with membrane-bound receptor for available ligand. The control of the expression of soluble RAGE variants, in concert with the control of various modification to carbohydrate expressed on the receptor, adds a level of complexity to ligand specificity. This may ultimately result in different paradigms of the inflammatory process.

Addendum pasted inside the back end-paper. / x, 179 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / This thesis presents a hypothesis that there are abnormalities of N-glycosylation in classical galactosaemia and that these abnormalities could contribute to the long-term complications. The aim of the thesis is to characterise and model N-glycosylation in skin fibroblasts from patients with galactosaemia. The study identifies a disturbance in the synthesis and processing of dolichol-linked oligosaccharides. It is anticipated that the serum glycoproteins in untreated galactosaemia may contain N-glycans that are partly absent or truncated. / Thesis (M.D.)--University of Adelaide, Dept. of Paediatrics, 1997

The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

La fibrose kystique (FK) est la maladie génétique mortelle la plus répandue chez les jeunes Canadiens. La colonisation des poumons par une bactérie opportuniste, Pseudomonas aeruginosa, est la principale cause de morbidité et de mortalité chez les patients FK. La maladie est causée par des mutations du gène codant pour la protéine CFTR (Cystic Fibrosis Transmembrane conductance Regulator) qui agit comme canal à ions chlorures. Ces modifications entraînent notamment une surexpression d'oligosaccharides fucosylés à la surface de l'épithélium pulmonaire. Le processus d'adhésion de la bactérie à la surface des cellules de l'épithélium pulmonaire est causé par la présence de deux lectines à la surface de la bactérie. Nous nous intéressons principalement à l'une d'entre elles, une lectine calcium dépendante qui reconnaît particulièrement le L-fucose (PA-IlL). Des études cristallographiques menées sur PA-IlL, en complexe avec divers ligands naturels tel que le Lewis a, ont permis d'identifier plusieurs éléments essentiels à l'obtention d'une forte interaction ligand-lectine. Basé sur ces études, le projet de recherche a consisté en la synthèse de différents analogues d'un disaccharide composé d'une unité fucose et glucosamine du type L-Fuc-a(1→4)-D-GIcNAcβ intimement impliqué dans le site de liaison de la protéine. Différents glycoclusters du disaccharide ont été synthétisés en utilisant la 'Click Chemistry'. De plus, quelques disaccharides modifiés en position C-2 et C-6 ont aussi été synthétisés. Les disaccharides ainsi que les glycoclusters ont été testés sur la PA-IlL en utilisant un test d'inhibition compétitive (ELLA). Les dérivés disaccharidiques ont montrés une constante de dissociation (Kd = 310 nM) dans le même ordre de grandeur que celle du meilleur ligand naturel Lewis a (Kd = 210 nM) connu jusqu'à maintenant pour la PA-IlL. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : Pseudomonas aeruginosa, Fibrose kystique, Lectine PA-IlL, Lewis a, Glycosylation, «click chemistry», Glycoclusters.

La présence des sucres dans le milieu biologique est indéniable. Ils font partie intégrante des mécanismes vitaux du corps humains mais aussi des autres êtres vivants comme les arbres, les plantes et les bactéries. Les produits naturels comportant une ou même plusieurs unités glycosidiques sont connus depuis longtemps. Cependant, le rôle biologique que jouent ces segments n'est pas toujours clair et mérite une attention particulière. Un exemple du rôle remarquable des sucres dans les rouages de la vie est celui des galectines-l et -3 qui influencent entre autre l'apoptose des cellules. Plus précisément, les glycosides phénoliques dont font partie les familles des flavonoïdes et des flavonoles présentent une pléiade de composés actifs affichant de fortes capacités antioxydantes ainsi que d'innombrables propriétés biologiques intéressantes. De leur côté, les 5,6-dihydropyran-2-ones, un hétérocycle de la catégorie des lactones, est un motif que l'on retrouve aussi dans une panoplie d'agents thérapeutiques. La (-)-psilotine est un produit naturel dont la structure est composée d'un glycoside phénolique et où l'aglycone est muni d'un cycle 5,6-dihydropyran-2-one. Jusqu'à maintenant, son effet connu est le ralentissement de la croissance des insectes et des plantes. Seule une synthèse racémique de ce composé avait été accomplie avant le début de ce projet. Des stratégies de synthèse ont donc été planifiées afin d'obtenir la (-)-psilotine de façon énantiosélective et de produire des dérivés analogues afin d'étudier le comportement biologique complet de cette famille de composés. La première voie, qui avait comme étapes clés une allylation stéréosélective de Keck, l'utilisation de groupements protecteurs de type chloroacétyle et une glycosidation avec assistance anchimérique, n'a pas porté fruit mais elle a tout de même permis de synthétiser efficacement le segment aglycone de façon énantiosélective. La deuxième voie se vouait à la glycosylation de ce même segment aglycone mais avec un époxyde de glucal formé notamment grâce à une élimination réductrice au zinc et une époxydation par l’OXONE. La synthèse s'est arrêtée à la formation d'un glucal silylé qui pourra toujours être utilisé pour d'autres travaux. La troisième stratégie employée représente la première synthèse achevée de la (-)-psilotine en ayant comme étapes clés plusieurs réactions déjà employées dans la première tentative mais aussi la glycosylation par catalyse de transfert de phase et l'utilisation d'éthers silylés comme groupements protecteurs. La quatrième voie s'avère être la deuxième synthèse de la (-)-psilotine. Cette fois, le segment aglycone est glycosylé efficacement et une réaction cruciale de clivage sélectif aux groupements acétyles situés sur une unité glycosidique est employée avec succès pour une seconde synthèse très avantageuse de la (-)-psilotine. En employant cette approche, la production d'analogues s'est enchaînée avec des modifications au niveau saccharidique et au niveau du segment aglycone afin de générer une gamme de molécules pour une batterie de tests biologiques. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : produit naturel, glycoside phénolique, 5.6-dihydropyran-2-one, (-)-psilotine, synthèse énantiosélective, allylation de Keck, glycosylation avec assistance anchimérique, glycosylation par transfert de phase, clivage sélectif aux sucres des groupements acétyles.

