Lau Kin Chong. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (leaves 149-158). / Abstracts in English and Chinese. / Abstract --- p.i / 摘要 --- p.iii / Acknowledgements --- p.v / Table of Contents --- p.vi / List of Tables --- p.xii / List of Figures --- p.xiii / List of Appendices --- p.xv / Abbreviations --- p.xvi / Chapter 1 --- Introduction / Chapter 1.1 --- Mucopolysaccharidosis type II as a lysosomal storage disease --- p.1 / Chapter 1.1.1 --- Prevalence of MPS II --- p.2 / Chapter 1.1.2 --- Pathophysiology of MPS II --- p.4 / Chapter 1.1.3 --- Clinical features of MPS II --- p.4 / Chapter 1.1.4 --- Clinical management of MPS II --- p.6 / Chapter 1.1.4.1 --- Diagnostic methods for MPS II --- p.6 / Chapter 1.1.4.2 --- Treatments for MPS II --- p.7 / Chapter 1.2 --- Iduronate-2-sulfatase protein (IDS) --- p.9 / Chapter 1.2.1 --- Role in GAG degradation --- p.9 / Chapter 1.2.2 --- Post-translational modifications --- p.11 / Chapter 1.2.2.1 --- Formylglycine formation --- p.11 / Chapter 1.2.2.2 --- Glycosylation --- p.12 / Chapter 1.2.2.3 --- Proteolysis --- p.12 / Chapter 1.2.3 --- Iduronate-2-sulfatase gene (IDS) --- p.14 / Chapter 1.2.3.1 --- Properties of IDS mutations --- p.15 / Chapter 1.2.3.2 --- Methylation patterns are correlated with transitional mutations --- p.17 / Chapter 1.2.3.3 --- Genotype-phenotype correlations between IDS gene and MPS II --- p.19 / Chapter 1.3 --- In this study --- p.21 / Chapter 1.3.1 --- Mutational analysis --- p.21 / Chapter 1.3.2 --- In vitro expression of mutant IDS --- p.22 / Chapter 1.3.3 --- Maturation of IDS polypeptides --- p.23 / Chapter 2 --- Materials & Methods / Chapter 2.1 --- Mutation screening for MPS II patients --- p.24 / Chapter 2.1.1 --- Patients --- p.24 / Chapter 2.1.2 --- Genomic DNA extraction --- p.24 / Chapter 2.1.2.1 --- Materials --- p.24 / Chapter 2.1.2.2 --- Methods --- p.25 / Chapter 2.1.3 --- IDS exons amplification by Polymerase Chain Reaction (PCR) --- p.26 / Chapter 2.1.3.1 --- Materials --- p.26 / Chapter 2.1.3.1.1 --- PCR --- p.26 / Chapter 2.1.3.1.2 --- Agarose gel electrophoresis --- p.27 / Chapter 2.1.3.1.3 --- PCR fragments purification --- p.29 / Chapter 2.1.3.2 --- Methods --- p.29 / Chapter 2.1.3.2.1 --- Amplifying IDS exons by PCR --- p.29 / Chapter 2.1.3.2.2 --- Purifying PCR fragments --- p.30 / Chapter 2.1.4 --- DNA sequencing for detecting IDS mutations --- p.30 / Chapter 2.1.4.1 --- Materials --- p.30 / Chapter 2.1.4.2 --- Methods --- p.30 / Chapter 2.1.4.2.1 --- Sequencing reaction --- p.30 / Chapter 2.1.4.2.2 --- Purifying sequencing products --- p.31 / Chapter 2.1.4.2.3 --- Analyzing sequencing results --- p.31 / Chapter 2.1.5 --- Fragment restriction endonuclease analysis --- p.31 / Chapter 2.1.5.1 --- Materials --- p.31 / Chapter 2.1.5.2 --- Methods --- p.32 / Chapter 2.2 --- Isolation of IDS cDNA from peripheral blood --- p.34 / Chapter 2.2.1 --- Materials --- p.34 / Chapter 2.2.1.1 --- Total RNA extraction --- p.34 / Chapter 2.2.1.2 --- Reverse-transcriptase PCR (RT-PCR) --- p.35 / Chapter 2.2.1.3 --- PCR for amplifying IDS cDNA --- p.35 / Chapter 2.2.2 --- Methods --- p.37 / Chapter 2.2.2.1 --- Extracting total RNA by QIAamp RNeasy Mini Kit --- p.37 / Chapter 2.2.2.2 --- Converting IDS mRNA into cDNA by RT-PCR --- p.38 / Chapter 2.2.2.3 --- Isolating IDS cDNA by PCR --- p.39 / Chapter 2.2.2.4 --- Isolating firefly luciferase gene by PCR --- p.39 / Chapter 2.3 --- Introducing IDS cDNA into Gateway Cloning System --- p.40 / Chapter 2.3.1 --- Materials --- p.40 / Chapter 2.3.1.1 --- Directional cloning --- p.40 / Chapter 2.3.1.2 --- LB medium/ agar with antibiotics preparation --- p.42 / Chapter 2.3.1.3 --- Plasmids purification from transformed cells --- p.42 / Chapter 2.3.1.4 --- Validation of IDS inserted plasmids --- p.43 / Chapter 2.3.2 --- Methods --- p.43 / Chapter 2.3.2.1 --- TOPO cloning reaction --- p.43 / Chapter 2.3.2.2 --- Transformation --- p.44 / Chapter 2.3.2.3 --- Small-scale plasmids preparation by QIAprep Miniprep Kit --- p.44 / Chapter 2.3.2.4 --- Sequencing the plasmids --- p.45 / Chapter 2.3.2.5 --- QuikChange II XL site-directed mutagenesis --- p.46 / Chapter 2.3.2.5.1 --- Synthesizing mutant strand with desired mutations --- p.46 / Chapter 2.3.2.5.2 --- Digesting parental strand --- p.46 / Chapter 2.3.2.5.3 --- Transformation --- p.47 / Chapter 2.3.2.6 --- Swapping IDS gene from entry clone to expression vectors --- p.47 / Chapter 2.3.2.6.1 --- LR clonase reaction --- p.47 / Chapter 2.3.2.6.2 --- Transformation --- p.48 / Chapter 2.4 --- Introducing IDS cDNA into RTS pIVEX Wheat Germ vector --- p.49 / Chapter 2.4.1 --- Materials --- p.49 / Chapter 2.4.1.1 --- Restriction digestion --- p.49 / Chapter 2.4.1.2 --- Purification of digested products --- p.50 / Chapter 2.4.1.3 --- Ligation of the IDS insert into pIVE´Xؤ1.3_WG --- p.50 / Chapter 2.4.2 --- Methods --- p.50 / Chapter 2.4.2.1 --- Restriction digestion to create sticky ends --- p.50 / Chapter 2.4.2.2 --- Purifying the digested products --- p.51 / Chapter 2.4.2.3 --- Ligating the IDS insert into pIVE´Xؤ1.3_WG --- p.51 / Chapter 2.4.2.4 --- Transformation --- p.51 / Chapter 2.5 --- Transient expression study of IDS constructs --- p.53 / Chapter 2.5.1 --- Materials --- p.53 / Chapter 2.5.2 --- Methods --- p.55 / Chapter 2.5.2.1 --- Cell culturing --- p.55 / Chapter 2.5.2.2 --- Transfecting IDS constructs by lipofection procedures --- p.55 / Chapter 2.5.2.3 --- Harvesting COS-7 cells --- p.56 / Chapter 2.5.2.4 --- Total RNA extraction from transfected COS-7 cells --- p.57 / Chapter 2.5.2.5 --- RT-PCR showing IDS mRNA stability --- p.58 / Chapter 2.5.2.6 --- Endocytosis of expressed IDS products into COS-7 cells --- p.58 / Chapter 2.6 --- Synthesizing IDS by cell-free in vitro expression systems --- p.59 / Chapter 2.6.1 --- Materials --- p.59 / Chapter 2.6.1.1 --- DNA templates for expression --- p.59 / Chapter 2.6.1.2 --- Commercial cell-free expression kits --- p.60 / Chapter 2.6.1.3 --- Supplements --- p.61 / Chapter 2.6.2 --- Methods --- p.64 / Chapter 2.6.2.1 --- Cell-free expression by ExpressWay plus expression system --- p.64 / Chapter 2.6.2.2 --- Cell-free expression by RTS 100 E.coli HY Kit --- p.64 / Chapter 2.6.2.3 --- Cell-free expression by RTS 100 Wheat Germ CECF Kit --- p.64 / Chapter 2.6.2.4 --- Cell-free expression by TnT Coupled Wheat Germ Extract Systems --- p.65 / Chapter 2.6.2.5 --- Cell-free expression by TNT Coupled Reticulocyte Lysate Systems --- p.66 / Chapter 2.7 --- Investigations of IDS protein expression --- p.67 / Chapter 2.7.1 --- Materials --- p.67 / Chapter 2.7.1.1 --- Isolation of Histidine-tagged proteins --- p.67 / Chapter 2.7.1.2 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis/ SDS-PAGE --- p.67 / Chapter 2.7.1.3 --- Fluorometric activity assay for IDS --- p.69 / Chapter 2.7.1.4 --- Luciferase activity assay --- p.72 / Chapter 2.7.2 --- Methods --- p.72 / Chapter 2.7.2.1 --- Isolating His-tagged IDS from cell-free expression products --- p.72 / Chapter 2.7.2.2 --- Protein staining of expression products --- p.73 / Chapter 2.7.2.2.1 --- Preparation of protein separating gel --- p.73 / Chapter 2.7.2.2.2 --- Preparation of proteins for SDS-PAGE --- p.73 / Chapter 2.7.2.2.3 --- SDS-PAGE analysis --- p.73 / Chapter 2.7.2.3 --- Fluorometric enzyme assay for IDS proteins --- p.74 / Chapter 2.7.2.4 --- Luciferase activity assay --- p.75 / Chapter 3 --- Results / Chapter 3.1 --- Mutational analysis of MPS II and carrier detection --- p.76 / Chapter 3.2 --- Investigating IDS mutants by transient expression --- p.86 / Chapter 