Seto Tai Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 122-138). / Abstracts in English and Chinese. / Thesis Committee --- p.ii / Statement --- p.iii / Acknowledgements --- p.iv / Abstract (in English) --- p.v / Abstract (in Chinese) --- p.vii / Table of Contents --- p.ix / List of Tables --- p.xvi / List of Figures --- p.xv / Chapter Chapter 1 --- General Introduction and Literature Review --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tobacco seed as bioreactor --- p.4 / Chapter 1.2.1 --- Advantages of using tobacco seed to produce bioactive human lysosomal enzyme --- p.4 / Chapter 1.2.2 --- Disadvantages and potential problems of using tobacco seed to produce bioactive human lysosomal enzyme --- p.5 / Chapter 1.2.2.1 --- Difference of asparagine-linked N-glycosylation between plant and human protein --- p.8 / Chapter 1.2.2.2 --- Immunogenicity of recombinant protein with plant-derived N-glycan to human --- p.10 / Chapter 1.2.2.3 --- "Strategy to ""humanize"" plant-derived recombinant human lysosomal enzyme" --- p.10 / Chapter 1.2.2.4 --- Lack of specific glycan structure一mannose-6-phosphate (M6P) tag addition --- p.11 / Chapter 1.2.2.5 --- Strategy for M6P tag addition on plant-derived human lysosomal enzyme --- p.12 / Chapter 1.3 --- The plant secretory pathway --- p.13 / Chapter 1.3.1 --- Plant vacuole in tobacco seed --- p.16 / Chapter 1.3.2 --- Soluble protein trafficking in plant cell --- p.17 / Chapter 1.3.3 --- Integral membrane protein trafficking in plant cell --- p.17 / Chapter 1.3.4 --- Components involved in integral membrane protein trafficking to PSV crystalloid --- p.19 / Chapter 1.3.4.1 --- BP-80 (80-kDa binding protein) --- p.19 / Chapter 1.3.4.2 --- α-TIP (α-tonoplast intrinsic protein) --- p.20 / Chapter 1.3.5 --- Using specific integral membrane protein trafficking system to target recombinant human lysosomal enzyme to tobacco seed PSV --- p.21 / Chapter 1.4 --- Homo sapiens α-L-iduronidase (hIDUA) --- p.21 / Chapter 1.4.1 --- Global situation of lysosomal storage disease一hIDUA deficiency --- p.21 / Chapter 1.4.2 --- Physiological role --- p.22 / Chapter 1.4.3 --- Molecular property --- p.24 / Chapter 1.4.3.1 --- Mutation and polymorphism --- p.24 / Chapter 1.4.4 --- Lysosomal secretory pathway --- p.24 / Chapter 1.4.5 --- Biochemical property --- p.25 / Chapter 1.4.6 --- Clinical application --- p.27 / Chapter 1.4.6.1 --- Enzyme replacement therapy (ERT) --- p.27 / Chapter 1.4.6.2 --- Clinical trial --- p.28 / Chapter 1.4.6.3 --- Economic value --- p.29 / Chapter 1.4.7 --- Expression system --- p.29 / Chapter 1.4.7.1 --- Production (overexpression) of rhIDUA in CHO cell system --- p.30 / Chapter 1.4.7.2 --- Production of rhIDUA in tobacco plant leaf --- p.30 / Chapter 1.5 --- Project objective and long-term significance --- p.30 / Chapter 1.5.1 --- Project objective --- p.30 / Chapter 1.5.2 --- Long-term significance --- p.31 / Chapter Chapter 2 --- Generation and Characterization of Anti-IDUA Antibodies --- p.32 / Chapter 2.1 --- Introduction --- p.33 / Chapter 2.2 --- Materials --- p.33 / Chapter 2.2.1 --- Chemical --- p.33 / Chapter 2.3 --- Methods --- p.35 / Chapter 2.3.1 --- Generation of polyclonal anti-IDUA antibody --- p.35 / Chapter 2.3.1.1 --- Design of synthetic peptide --- p.35 / Chapter 2.3.1.2 --- Conjugation of synthetic peptide to carrier protein --- p.39 / Chapter 2.3.1.3 --- Immunization of rabbit --- p.39 / Chapter 2.3.2 --- Characterization of polyclonal anti-IDUA antibody in rabbit serum --- p.40 / Chapter 2.3.2.1 --- Dot-blot analysis --- p.40 / Chapter 