The use of reduced oxygen-packaging techniques has resulted in anaerobic bacteria emerging as a problem for the brewing industry over the last twenty-five years. The genus Pectinatus, consisting of the species<i>P. cerevisiiphilus</i> and<i> P. frisingensis</i>, is a concern for producers of unpasteurized beer. As a result, there is an ongoing need to both understand this genus and develop rapid detection methodologies to combat its presence in the brewery. The objectives of this study were to sequence and characterize the flagellin genes from both Pectinatus species and evaluate the genes and proteins from a taxonomic and detection-suitability standpoint. <p>A combination of micro-protein sequencing, polymerase chain reaction (PCR) and Bubble-PCR was used to completely sequence one flagellin gene from each Pectinatus species. This knowledge was then utilized to sequence the flagellin gene from four additional Pectinatus isolates, two from each species. To confirm the identity of the flagellin genes, one flagellin gene from each species was cloned, expressed and detected with Pectinatus-specific antibodies. A discrepancy between of the predicted protein size and the actual protein size led to tests for glycosylation, a post-translational modification. Taxonomic analyses, based on the flagellin genes, were conducted at both the superkingdom and genus levels. Finally, genus- and species-specific PCR primer sets were designed and tested for the specific detection of Pectinatus in the brewery. <p>Cloning and expression data confirmed the identity of the sequenced genes as Pectinatus flagellin genes. Glycosylation was positively confirmed to be a post-translational modification for five of the six strains tested. Phylogenetic analysis revealed that both of the Pectinatus species grouped with the phylum Firmicutes (low G+C, Gram-positive bacteria) and that there was more diversity at the species level within the <i>P. frisingensis</i> flagellin gene than the <i>P. cerevisiiphilus</i> flagellin gene. As a final point, the detection of most Pectinatus isolates was achieved with the preliminary PCR primer sets designed, however, some non-Pectinatus beer spoilage organisms, primarily wort spoilage organisms, were also detected. Both the basic science and the applied results generated from this study will aid the brewing industry in its ongoing battle to control Pectinatus contamination.

Generation of alcohol for biofuels from fermentation of sugar or starch has several economic disadvantages such as high cost of sugar processing and land usage competing with staple food. The solution may reside in hydrolysis of cellulose from crop waste such as stalks of rice and corn or non-crop plants such as weeds or wood. Our goal is to identify cellulases that can degrade cellulosic biomass more efficiently. Studies of microbial Family 9 glycoside hydrolase (GH9) proteins, including both endo-glucanases (EC 3.2.1.4) and cellobiohydrolases (EC 3.2.1.91), have shown that they function through an inverting mechanism to cleave the 1, 4-£]-glucosidic bond between two unsubstituted Glc units. The main function of plant glycoside hydrolases are involved in polysaccharide metabolism of cell wall during cell growth. Twelve Arabidopsis thaliana (Columbia) endo-1,4-£]-glucanases that belong to the GH9, were cloned and expressed in Pichia pastoris in order to produce cellulases to facilitate efficient bio-alcohol production. The recombinant proteins do not show in vitro endo-1, 4-£]-glucanase activity, but we can detect the recombinant proteins expression in supernatant or in pellet. The lack of enzymatic activity from recombinant proteins is probably due to improper folding or glycosylation, or fast degradation resulted from the above reasons. Other bioreactor will be tested in the future. Genetic engineering to modify Arabidopsis thaliana (Columbia) endo-£]-1, 4-glucanases is another approach to produce functional cellulases with economic efficiency that can be adapted to industrial scale for alcohol generation. On the other hand, we use semi-quantitative PCR method to study the Arabidopsis GH9 genes expression level in different tissue. At4g39000 and At3g43860 were found only in flowers and inflorescence, and At1g65610 expression in roots and shoots of the amount of more. Other genes in different tissues, was no found significant difference.

