Hyaluronan, a member of the glycosaminoglycan family, is a critical component of the extracellular matrix. A model for hyaluronan degradation has been advanced that invokes the activity of both hyaluronidases and exoglycosidases. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to hyaluronan breakdown. Herein, we have used several mouse models to investigate the contributions of the exoglycosidase, β-hexosaminidase, and the endoglycosidase, hyaluronidase 1, to the lysosomal degradation of hyaluronan. We employed immunohistochemistry and fluorophore assisted carbohydrate electrophoresis to determine the degree of hyaluronan accumulation in mice deficient in one or both enzyme activities. β-Hexosaminidase-deficient mice displayed only low levels of hyaluronan accumulation, limited to the liver and lymph node. The distribution and levels of hyaluronan in hyaluronidase 1-deficient mice were similar to that observed in β-hexosaminidase-deficient mice. Interestingly, extensive global hyaluronan accumulation was present in mice deficient in both enzymes, with the highest levels found in liver and lymph node. Chondroitin, a glycosaminoglycan similar in structure to hyaluronan, was also broadly accumulating in mice deficient in both enzymes. Further, the accumulation of chondroitin sulphate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulphate breakdown. Extensive accumulation of hyaluronan and chondroitin when both enzymes are lacking suggests β-hexosaminidase and hyaluronidase 1 are functionally redundant in hyaluronan and chondroitin breakdown, as broad extensive accumulation was not observed in mice deficient in only one of these enzymes.

Hyaluronan, a member of the glycosaminoglycan family, is a critical component of the extracellular matrix. A model for hyaluronan degradation has been advanced that invokes the activity of both hyaluronidases and exoglycosidases. However, no in vivo studies have been done to determine the extent to which these enzymes contribute to hyaluronan breakdown. Herein, we have used several mouse models to investigate the contributions of the exoglycosidase, β-hexosaminidase, and the endoglycosidase, hyaluronidase 1, to the lysosomal degradation of hyaluronan. We employed immunohistochemistry and fluorophore assisted carbohydrate electrophoresis to determine the degree of hyaluronan accumulation in mice deficient in one or both enzyme activities. β-Hexosaminidase-deficient mice displayed only low levels of hyaluronan accumulation, limited to the liver and lymph node. The distribution and levels of hyaluronan in hyaluronidase 1-deficient mice were similar to that observed in β-hexosaminidase-deficient mice. Interestingly, extensive global hyaluronan accumulation was present in mice deficient in both enzymes, with the highest levels found in liver and lymph node. Chondroitin, a glycosaminoglycan similar in structure to hyaluronan, was also broadly accumulating in mice deficient in both enzymes. Further, the accumulation of chondroitin sulphate derivatives was detected in mice deficient in both enzymes, as well as in β-hexosaminidase-deficient mice, indicating that both enzymes play a significant role in chondroitin sulphate breakdown. Extensive accumulation of hyaluronan and chondroitin when both enzymes are lacking suggests β-hexosaminidase and hyaluronidase 1 are functionally redundant in hyaluronan and chondroitin breakdown, as broad extensive accumulation was not observed in mice deficient in only one of these enzymes.

No description available.

No description available.

No description available.

Bacterial capsules are important virulence factors for a number of different pathogens, including Escherichia coli, Neisseria meningitidis, Haemophilus influenzae, and Pasteurella multocida. Capsular polysaccharides (CPSs) synthesized via the ATP-binding cassette (ABC) transporter-dependent pathway protect these bacteria from complement-mediated killing and phagocytosis, and consist of long polysaccharide chains attached to the cell surface via a phospholipid. CPSs are synthesized on the cytoplasmic face of the inner membrane before transport to the cell surface. While the enzymes that synthesize the polysaccharide have been studied in detail, very little is known about the structure and biosynthesis of the phospholipid terminus. To determine the structure of the reducing terminal glycolipid, CPS from E. coli K1, K5, and N. meningitidis group B was purified using a novel strategy and its structure was determined by mass spectrometry, nuclear magnetic resonance and chemical methods. All three polysaccharides possess terminal lyso-phosphatidylglycerol, which is connected to the CPS repeat unit by a linker consisting of multiple 3-deoxy-D-manno-octulosonic acid (Kdo) residues, forming an alternating β-2,4/β-2,7-linked structure. In addition to describing its structure, the biosynthesis of the glycolipid terminus was also investigated. KpsC and KpsS are conserved proteins encoded in the capsule loci from different bacteria with ABC transporter-dependent capsule assembly pathways but have no previously assigned function. An in vitro assay was developed to characterize KpsSC activities, leading to the finding that they are the Kdo transferases responsible for synthesis of the poly-Kdo linker. This research has contributed significantly to the understanding of the structure and biosynthesis of capsular polysaccharides.

The proposed thesis brings new information on several aspects of tick glycobiology - tick N-glycans, tick lectins, and glycosylation of the tick-borne pathogen, Lyme disease spirochetes Borrelia burgdorferi s.l.