2.3.3 --- Purification of polyclonal anti-IDUA antibody --- p.42 / Chapter 2.3.3.1 --- Construction of anti-IDUA antibody affinity column --- p.42 / Chapter 2.3.3.2 --- Affinity-purification of anti-IDUA antibody --- p.42 / Chapter 2.3.4 --- Western blot detection of denatured rhIDUA --- p.42 / Chapter 2.4 --- Results --- p.43 / Chapter 2.4.1 --- Characterization of polyclonal anti-IDUA antibody --- p.43 / Chapter 2.5 --- Discussion --- p.51 / Chapter 2.6 --- Conclusion --- p.51 / Chapter Chapter 3 --- Generation and Characterization of Transgenic Tobacco Plants Expressing rhIDUA Fusions --- p.52 / Chapter 3.1 --- Introduction --- p.53 / Chapter 3.1.1 --- Signal peptide of hIDUA (hIDUA SP) --- p.54 / Chapter 3.1.2 --- Signal peptide of proaleurain (Pro. SP) --- p.54 / Chapter 3.1.3 --- Hypothesis to be tested in this study --- p.54 / Chapter 3.2 --- Materials --- p.55 / Chapter 3.2.1 --- Chemical --- p.55 / Chapter 3.2.2 --- Primers --- p.55 / Chapter 3.2.3 --- Bacterial strain --- p.58 / Chapter 3.2.4 --- The insert-Homo sapiens α-L-iduronidase (hIDUA) cDNA used in this study --- p.58 / Chapter 3.2.5 --- The vector-pLJ526 used in this study --- p.59 / Chapter 3.3 --- Methods --- p.61 / Chapter 3.3.1 --- Construction of chimeric gene construct --- p.61 / Chapter 3.3.1.1 --- Restriction endonuclease´ؤPfIMIl --- p.61 / Chapter 3.3.1.2 --- Recombinant DNA and molecular cloning techniques used in this study --- p.61 / Chapter 3.3.1.3 --- Cloning of pSPIDUA-FLAG --- p.62 / Chapter 3.3.1.4 --- Cloning of pSPIDUA-control --- p.62 / Chapter 3.3.1.5 --- Cloning of a universal construct (pUniversal) --- p.62 / Chapter 3.3.1.6 --- Cloning of pSP-IDUA-T7 --- p.66 / Chapter 3.3.1.7 --- Cloning of pSP-IDUA-control --- p.66 / Chapter 3.3.1.8 --- Cloning of chimeric gene construct into Agrobacterium binary vector --- p.66 / Chapter 3.3.2 --- Expression of chimeric gene construct in tobacco plant --- p.73 / Chapter 3.3.2.1 --- Tobacco plant --- p.73 / Chapter 3.3.2.2 --- Electroporation of Agrobacterium --- p.73 / Chapter 3.3.2.3 --- Agrobacterium-mediated transformation of tobacco plant --- p.74 / Chapter 3.3.2.4 --- Selection and regeneration of tobacco transformant --- p.75 / Chapter 3.3.3 --- Characterization of transgenic tobacco plant expressing rhIDUA fusion --- p.75 / Chapter 3.3.3.1 --- Genomic DNA polymerase chain reaction (PCR) --- p.75 / Chapter 3.3.3.2 --- Southern blot analysis --- p.76 / Chapter 3.3.3.3 --- Total RNA reverse transcription-PCR (RT-PCR) --- p.77 / Chapter 3.3.3.4 --- Northern blot analysis of tobacco leaf --- p.78 / Chapter 3.3.3.5 --- Western blot analysis --- p.79 / Chapter 3.3.4 --- Purification of plant-derived rhIDUA fusion --- p.81 / Chapter 3.3.4.1 --- Construction of affinity column with anti-IDUA antibody --- p.81 / Chapter 3.3.4.2 --- Affinity-purification of rhIDUA fusion from tobacco mature seed --- p.81 / Chapter 3.3.5 --- Confocal immunoflorescence study --- p.82 / Chapter 3.3.5.1 --- Preparation of paraffin section --- p.82 / Chapter 3.3.5.2 --- Single immunocytochemical labeling --- p.82 / Chapter 3.3.5.3 --- Double labeling with one monoclonal and one polyclonal antibodies --- p.83 / Chapter 3.3.5.4 --- Double labeling with two polyclonal antibodies --- p.83 / Chapter 3.3.5.5 --- Image collection --- p.84 / Chapter 3.4 --- Results --- p.85 / Chapter 3.4.1 --- Chimeric gene construction and confirmation --- p.85 / Chapter 3.4.2 --- Selection and regeneration of tobacco transformant with kanamycin- resistance --- p.86 / Chapter 3.4.3 --- Genomic DNA PCR screening of tobacco transformant --- p.88 / Chapter 3.4.4 --- Southern blot analysis of tobacco transformant --- p.91 / Chapter 3.4.5 --- Total RNA RT-PCR screening of tobacco transformant --- p.93 / Chapter 3.4.6 --- Northern blot analysis of tobacco transformant --- p.93 / Chapter 3.4.7 --- Western blot analysis --- p.96 / Chapter 3.4.7.1 --- Western blot analysis of pSP-IDUA-T7-121 transformant leaf --- p.96 / Chapter 3.4.7.2 --- Western blot analysis of pSP-IDUA-T7-121 transformant mature seed --- p.98 / Chapter 3.4.8 --- Affinity-purification of rhIDUA fusion --- p.98 / Chapter 3.4.9 --- Expression level of rhIDUA fusion --- p.102 / Chapter 3.4.10 --- Subcellular localization of rhIDUA fusion --- p.102 / Chapter 3.5 --- Discussion --- p.111 / Chapter Chapter 4 --- Summary and Future Perspectives --- p.117 / References --- p.122 / Appendix 1 --- p.139 / Appendix II (List of Abbreviations) --- p.141

In response to extra-cellular cues, cells activate signal transduction pathways to elicit a biological response. Cell surface growth factor receptors such as the Met receptor tyrosine kinase (RTK) activate signals that result in cellular proliferation, survival, migration, as well as epithelial morphogenesis. In order for signal transduction to occur, docking proteins are recruited to the activated RTK, become phosphorylated on tyrosine residues, which then serve as docking sites for the recruitment of other signaling proteins. Docking proteins function to diversify the signal by assembling multi-protein complexes. The Gab1 docking protein is the most tyrosine phosphorylated protein upon Met receptor activation and is required for Met mediated signaling and biology. / Gab1 belongs to a family of docking proteins including the highly related Gab2 protein. Gab1 promotes signals for epithelial morphogenesis downstream of the Met receptor, however Gab2 is unable to do so. Insertion of the Gab1 Met binding Motif (MBM) which confers direct binding to the Met receptor, as well as membrane targeting of Gab2 is sufficient to switch the capacity of Gab2 to activate the morphogenic program, cell scatter and lamellipodia formation. This is achieved via activation of sustained signaling pathways, and redistribution of the Gab protein, and associated molecules to sites of lamellipodia formation at the peripheral edge of the cell. / Activation of the Met RTK, promotes the formation of dorsal ruffles on the apical surface of epithelial cells. The Met receptor, Gab1 and Gab1 associated molecules Shp2, Crk, and p8S subunit of PI3K, are localized to these structures, however only the Gab1erk complex is required to drive dorsal ruffle formation. Gab1 is required for Met induced dorsal ruffles as well as downstream the PDGF and EGF RTKs. These are a signaling micro-environment which results in enhanced receptor degradation. Inhibition or enhancement of Met mediated dorsal ruffle formation correlates with receptor stability. / Dorsal ruffle formation downstream of Met requires the enzymatic activity of PI3K and PLCgamma, both enzymes that metabolize PIP2, and form complexes with Gab1 downstream of Met. PLCgamma and the PIP3 lipid product of PI3K are co-localized with Gab1 in dorsal ruffles. Gab1 engages with elements of the cytoskeleton, actin and cortactin, providing a link between growth factor signaling and remodeling of the actin cytoskeleton. Gab1 is localized to membrane protrusions of the basal surface in organoid cultures and is required for actin protrusions of the basal surface of breast cancer cells.

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