In this work, we examine two disparate aspects of glycosylation. The first project involves the elucidation of the glycosylation of the novel tetronolide natural product, kijanimicin. The biosynthesis of the deoxysugar TDP-L-digitoxose from the kijanimicin natural product pathway was achieved in vitro. The genes were identified from the cluster, cloned, expressed and the products were purified. Activity was demonstrated for the novel enzymes and the pathway was reconstructed in vivo using Streptomyces lividans. These strains of S. lividans were used to examine kijanimicin glycosyltransferase activity. We were able to demonstrate activity for 3 of 4 digitoxosyltransferases in the biosynthetic pathway and propose a biosynthetic scheme by which the tetrasaccharide chain is formed. We identified two putative glycosidases with novel folds, and one glycosyltransferase that appears to have unprecedented activity, attaching 2 if not 3 sugars in sequence. In the second portion of this work, we attempted to identify the eukaryotic C-mannosyltransferase enzyme and demonstrate its activity in vitro and in vivo. Here, we describe our efforts to identify the CMT. Through in silico analysis, putative C-mannosyltransferase genes were identified. These genes were expressed in E. coli and S. cerevisiae, however gene expression was apparently toxic to E. coli. S. cerevisiae expression was acceptable, but extraction proved to be somewhat problematic. We describe our efforts to develop a CMT assay for use in vitro by expressing the putative CMT in insect cells, which was much more promising. We also attempted to knock down the putative CMT genes using shRNA, which demonstrated that the genes of unknown function that were identified were essential for cellular viability. This work has contributed to the fields of both C-mannosylation and natural product glycosylation. We have elucidated the biosynthetic pathway of a novel deoxysugar, and identified potentially valuable tools for glycoengineering including a glycosyltransferase that appears to exhibit novel polymeric activity, as well as identifying two glycosyltransferase proteins that are apparent glycosidases. Our attempts to identify the CMT provided valuable insight into the future development of a C-mannosylation assay, and we have identified several promising protein candidates that are apparently essential for H. sapiens cellular viability.

Glycosylation, the addition of sugar moieties to nascent proteins, is one of the most common posttranslational modifications. Glycosylation regulates protein structure, function and localization. Most cell surface proteins and secreted proteins are glycosylated by the addition of Asparagine(N)-linked glycans (N-glycans). Aberrant N-glycosylation is a well-accepted feature of malignancy and is a potential prognostic marker for some types of cancer. For example, increased expression of complex N-glycans has been detected in cancers of breast, colon and has been correlated with reduced survival of the patients. Therefore, understanding the role of N-glycosylation in malignancy could be beneficial for developing novel therapeutic and prognostic strategies. To examine the role of N-glycosylation in malignancy, we applied chemical biology and genetic approaches. First, we conducted a high throughput screen to identify compounds that could block L-PHA-induced cell death. Our screen identified the cardiac glycoside Na+/K+-ATPase inhibitors as novel inhibitors of N-glycosylation. Further analysis of N-glycans consistently confirmed that inhibition of Na+/K+-ATPase impairs the N-glycosylation, as well as migration and invasion. Interestingly, other studies have shown antimetastatic effects of cardiac glycosides in patients. Thus, our high throughput screen identified Na+/K+-ATPase inhibition as a novel strategy to target the N-glycosylation pathway. In addition, we used a genetic approach to investigate the role of N-acetylglucosaminyltransferase I (GlcNAc-TI/Mgat1) in malignancy. Knockdown of GlcNAc-TI decreased the cell-surface expression of complex N-glycans. By confocal microscopy, knockdown of GlcNAc-TI decreased cell surface expression of β1 integrins and increased their localization around the nucleus. Moreover, GlcNAc-TI knockdown decreased the migration and invasion of malignant cells. Next, we investigated the effect of GlcNAc-TI in an orthotopic xenograft mouse model of metastasis. GlcNAc-TI knockdown significantly decreased the lung colony formation of the highly metastatic PC3N7 human prostate cancer cell line in mice. Our results suggest an important role for GlcNAc-TI in tumor metastasis. Interestingly, breast cancer patients with lower expression levels of Mgat1 had lower risk of disease relapse after therapy. Thus, GlcNAc-TI plays an important role in cancer progression and metastasis and GlcNAc-TI inhibitors could have therapeutic benefits for cancer patients. Moreover, expression levels of GlcNAc-TI could be used as a prognostic marker in patients with cancer.