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

Orientador: Carlos Eduardo Steiner / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T08:47:23Z (GMT). No. of bitstreams: 1 Morini_SandraRegina_M.pdf: 2171982 bytes, checksum: 43545a3cec722564439f5853d48c9de2 (MD5) Previous issue date: 2007 / Resumo: A mucopolissacaridose tipo II (MPS II) é uma doença de depósito lisossômico rara causada pela deficiência da atividade da enzima iduronato-2-sulfatase. Essa enzima é responsável pelo catabolismo de dois Glicosaminoglicanos (GAGs) diferentes, o sulfato de dermatan e o sulfato de heparan. O acúmulo lisossômico desses GAGs causa disfunção de células, tecidos e órgãos, sendo que o envolvimento do Sistema Músculo-esquelético se deve ao acúmulo essencial em articulações e no tecido conectivo. A MPS II abrange muitos aspectos e dois fenótipos são reconhecidos, o leve e o grave, que representam os dois pontos extremos de um vasto espectro da gravidade clínica. O objetivo desse estudo foi caracterizar o Sistema Músculo-esquelético de uma amostra de sujeitos com MPS II, relacionando as conseqüências biomecânicas e funcionais. Foram encontradas alterações biomecânicas, principalmente em articulações dos membros superiores, que interferiram em algumas atividades funcionais dos sujeitos do estudo. Através da goniometria, observou-se uma defasagem nas amplitudes de movimentos de várias articulações, especialmente em ombros e cotovelos. Os sujeitos apresentaram perda de força muscular em diversos grupos musculares, principalmente flexores e extensores dos dedos das mãos e pronadores e supinadores do antebraço. Foram observadas algumas alterações posturais características e disostose múltipla em todos os indivíduos. Tais alterações contribuíram para os déficits encontrados nas atividades de vida diária. As escalas PEDI e FIM foram aplicadas e os resultados demonstraram uma relação de maiores déficits em tarefas ligadas com as funções de membros superiores, tais como: pentear os cabelos, vestir uma camiseta e amarrar os sapatos / Abstract: Mucopolysaccharidosis type II (MPS-II) is a rare lysosomal storage disorder caused by deficiency in the activity of the enzyme iduronate-2-sulphatase. This enzyme is responsible for the catabolism of two different glycosaminoglycans (GAGs), dermatan sulfate and heparan sulfate. Lysosomal accumulation of these glycosaminoglycan molecules results in cell, tissue and organ dysfunction. The musculoskeletal system involvement is due to essential accumulated glycosaminoglycans in joints and connective tissue. MPS-II has many clinical features and includes two recognized clinical entities, mild and severe, that represent two ends of a wide spectrum of clinical severity. The aim of this study was to describe the musculoskeletal system in a sample of subjects with MPS-II relating the biomechanics and functional consequences. Biomechanical alterations were found, essentially in superior limbs joints that interfered in some functional activities of the subjects in the study. Deficit in movement amplitude in many joints, especially shoulders and elbows, was observed through the goniometry. The subjects showed force loss in several muscular groups, especially the flexors and extensors of the fingers as well as pronators and supinators. Dysostosis multiplex was seen in all individuals and typical postural changes were noted. These alterations contributed with the deficits in daily activities. PEDI and FIM scales was applied and the results demonstrated larger deficits in tasks with the functions of superior members, such as: to comb the hair, to dress a shirt and to tie the shoes / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente