by Ngai Yuk Keung. / Thesis (M.Sc.)--Chinese University of Hong Kong, 1996. / Includes bibliographical references (leaves 69-75). / SUMMARY --- p.1 / Chapter 1. --- INTRODUCTION --- p.3 / Chapter 2. --- LITERATURE REVIEW --- p.4 / Chapter 2.1 --- Properties of GAGs --- p.4 / Chapter 2.1.1 --- FUNCTION OF GAGs --- p.6 / Chapter 2.1.2 --- METABOLISM OF GAGs --- p.6 / Chapter 2.2 --- Mucopolysaccharidoses --- p.12 / Chapter 2.2.1 --- CLINICAL PRESENTATION AND GENETICS OF MPS --- p.13 / Chapter 2.2.2 --- Treatment --- p.13 / Chapter 2.3 --- Laboratory tests for the diagnosis of MPS --- p.17 / Chapter 2.3.1 --- SCREENING --- p.17 / Chapter 2.4 --- Confirmation method --- p.20 / Chapter 2.4.1 --- Enzyme assay --- p.20 / Chapter 2.4.2 --- Electrophoresis --- p.21 / Chapter 2.4.3 --- Chromatography --- p.22 / Chapter 2.5 --- Conclusion of Literature Review --- p.23 / Chapter 2.6 --- Choice of method --- p.24 / Chapter 2.7 --- The present study --- p.25 / Chapter 3. --- MATERIALS --- p.26 / Chapter 3.1 --- Material --- p.26 / Chapter 3.2 --- Stock solution --- p.26 / Chapter 3.3 --- Stock standards --- p.26 / Chapter 3.4 --- Working standards --- p.27 / Chapter 3.5 --- Working DMB solution --- p.27 / Chapter 3.6 --- Instruments --- p.27 / Chapter 3.7 --- Statistics --- p.27 / Chapter 3.8 --- Urine samples --- p.28 / Chapter 4. --- METHODS --- p.29 / Chapter 4.1 --- Studies on spectral characteristics --- p.29 / Chapter 4.1.1 --- Spectrum of DMB --- p.29 / Chapter 4.1.2 --- Spectra of DMB-GAG products --- p.29 / Chapter 4.2 --- Studies on stability --- p.29 / Chapter 4.2.1 --- Stability of DMB dye in assay condition --- p.29 / Chapter 4.2.2 --- Stability of DMB-GAG products in assay condition --- p.30 / Chapter 4.3 --- Linearity studies --- p.30 / Chapter 4.3.1 --- Linearity of DMB dye --- p.30 / Chapter 4.3.2 --- Linearity of GA G assay --- p.32 / Chapter 4.4 --- Detection limit --- p.32 / Chapter 4.5 --- Precision study --- p.32 / Chapter 4.5.1 --- Within-run precision --- p.32 / Chapter 4.5.2 --- Between-day precision --- p.32 / Chapter 4.6 --- Interference study --- p.33 / Chapter 4.6.1 --- Haemoglobin --- p.33 / Chapter 4.6.2 --- Protein --- p.33 / Chapter 4.7 --- Recovery study --- p.34 / Chapter 4.8 --- Correlation study --- p.34 / Chapter 4.9 --- Automated measurement of urinary GAG --- p.35 / Chapter 4.10 --- Reference range --- p.35 / Chapter 4.11 --- GAG in mentally retarded patients --- p.36 / Chapter 5. --- RESULTS --- p.37 / Chapter 5.1 --- Studies on spectral Characteristics --- p.37 / Chapter 5.1.1 --- DMB Spectrum --- p.37 / Chapter 5.1.2 --- Spectra of DMB-GAG products --- p.37 / Chapter 5.2 --- Studies on stability --- p.43 / Chapter 5.2.1 --- Stability of DMB dye in assay condition --- p.43 / Chapter 5.2.2 --- Stability ofDMB-GAG reaction mixture in assay condition --- p.43 / Chapter 5.3 --- Linearity studies --- p.43 / Chapter 5.3.1 --- Linearity of DMB dye --- p.43 / Chapter 5.3.2 --- Linearity of GAG assay --- p.47 / Chapter 5.3.3 --- Detection limit --- p.50 / Chapter 5.4 --- Precision study --- p.50 / Chapter 5.5 --- Interference study --- p.52 / Chapter 5.5.1 --- Haemoglobin --- p.52 / Chapter 5.5.2 --- Protein --- p.52 / Chapter 5.6 --- Recovery study --- p.52 / Chapter 5.7 --- Correlation study --- p.52 / Chapter 5.8 --- Reference range --- p.55 / Chapter 5.9 --- GAG in mentally retarded patients --- p.55 / Chapter 6. --- DISCUSSION --- p.60 / Chapter 6.1 --- Analytical Performance --- p.62 / Chapter 6.2 --- Clinical performance --- p.64 / Chapter 6.2.1 --- Reference population --- p.64 / Chapter 6.2.2 --- Mentally retarded patients --- p.65 / Chapter 6.2.3 --- Suggestion for future studies --- p.66 / Chapter 7. --- CONCLUSION --- p.67 / Chapter 8. --- APPENDIX --- p.68 / Chapter 9. --- REFERENCE --- p.69

No description available.

