Altered glycosylation is a hallmark of cancer and can be used to distinguish cancer cells from normal cells. This thesis shows a comprehensive study of glycosylation on a variety of human prostate cell lines. Cell surface glycans were analyzed using lectins and antibodies. Glycans were also released from glycoproteins, purified and analyzed by tandem mass spectrometry. The enzymatic activities and expression levels of glycosyltransferases responsible for the syntheses of N- and O-glycans have been determined and correlated with glycan structures. We found that prostate cells are capable of synthesizing complex N-glycans and core 1 based O-glycans, and that each cell line has characteristic glycosylation pathways. Elevated levels of truncated O-glycans including sialyl-T and di-sialyl-T antigens were detected in prostate cancer cell lines. Complex O-glycans without sialic acid residues constituted the major O-glycans in normal prostate cells, but sialyl-T antigens were prevalent in the most aggressive prostate cancer cells derived from bone metastasis (PC-3). The expression and activity of sialyltransferase ST3Gal-I, responsible for the synthesis of sialyl-T antigens, were up-regulated in PC-3 cells, providing a mechanism for sialyl-T expression. Forced down-regulation of ST3Gal-I by RNA interfering technology as well as neuraminidase treatments of cells were employed to modify cellular glycosylation, and indicated a critical role of sialic acid in the regulation of apoptosis. These observations facilitate the identification of prostate cancer markers and help to understand the importance of glycans in cancer progression. To elucidate mechanisms of glycosylation and develop glycosyltransferase inhibitors, we characterized recombinant soluble human Gal- and GlcNAc-transferases that synthesize O-glycan cores 1 to 4, critical for the overall structures of O-glycans. The biochemical properties and substrate specificities of these enzymes were determined using synthetic acceptor substrate analogs. These collective results were used to develop novel glycosyltransferase inhibitors, including acceptor substrate analogs and bivalent imidazolium salts. These compounds inhibited selected glycosyltransferases, and thus have the potential to modify cellular glycans for functional studies. Glycosyltransferase inhibitors could also be used in biotechnology and in therapeutic applications to increase the apoptotic potential of prostate cancer cells. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2013-07-23 21:19:36.197

Leung Yuet Kin. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (p. 147-162). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstracts in English and Chinese.

The lumen of the endoplasmic reticulum (ER) provides a specialized environment to assure the folding and oligomerization of secretory proteins to their native conformations. UDP-glucose:glycoprotein glucosyltransferase (UGGT) is a biosensor in the ER that detects the folding state of glycoproteins. UGGT-catalyzed monoglucosylation of incompletely folded glycoproteins leads to their continued retention in the ER through their association with the lectins calnexin and calreticulin for further folding or for degradation. Purified recombinant UGGT from rat liver and glycoprotein substrates from a mutant strain of Saccharomyces cerevisiae were used in an in vitro system to examine the peptide components recognized by UGGT in unfolded glycoproteins and glycopeptides. Mass spectrometry was used to measure and quantitate the levels of glucose incorporation into these substrates that was directly related to their level of recognition by UGGT. To assess the capacity of UGGT for sensing non-native structures in glycoprotein substrates, Exo-1,3-beta-glucanase (beta-Glc) from S. cerevisiae was crystallized and its structure determined. A mutagenesis strategy was used to mutate solvent-exposed residues to yield the beta-Glc F280S point mutant that retained enzymatic activity while still being recognized by UGGT. These data suggest that UGGT recognizes solvent-exposed hydrophobic patches in the primary and tertiary structure of glycoproteins even in near-native conformations.

Synthesis from UDP-($ sp{14}$C) glucose of charged lipid-linked glucosyl compounds by pea membranes was short-lived, and of very limited magnitude compared to the synthesis of 1,4- and 1,3-linked B-glucans. Lipid-linked monophosphoryl glucose was the only charged lipid formed at initial stages, and had properties similar to that of dolichol-monophosphoryl glucose. It exhibited no turnover during pulse-chase experiments. Lipid-linked pyrophosphoryl-glucose or -oligosaccharides were not detected. Coumarin inhibited the synthesis of SDS-soluble products and glucans, but not of the lipid-P-glucose. Transfer of the label from endogeneous lipid-P-($ sp{14}$C) glucose or from dolichol-P-($ sp3$H) glucose into non-lipid products was minimal. It was concluded that the lipid-linked phosphoryl saccharide formed from UDP-glucose was not an obligate intermediate in the formation of B-glucans in pea membranes. / Fucose-containing lipid-linked intermediates were not involved in the biosynthesis of xyloglucans. However, pea microsomal membranes catalysed the transfer of $ lbrack sp{14}{ rm C} rbrack$-fucose from GDP-$ lbrack sp{14}$C) fucose, with or without added unlabelled UDP-glucose, UDP-xylose or UDP-galactose, to an insoluble product with properties characteristic of xyloglucan. After digestion of the ethanol-insoluble pellet with Streptomyces griseus endocellulase, $ lbrack sp{14}$C) fucose residues occurred exclusively in a fragment identified as the xyloglucan nonasaccharide, Glc$ sb4$ Xyl$ sb3$ Gal Fuc. By comparison, in incubations with UDP-$ lbrack sp3$H) xylose, the maximum size of labeled oligosaccharide found following cellulase digestion of products was an octasaccharide. In the presence of both GDP-$ lbrack sp{14}$C) -fucose and UDP-$ lbrack sp3$H) xylose, a nonasaccharide containing both labels was produced. Fucose and xylose residues were transferred rapidly to acceptor molecules of MW up to 300,000. Such products did not elongate detectably over 60 min of incubation. We concluded that the nonasaccharide subunit of xyloglucan was generated in vitro by transfucosylation to preformed acceptor chains, and that its synthesis was dependent on exogenous GDP-fucose. / Microsomal membranes were separated by rate-zonal centrifugation on renografin gradients. Transfer to xyloglucan of labelled fucose and xylose from GDP- ($ sp{14}$C) fucose and UDP- ($ sp{14}$C) xylose occurred mainly in dictyosome-enriched fractions. No transferase activity was detected in secretory vesicle fractions. Pulse-chase experiments using pea stem slices incubated with ($ sp3$H) fucose suggested that xyloglucan chains are fucosylated and their structure completed within the dictyosomes, before being transported to the cell wall by secretory vesicles.

Thesis (M.S.)--Youngstown State University, 2008. / Includes bibliographical references (leaves 60-63). Also available via the World Wide Web in PDF format.

Formation of L-cysteine, from L-serine and sulphide, represents the principal route of Sulphur incorporation into organic compounds in living organisms. Cysteine biosynthesis in plants is achieved by two enzymes, serine acetyltransferase (SAT) and 0-acetylserine (thiol) lyase (OASTL), which form a cysteine synthase complex. Three novel cDNA species, Sat-52, Sat-53 and Sat-106, encoding SAT isoforms from A. thaliana were isolated from a collection of cDNAs previously cloned by functional complementation of the E. coli cysE mutant strain JM15, which is defective in serine acetyltransferase. Deduced amino acid sequence analysis suggests that Sat-52 encodes a putatively mitochondrial isoform whilst Sat-53 and Sat-106 encode proteins with cytoplasmic locations. Sequence information derived from the Arabidopsis Genome Initiative allows mapping of Sat-52, Sat-53 and Sat-106 genes to locations on chromosomes V, I and II respectively. A fourth SAT cDNA from A. thaliana, Sat-1, was cloned prior to the work detailed here and encodes a putatively plastidic isoform of the enzyme. Southern hybridisation against digested genomic DNA suggests that each SAT gene is represented by a single copy in the A. thaliana genome. DNA probes specific to the SAT gene-family members were designed and used in various studies to examine expression of SAT genes. Northern blotting and hybridisation was used to determine transcript distribution between root, leaf, stem, flower and silique tissues and to study the expression of the gene-family in response to sulphate and nitrate nutrition. Spatial distribution of Sat-52 and Sat- 53 transcript in root, leaf and stem tissue was also examined by in situ hybridisation using specific riboprobes. Both genes are highly expressed in leaf trichomes. The Sat-52 transcript was also localised to the vascular- bundles of root, leaf and stem tissue. The isoforms encoded by Sat-52 and Sat-53 are hypothesised to have specific roles in these cell-types.

No description available.

During tumor progression and development of distant metastases, a subset of cancer cells undergoes transformation programs, such as epithelial-mesenchymal transition (EMT), to acquire enhanced migratory attributes to commence the metastatic cascade with the intension of achieving an active cell adhesion molecule-mediated organ-specific homing. Similarly, naive T cells reform the assemblage of their surface adhesion molecules during differentiation to activated T cells in order to successfully home to sites of inflammation and other extra-lymphoid organs for surveillance purposes. Sialyl-Lewis X (sLex) is well-known for mediating the homing of epithelial circulating tumor cellss (CTCs) and activated T cells to target sites through the interaction with endothelial selectins. Since glycan structures are not directly encoded by the genome, their expression is dependent on the glycosyltransferase (GT) expression and activity. Yet, the modulation of GTs during breast cancer transformation and in different molecular subtypes is still unknown. In addition, although the regulation of GTs during T cell activation is well-understood, the regulation at the epigenetic level is lacking. O-glycan-type sLex expression and E-selectin binding under static and flow conditions varies among molecular subtypes of breast cancer and upon the induction of EMT which is linked to the expression patterns of GTs. GTs displayed a significant prognostic value of in the association with the patients' survival profiles and in the ability to predict the breast cancer molecular subtypes from the expression data of a random patient sample. Also, GTs were able to differentiate between tumor and their normal counterparts as well as cancer types and glioblastoma subtypes. On the other hand, we studied the regulation of GTs in human CD4+ memory T cells compared to the naive cells at the epigenetic level. Memory T cell subsets demonstrated differential chromatin accessibility and histone marks within the promoters of the GTs genes. Moreover, they showed differential binding of pioneer and nonpioneer transcription factors (TFs). We proposed a model for the regulation of FUT7 during T cell activation that relies on the interplay between chromatin-remodeling and cell-fate-specifying TFs. Furthermore, we developed a fluorescent multiplex cell rolling (FMCR) assay to study the cell adhesion properties under physiological conditions. Compared to the conventional parallel plate flow chamber (PPFC) assay, the novel technique posses a high-throughput capacity which helps eliminate the inter-experimental variation problem by running multiple samples simultaneously and under competitive settings. We also developed a real-time analysis pipeline that enhanced the statistical power of the assay. Overall these modifications to the traditional parallel plate assay improves the reliability and results along with saving time and effort.

No description available.

No description available.