The HIV-1 surface glycoprotein, gp120, is made of a rapidly mutating protein core and an extensive carbohydrate shield which are, respectively, encoded by the viral genome and synthesised by the host cell. In contrast to host cell glycoproteins however, gp120 contains a population of unprocessed oligomannose-type glycans that interact with host lectins, promote HIV infection, and alter cell signalling. They also form the basis of the epitopes of several broadly neutralising antibodies isolated against HIV, making them a key feature for immunogen design. The mechanistic basis of how HIV glycans are differentially processed by the host cell was demonstrated on a recombinant gp120 model, suggesting that steric occlusion within the patch of densely packed glycans lead to lack of processing by ER and Golgi α-mannosidases. Furthermore, an elevated level of oligomannose-type glycans was evidenced on gp120 isolated from HIV-1_JRCSF virions produced in PBMCs, compared to recombinant material (respectively ~79% and ~29% of total N-linked glycans), along with a subset of highly processed and sialylated, bi-, tri- and tetra-antennary complex-type glycans, which could be involved in direct interaction with key host cell immune receptors and strongly suppress both antibody and T-cell immune responses. The effect of variation in viral production systems was analysed, with envelope glycoprotein derived from pseudoviral particles produced in HEK 293T cells exhibiting predominantly an oligomannose population (98%), compared to gp120 isolated from a single-plasmid infectious molecular clone (56%). Finally, mutation of one or several glycosylation site(s), known to be required for oligomannose-restricted neutralizing antibodies, was shown to induce a subtle redistribution within the oligomannose series whilst maintaining overall oligomannose levels. The gp120 glycan profile is therefore robust to mutations and also remarkably similar across primary viral isolates from Africa, Asia and Europe and consequently represents an attractive target for vaccine development.

The biosynthesis of GDP-Fucose, the activated donor for fucose, has been recently shown to be essential in the parasite Trypanosoma brucei. Fucose is a common sugar modification on eukaryotic glycan structures, but it has not been well described in trypanosomatids. To elucidate the role of fucose in T. brucei we searched for putative fucosyltransferases in this parasite. A single putative T. brucei fucosyltransferase (TbFT) was identified and recombinantly expressed in Escherichia coli. The protein was active and structural characterization of its reaction product identified it as a GDP-Fuc: ß-D-galactose a-1,2-fucosyltransferase with preference for Galß1,3GlcNAc containing structures as glycan acceptors. A procyclic form conditional null mutant for TbFT was generated and this glycosyltransferase shown to be essential for parasite growth in vitro, with the mutant cells displaying a slightly abnormal morphology and an apparent reduction in the surface high molecular weight glycoconjugate complex. Here we also describe the various experimental approaches that were used to try to identify the fucosylated glycocojugates in T. brucei. Lastly, to better understand the biosynthesis of GDP-Mannose, the starting metabolite for the biosynthesis of GDP-Fuc, we biochemically characterized T. brucei phosphomannomutase (TbPMM). Here we show this enzyme could interconvert not only mannose-phosphates, but also glucose-phosphates.

N-linked glycosylation is the most common form of post-translational modification in nature and is essential to almost all enveloped viruses, including members of the Flaviviridae family. The host cell N-linked glycoprotein processing pathway is utilised by these viruses and as such has long been identified as a potential target for the development of antiviral drugs. Here, the antiviral mechanisms of three classes of small molecules targeting the secretory pathway and altering viral envelope glycosylation are investigated, using the HCV surrogate model, BVDV. The antiviral activity of imino sugars, principally through α-glucosidase inhibition, is well-characterised and here, a group of novel adamantyl coupled imino sugars are investigated and demonstrated to inhibit ER α glucosidases, which correlates with their antiviral activity against BVDV. Additionally, BVDV is used to study the antiviral mechanism of action of nitazoxanide. Nitazoxanide, the parent compound of the thiazolide class of structures, is a broadly antimicrobial compound with antiviral activity against HBV, HCV, influenza, JEV and others. Here, nitazoxanide is shown to be antiviral against BVDV by inducing Ca²⁺ release from ATP-sensitive intracellular calcium stores, disrupting ER-Golgi trafficking and inhibiting complex glycan formation. Finally, the potential of Golgi endo-α-mannosidase as an antiviral target is explored, using the endomannosidase inhibitor glucose-isofagomine in conjunction with the imino sugar α-glucosidase inhibitor NAP-DNJ. Endomannosidase is shown to be a valid antiviral target for BVDV, both alone and in combination with α-glucosidase inhibition, and is utilised by viral glycoproteins to acquire complex glycan structure, even in the absence of α-glucosidase inhibition. Altogether, this work furthers our understanding of the varied antiviral mechanisms of small molecules targeting the secretory pathway, enhancing the search for novel antiviral drugs directed against host cell machinery.