published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Glycosaminoglycans (GAGs) are linear polysaccharides expressed ubiquitously on animal cell surfaces and within extracellular matrices. GAGs usually occur as parts of proteoglycans and often accomplish their biological functions through their interactions with proteins. GAG oligosaccharides for this work were produced via enzymatic digest of heparin, followed by gel filtration and ion exchange chromatography. Two tetrasaccharide species obtained from this digest were characterised using 1H and 13C NMR spectroscopy. Complement factor H (fH) is a regulatory protein of the alternative pathway of the complement system, a major component of human innate immunity. Acting as a cofactor to factor I, fH inhibits C3b-initiated complement activation on host cells, protecting cells from auto immune attack. This study focused on the interaction of factor H with GAGs, which are thought to be among the markers allowing factor H to distinguish between self and non self surfaces. Binding studies of two heparin-binding sites in fH are presented. These include the C-terminal modules 19 and 20 (fH~19-20) and fH~7-8. FH~7, fH~7-8 and fH~19-20 were produced recombinantly in various isotope forms. The techniques used to study the protein-GAG interactions in this work encompass NMR spectroscopy, mass spectrometry, gel mobility shift assays (GMSA) and chemical cross linking. Several genetic studies suggest that a common polymorphism in the heparin-binding module fH~7, Y402H, plays a role in the development of age-related macular degeneration (AMD). The work presented here included preparation and backbone resonance assignment of a 13C, 15N- labelled sample of fH~ 7-8 via triple resonance NMR experiments. Further NMR experiments were employed to investigate the role of the lysine and arginine sidechains of fH~7 in GAG binding. These studies were combined with the preparation and characterisation of a covalently cross linked GAG-protein complex using NMR and mass spectrometry. A range of fH~19-20 mutations that are linked to a severe kidney disease, atypical haemolytic uraemic syndrome (aHUS), were characterised using GMSA. No correlation between the disease and the heparin binding properties of the aHUS mutants was observed. The mutant proteins were also characterised with respect to their ability to compete with full-length fH in a physiological complement assay. Simultaneous binding of WT fH~19-20 to GAGs and C3d, the relevant fragment of C3b, was assessed using NMR. NMR experiments were also conducted with NK1, which comprises the two N-terminal heparin-binding modules of hepatocyte growth factor/scatter factor (HGF/SF), and heparin as well as dermatan sulfate-derived GAGs. Relaxation studies on a human defensin, HBD2, were performed to assess the role of GAGs in HBD2 self-association.

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.

The structural complexity of glycosaminoglycans (GAGs) such as heparin and heparan sulfate (HS) and their numerous biological roles, brings forth the need to develop new methods, capable of studying GAGs and their interactions with peptides and proteins under native settings. This thesis explores the development of chemical tools to study heparin/HS binding interactions under physiologically relevant conditions using fluorescence. In chapter 2, we designed peptide-based quinolinium probes to study the structural requirements of cationic peptides required for high affinity peptide-heparin interactions. These fluorescent probes enabled the study of peptide-heparin interactions at nM concentrations allowing the calculation of peptide-heparin binding constants. It was observed that peptides with positive charge displayed on one face of an α-helix in a continuous arrangement bound to heparin with the highest affinity and that heparin likely prefers to bind to these peptides while remaining in an extended conformation. In chapter 3, we set out to study an important biological role of HS which involves the binding and sequestering of proteins at the cell surface, facilitating endocytosis. HS has been implicated in the mechanism of cell penetrating peptide (CPP) cell uptake, with different CPPs showing different degrees of HS dependence on uptake as well as different mechanisms of entry. The role of HS in the mechanism of CPP uptake was investigated in chapter 3 using fluorescent peptide-based probes incorporating fluorophore/quencher pairs. These were used to identify and characterize the ability of heparin/HS to bind and cluster with CPPs to form colloidally stable aggregates. It was shown that the CPP Antp formed much more stable clusters with heparin than the TAT peptide despite both peptides having similar binding affinity for a single heparin chain. These findings were used to explain the cell surface HS dependence of Antp on cell uptake via endocytosis in contrast to the low dependance of TAT on HS and its uptake via translocation. A general model relating the ability of a CPP to cluster surface HS to its preferred mechanism of cell entry was proposed. In chapter 4, a strategy to selectively, and site specifically acylate carbohydrate binding proteins was developed using thioester-based affinity conjugates. It was possible to label maltose binding protein, a periplasmic protein, with high yield and selectivity at a single lysine residue proximal to the maltose binding site. Selective protein labeling could be carried out in bacterial cell extracts and in live bacterial cells. This strategy can potentially be applied to develop protein-based carbohydrate biosensors as well as profile carbohydrate binding proteins in biological samples.

Thesis (Ph. D.)--University of Hong Kong, 1987.

Interactions between serum lipoproteins and human cells in the secretion of glycosaminoglycans (GAG) were investigated in cultures of arterial smooth muscle cells (SMC) and fibroblasts. Lipoprotein free serum (LFS) supported cell proliferation but caused a dose-dependent decrease in GAG secretion and cell cholesterol. Addition of low density lipoproteins (LDL) to 10% LFS in the medium caused increases in GAG secretion and cell cholesterol but a net decrease in cell population. LDL effects were positively correlated with the rate of cell proliferation independent of serum concentration. High density lipoproteins (HDL) decreased GAG secretion in slowly but not in actively proliferating cultures; however in the latter, it reduced all LDL-induced changes. Cells from non insulin-dependent diabetics had an increased GAG response to LDL. Low concentrations of platelet factors enhanced GAG secretion. The addition of a GAG mixture inhibited all LDL effects. On the basis of these observations, a hypothetical model is presented indicating that GAGs play a significant role in the development of atherosclerosis.

Thesis (Ph. D.)--Virginia Commonwealth University, 2008. / Prepared for: Dept. of Medicinal Chemistry Bibliography: leaves 149-183 . Also available online via the Internet.