Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2012 / Various cytomorphologic and biochemical markers of apoptosis are found in different compartments (plasma membrane, cytoplasm, nucleus, and mitochondria) of target cells. Although the plasma membrane is an easily accessible cellular compartment, relatively little is known about the changes in the expression of plasma membrane glycoproteins during apoptosis, and whether these changes could be used for detection of apoptosis. A critical element of this study was to purify lectins from crude homogenate on glycoprotein-cryogel affinity matrices, and later use the lectins to detect changes on the cell surface of apoptotic cells. Pterocarpus angolensis seed lectin was extracted and fractionated using ammonium sulphate precipitation. The 60 % ammonium sulphate pellet was dissolved in saline azide and purified using Sephadex G-75 affinity chromatography. A 28 kDa lectin was retarded within the column and appeared as a short and broad peak on the chromatogram. Traditionally, Sephadex G-75 column are used predominantly for size exclusion, in this study, the column was used in a non-traditional way for affinity chromatography, as the purified protein is able to bind sugar moieties existing in the structure of Sephadex G-75. A single-step purification of P. angolensis seed lectin was achieved by directly applying unclarified P. angolensis crude extract to the pAAm-cryogel using fetuin as the affinity ligand. Pterocarpus angolensis extract fractionated into 2 peaks, which revealed a highly concentrated band on SDS-PAGE. The results also revealed that an increased binding of the lectin to the fetuin-cryogel matrices was also dependent on the time of incubation. This study suggested very low capacities of the cryogels for the protein due to low coupling sites on the matrix. Taking into account that lectins serve as invaluable tools in diverse area of biomedical research, this study proposed using specific plant lectins to follow the expression of plasma membrane glycoproteins during programmed cell death. Treatment of HL-60 cells with lithium and actinomycin D confirmed a time- and dose-dependent inhibition of proliferation and a decrease in proliferation, which suggest cell death of the treated cells. The observed cell death was further investigated for cellular and biochemical hallmark features of apoptosis, which has shown preferential binding of annexin V-FITC to phosphatidylserine and low molecular DNA ladder. Several FITC labelled lectins were used to detect changes in cell surface glycosylation that accompany apoptosis. This study xvii has shown amongst several FITC-labelled lectins that T. vulgaris lectin could intensively stain the membrane area of apoptotic cells suggesting that the expression of N-acetylglucosamine was significantly increased during actinomycin D induced apoptosis of HL-60 cells. Binding was shown to be specific because it was blocked by the corresponding inhibitory sugar. Thus, the method described in this study could be suitable for the detection of very early stages of apoptosis by recognizing the cell surface carbohydrates of apoptosis.

Includes bibliographical references (leaves 355-403)

The production of recombinant glycoproteins from mammalian cell cultures requires robust processes that can achieve high protein yield while ensuring the efficacy of these proteins as human therapeutics. We describe two approaches currently being developed in our group to genetically engineer cell lines with desirable characteristics for recombinant protein production. To enhance the degree of sialylation in the glycoprotein product, we propose to increase intracellular sialic acid availability by overexpressing the CMP-sialic acid transporters. We are also interested in engineering mammalian cells that can proliferate at reduced cultivation temperatures. Low temperature cultivation of mammalian cells has been shown to enhance glycoprotein production but reduces cell growth. It is hypothesized that a mutant cell line that can proliferate at low temperatures may be coupled with low temperature cultivation to improve recombinant protein production. / Singapore-MIT Alliance (SMA)

This thesis describes the application of mass spectrometry (MS) to glycoprotein and oligosaccharide analysis. Glycosylated proteins are involved in cell-cell and cell-matrix recognition. Applications of trypsin and proteinase K to hydrolyze glycoproteins into glycopeptides that are compatible with MS and MS/MS analysis are investigated. For successful site-specific analysis of glycans, glycopeptides with short peptide (3-8 residues) are needed. Although trypsin is an important enzyme for protein identification, proteinase K is superior for site-specific glycan analysis due to its potential to hydrolyze every glycoprotein to short glycopeptides. The gas-phase dissociation pathways, kinetics and energetics of protonated oligosaccharides are described. The oligosaccharides dissociate via cleavage at the glycosidic linkages during thermal activation. Using double resonance experiments, it was established that oligosaccharides undergo sequential and parallel fragmentation reactions. Furthermore, dissociation of product ions to secondary ions was confirmed. Arrhenius activation parameters, Ea and A for protonated alpha- and beta-linked D-glucopyranose oligosaccharides are reported.

Thesis (Ph. D.)--University of Washington, 2002. / Vita. Includes bibliographical references (leaves 77-99).

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

The Paramyxoviridae family includes several viruses that are important to human health, including measles virus. The envelope glycoproteins are essential for attachment and entry of the virus into the host cell [1, 2]. To develop novel therapeutics against the virus, detailed knowledge of envelope glycoprotein protein-protein interaction is important. The goal of this study is to characterize the MeV entry machinery on a molecular level. Interaction of haemaglutinin (H) and fusion (F) protein in pre-fusion form can be biochemically detected with DTSSP. To map the interaction site of H and F protein in pre-fusion form, we have mutated lysine (K) to arginine (R) in the F protein, and examined surface expression. The mutated F was still expressed on the surface and amount of surface expression correlated with fusion activity. The altered F proteins produced in this study will be used to further characterize the H-F interaction.

published_or_final_version / abstract / toc / Surgery / Master / Master of Philosophy

No description available.

No description available.