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.

Neisseria meningitidis is a causative agent of meningitis and septicaemia. Pili are one of the major virulence factors that contribute to the pathogenicity of N. meningitidis. Pili of Neisseria are type IV fimbriae composed primarily of thousands of identical pilin subunits. Pilin of N. meningitidis is post-translationally modified by trisaccharide, phosphorylcholine and -glycerophosphate. The genes involved in pilin expression, pilin glycosylation and phosphorylcholine modification are phase variable (high frequency ON/OFF switching of expression). The function of pilin post-translational modifications and their phase variable expression in host:pathogen interactions is unknown. The phase variable expression of glycosylation in bacteria has been proposed to function in bacterial adherence and immune avoidance. However, the function of pilin glycosylation in N. meningitidis is unclear. Phosphorylcholine is expressed in a number of respiratory organisms including P. aeruginosa (on teichoic acid), S. pneumoniae (on lipoteichoic acid) and H. influenzae (on LPS). Phosphorylcholine in these organisms is important in colonisation of the nasopharynx and invasion of the epithelium. Studies on N. meningitidis pilin post-translational modifications have been restricted by difficulties in purification of pilin protein. In this thesis, we evaluated current pilin purification methods and established an efficient method of purifying pilin from N. meningitidis by Flag-tag purification system. Flag-tag purified pilin is post-translationally modified. The LC-ESI/MS/MS analysis performed in this thesis using Flag-tag purified pilin successfully determined the phosphorylcholine post-translational modification sites. Based on the MS data and the mutagenesis analysis, phosphorylcholine is covalently linked to serine 157 and serine 160 of pilin. The colony immunoblot of a serine 157/160 to alanine mutant revealed that phosphorylcholine modifications of these sites on pilin are the only surface exposed phosphorylcholine and is responsible for binding to TEPC-15 (the monoclonal antibody which binds to phosphorylcholine). In this thesis, molecular modelling demonstrated that surface exposure of pilin phosphorylcholine could be altered by the phase variation of pilin glycosylation on the adjacent pilin monomer. Furthermore, the sites for phosphorylcholine modification are commonly observed in N. meningitidis strains but not in N. gonorrhoeae indicating the importance of phosphorylcholine in pathogenisis of N. meningitidis. In addition, the biosynthesis of phosphorylcholine for pilin post-translational modification still remains a mystery. Bacteria generally obtain choline from the environment. In this thesis, we demonstrated that pilin phosphorylcholine post-translational modification could be endogenously synthesized in N. meningitidis. In summary, this thesis describes the purification method of obtaining pure post-translationally modified pilin from N. meningitidis. The phosphorylcholine post-translation modification sites on pilin have been determined and showed the importance of these sites in antibody binding specificity.

Thesis (M.S.)--University of Montana, 2008. / Title from title screen. Description based on contents viewed May 8, 2008. Includes bibliographical references.

Thesis (Ph.D.)--University of Melbourne, Dept. of Gastroenterology and the Immunology Research Centre St. Vincents Hospital & Dept of Medicine, 2004. / Typescript (photocopy). Includes bibliographical references (leaves 309-343).

Jena, Universiẗat, Diss., 2008.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 232-263). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (p. 162-202). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 232-263). Also available online.

Thesis (M.S.)--Northern Michigan University, 2009. / Bibliography: leaves 114-116.

Protein based contrast agents (ProCAs) developed by the Yang lab exhibit unique capabilities in enabling magnetic resonance imaging (MRI) with significantly improved sensitivity and targeting capabilities by utilizing biomarkers which can target various carcinomas in animals. Further clinical in vivo human applications require modifications of these designed contrast agents to further improve organ and tissue biodistribution, biomarker and cell targeting capabilities, and reduction of immunogenicity. The aim of this thesis is to develop a novel protein modification on ProCA by glycosylation to improve liver distribution by targeting liver receptor, asialoglycoprotein receptor (ASGPR). Rat and humanized first generation and humanized third generation ProCA were expressed and purified using either glutathione s-transferase (GST) tagged or taggless methods. Rat ProCA1, rProCA1, was then used to optimize glycan modification with glycosylation achieved at the highest level using a 100:1 molar ratio and three lysine residues. Similar to non-modified rProCA1 and PEGylated rProCA1, metal binding affinity of gadolinium for glycan modified rProCA1, Glyco-rProCA1, was found to be 9.49 x 10-12 M, and relaxivity was found to be greater than clinically available contrast agents with 34.08 and 42.67 mM-1s-1 for r1 and r2 respectively. Glycosylation of rProCA1 has significantly increased human serum stability, and we have achieved significant liver MRI enhancement via tail vein injection due to high ASGPR expression in the liver altering biodistribution of glycan modified ProCA, and we have also imaged uptake in the secretory glands. These biodistribution changes were noted by immunohistochemistry (IHC) staining which was found to stain liver sinusoid with spaces in between. The distribution to the liver was further confirmed via inductively coupled plasma optical emission spectrometry (ICP-OES) which shows Glyco-rProCA1 has significant uptake of gadolinium in the liver tissue. This study represents the first achievement of in vivo liver imaging by glycosylation using a lactose targeting moiety covalently bonded to protein contrast agents for MRI showing promise for future more specific targeting or whole body imaging capabilities.