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.

No description available.

The causative agent of AIDS (acquired immune deficiency syndrome), HIV (human immunodeficiency virus), is one of the most extensively studied pathogens in modern history. The virus has multiple mechanisms of persisting in the host including evading host immune response. Since HIV-1 depends heavily on the host machinery for various aspects of its life cycle, unraveling the complex interplay between the host and HIV-1 could provide new clues to therapeutic avenues. In T cells, HIV assembles and subsequently buds through the plasma membrane incorporating host derived proteins and lipids in the viral envelope. HIV is thought to utilize a pre-existing mechanism for the budding of normal cellular vesicles called microvesicles to exit host cells. The evidence for this theory comes from reports of similarities between HIV and microvesicles observed for a small subset of proteins and lipids, leading to controversies about its validity. To further test this hypothesis, we utilized lectin microarrays to obtain a comprehensive glycomic profile of HIV and microvesicles derived from a panel of T cell lines. Glycosylation is critical to protein sorting and has a crucial role in HIV-1 biology, making it an ideal marker to compare the particles and the host cell membrane. We observed similar glycomic profiles for HIV-1 and microvesicles strongly suggesting an analogous mode of egress. Glycosylation of both particles seems to vary based on the parent cell line, providing additional evidence for this hypothesis. Microvesicles are involved in immune response modulation; hence the incorporation of microvesicular proteins could influence interactions of HIV with the immune system. The differences in glycosylation between these two particles could be potentially explained by the heavily glycosylated viral envelope glycoprotein. I also demonstrated that these vesicles bud from particular glycan enriched domains of the plasma membrane. Additionally, this work sheds light on the potential mode of interaction between galectin, an immune lectin and HIV-1. This work strongly argues for a conserved mechanism of exocytosis for both particles and sets the stage for examining the role of glycosylation in trafficking of proteins to the sites of microvesicular and viral budding. / text

The conceptually simple process of linking carbohydrate units by glycosylation has proven to be one of the most difficult synthetic processes to control from a stereochemical perspective. In particular it is the stereocontrolled synthesis of 1,2-cis glycosyl linkages (e.g. α-glucosides, β-mannosides) which poses the most difficult challenge. The research presented in this thesis describes new ways in which stereocontrol in glycosylation reactions can be achieved. New methods of neighbouring group participation have been explored, utilising novel protecting groups at the 2-postion of a series of glycosyl donors. In particular the use of glucosyl donors bearing a 2-O-(2-(2,4,6- trimethoxyphenyl)thio)ethyl protecting group at the 2-hydroxyl, have shown exceptional α-selectivity especially when a completely armed donor was used. Work within this thesis also describes the use of chiral Brønsted acid catalysts in stereoselective glycosylation reactions. However the yields and stereoselectivity obtained were not very encouraging.

Diastereoselective control of glycosylation still remains a difficult task. Therefore, new glycosylation methods using asymmetric catalysis were developed to control the diastereoselectivity. Two systems were developed and each focused on a separate type of glycosyl donor. In the first system, glycosyl halides were subjected to reaction conditions inspired by Hamilton et al., who effectively had controlled the substitution of a racemic chloroamine by an alcohol. Asymmetric control of glycosylation was achieved through this adapted catalytic system. Both enantiomers of the catalyst ((R) and (S) TRIP) generally displayed b-selectivity with tertiary butyl methyl ether (TBME) as the solvent allowing almost exclusive formation of the β-anomer. However, low and inconsistent yields were obtained. The second system proposed the use of the same phosphoric acid catalyst (TRIP) to catalyse the glycosylation of glycals. However, this was ineffective as the catalyst was not a strong enough Brønsted acid. These studies then led to the development of two new chiral catalysts which then promoted the glycosylation of glycals, along with the formation of an undesired side product. Attempts were made to reduce the formation of the side product but unfortunately this proved unsuccessful. The diastereoselective outcome displayed between the different catalysts in separate trials was negligible, but the principles developed in this study should lead to the further development of new chiral catalysts for the glycosylation of glycals.

O-mannosylation is a specific form of glycosylation, a post-translational protein modification with O-linked mannose attached to serine or threonine residues. O-mannosylation is implicated in crucial biological processes such as neuronal and muscle development, cell adhesion and cell migration. Two O-mannosyltransferase genes have been described in mammalian genomes so far, POMT1 and POMT2. Disruptions of O-mannosylation result in congenital muscular disorders in humans. The most severe, the Walker-Warburg Syndrome is associated with mutations in POMT1 and POMT2. Just like vertebrates, Drosophila has two O-mannosyltrasferase genes, DmPOMT1 (rt) and DmPOMT2 (tw), which share significant similarities with their mammalian counterparts. Mutations in both DmPOMT1 and DmPOMT2 cause the "rotated abdomen" phenotype, a clockwise rotation of abdominal segments in adult flies. In my dissertation, I analyzed the expression patterns of rt and tw during development. Both genes have similar essentially overlapping expression patterns. Immunostaining revealed that RT and TW proteins are co-localized in the ER compartment. The analysis of double mutants revealed a mutual epistatic relationship between rt and tw, which could be evidence for RT and TW functioning in the same molecular complex. Also, I studied temporal and spatial requirements of tw during development. I found a broad "developmental window competent to fully rescue the abdomen rotation in adult flies." The spatial studies of tw requirements demonstrated that tw expression is pattern-dependent and the function of tw is cell-autonomous or it has a very short-range effect. The analysis of rescue results with different drivers suggested that the tw requirement is not strictly limited to larval epidermis or muscles alone, but required a contribution from epidermal and muscle cells with a possible involvement of CNS. I have shown that Drosophila Dystroglycan is modified with mannose in the presence of RT-TW enzymatic complex in vivo and in vitro. The co-expression of RT and TW is required to generate high-molecular-mass bands of DG. The lectin staining revealed differences in glycan compositions of DG purified from different genetic backgrounds. Overall, this research work established Drosophila as a model system to study mannosylation, which may shed light on mechanisms of muscular dystrophies in humans.

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Sydney, 2005. / Title from title screen (viewed 19 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the Heart Research Institute, Faculty of Medicine. Degree awarded 2005; thesis submitted 2004. Includes bibliographical references. Also available in print form.

Thesis (Ph. D.)--Ohio State University, 2003. / Title from first page of PDF file. Document formatted into pages; contains xiii, 279 p.: ill. (some col.). Includes abstract and vita. Advisor: Todd L. Lowary, Dept. of Chemistry. Includes bibliographical references (p. 150-154).

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.