3.2.1 --- Fluorometric enzyme assay for measuring IDS activity --- p.86 / Chapter 3.2.2 --- Source of IDS gene for transient expression in COS-7 cells --- p.89 / Chapter 3.2.3 --- In vitro expression of IDS and its mutants in COS-7 cells --- p.92 / Chapter 3.2.3.1 --- Analysis of transient expression in terms of IDS activity --- p.92 / Chapter 3.2.3.2 --- Analysis of IDS mRNA stability in COS-7 cells --- p.95 / Chapter 3.2.3.3 --- Analysis of IDS protein stability in COS-7 cells --- p.95 / Chapter 3.3 --- Cell-free in vitro expression for investigating the IDS mutants --- p.98 / Chapter 3.3.1 --- The five cell-free systems involved --- p.98 / Chapter 3.3.2 --- Source of IDS gene for cell-free in vitro expression --- p.98 / Chapter 3.3.3 --- SDS-PAGE analysis of IDS protein stability in cell-free systems --- p.100 / Chapter 3.3.3.1 --- Wheat germ-based cell-free expression system (Roche) --- p.100 / Chapter 3.3.3.2 --- E.coli-based cell-free expression system (Invitrogen) --- p.102 / Chapter 3.3.3.3 --- E.coli-based cell-free expression system (Roche) --- p.102 / Chapter 3.3.4 --- In Vision His-tag In-gel stain for wild-type IDS and its mutant --- p.103 / Chapter 3.3.5 --- Analysis of IDS activity in cell-free expression systems --- p.107 / Chapter 3.3.6 --- Analysis of the cellular uptake of IDS --- p.110 / Chapter 4 --- Discussions / Chapter 4.1 --- Mutational analysis --- p.113 / Chapter 4.1.1 --- Heterogeneity of IDS mutations --- p.113 / Chapter 4.1.2 --- Role of molecular diagnosis for MPS II --- p.113 / Chapter 4.1.3 --- Two novel mutations and one reported mutation were identified --- p.115 / Chapter 4.1.3.1 --- A novel nonsense mutation: Ser369term --- p.115 / Chapter 4.1.3.2 --- A reported nonsense mutation: Gln389term --- p.115 / Chapter 4.1.3.3 --- A novel missense mutation: Leu339Pro --- p.116 / Chapter 4.2 --- Expression studies of the IDS mutants --- p.117 / Chapter 4.2.1 --- Analysis of transient expression in COS-7 cells --- p.117 / Chapter 4.2.1.1 --- Stability of mutant mRNA --- p.119 / Chapter 4.2.1.2 --- IDS catalytic activity --- p.119 / Chapter 4.2.2 --- Analysis of mutant stability by cell-free expression systems --- p.120 / Chapter 4.2.3 --- Structural analysis of amino acids alterations --- p.121 / Chapter 4.2.3.1 --- p.L339P causes conformational change --- p.122 / Chapter 4.2.3.2 --- p.L339R changes overall charge balance --- p.122 / Chapter 4.2.3.3 --- Mutations at Leu339 residue affect substrate binding --- p.123 / Chapter 4.3 --- Analysis of IDS maturation processing --- p.124 / Chapter 4.3.1 --- Active IDS modifications are not completed in lysosomes --- p.124 / Chapter 4.3.2 --- C-terminal proteolysis is essential for active IDS --- p.125 / Chapter 4.3.3 --- Functional role of glycosylation during IDS processing --- p.126 / Chapter 4.4 --- Analysis of cell-free expression systems --- p.128 / Chapter 4.4.1 --- Microbial systems using E.coli cell extracts: insoluble IDS precursors --- p.128 / Chapter 4.4.2 --- Plant system using wheat germ extracts: soluble IDS precursors --- p.129 / Chapter 4.4.3 --- Mammalian system using rabbit reticulocytes extracts: undetectable --- p.129 / Chapter 4.5 --- Role of transfecting IDS constructs --- p.131 / Chapter 4.6 --- Conclusion --- p.132 / Appendices --- p.133 / Electronic-database and computing system --- p.149 / Bibliography --- p.149
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_325633 |
| Date | January 2006 |
| Contributors | Lau, Kin Chong., Chinese University of Hong Kong Graduate School. Division of Chemical Pathology. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | print, xviii, 158 leaves : ill. (some col.) ; 30 cm. |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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