Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 131-148).

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Myelin ist eine Membran von entscheidender Bedeutung. In Nervensystem von Wirbeltieren isoliert es die Axone und fördert so die schnelle Weiterleitung von Nervenimpulsen. Um als Isolator zu wirken, muss sich Myelin zu einer stabilen, Lipid-reichen Struktur zusammenlagern. Während die biochemische Zusammensetzung von Myelin gut beschrieben ist, sind Mechanismen, welche diese einzigartige Zusammensetzung regulieren, schlecht verstanden. In dieser Arbeit zeigen wir, dass Oligodendrozyten eine Art molekulares Sieb verwenden, um Membranproteine aus kompakten Myelin-Domain herrauszufiltern. Das Myelin Basische Protein (MBP) bildet eine Größen-abhängige Barriere und kontrolliert den Zugang von Proteinen in das kompakten Myelin, basierend auf der Größe ihrer zytoplasmatischen Domänen. Nur Proteine mit einer cytosolischen Domäne von weniger als 30 Aminosäuren können diese die Permeabilitätsbarriere überwinden. Im Hinblick auf den zugrundeliegenden Mechanismus zeigen wir, dass MBP nach Bindung an die innere Membran der Myelin-Doppelschicht mit sich selbst assoziiert und eine Phasentrennung auftritt. Diese Selbstorganisation erfordert hydrophobe Wechselwirkungen zwischen den Phenylalanin-Reste von MBP. Ein Ersetzen der Phenylalanin-Reste durch Serine hemmt die Selbst-Assoziation der MBP-Moleküle, ohne jedoch die Membranbindung zu beeinflussen. Wir zeigen weiter, dass die Selbst-Assoziation der MBP-Moleküle während der Entwicklung für die Verdrängung von sperrigen Proteinen aus den kompakten Myelin-Membranen benötigt wird. Dieses System könnte sich in Oligodendrozyten entwickelt haben, um eine anisotrope Membran-Organisation zu bilden, welche den Aufbau der der isolierenden, lipidreichen Membranen ermöglicht.

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The myelin basic protein (MBP) family arises from different transcription start sites of the Golli (gene of oligodendrocyte lineage) gene, with further variety generated by differential splicing. The “classic” MBP isoforms are peripheral membrane proteins that facilitate compaction of the mature myelin sheath, but also have multiple protein interactions. As an intrinsically disordered protein, MBP has proven to have complex structural and functional relationships with proteins in vitro including actin, tubulin, Ca2+-calmodulin, and multiple protein kinases. The investigations reported in this thesis were to further examine the multifunctionality, and protein:protein interactions of MBP with cytoskeletal and SRC homology 3 domain (SH3) proteins in cells using an oligodendrocyte (OLG) model system to better understand MBP’s contributions to membrane structure, formation, and elaboration in the developing OLG. A new function of MBP has been described showing that classic MBPs can modulate voltage operated calcium channels (VOCCs) by direct or indirect protein-protein interactions at the OLG cytoplasmic leaflet. These interactions contribute to global calcium homeostasis, and may play a complex developmental and spatiotemporal role in the regulation of oligodendrocyte precursor cell (OPC) migration and OLG differentiation. The importance of MBPs SH3 ligand binding domain within its central amino acid region was investigated with the protein-tyrosine kinase Fyn. Co-expression of MBP with a constitutively-active form of Fyn in OLGs resulted in membrane process elaboration, a phenomenon that was abolished by amino acid substitutions within MBP’s SH3-ligand domain. These results suggest that MBP’s SH3-ligand domain plays a key role, and may be required for proper membrane elaboration of developing OLGs. Lastly, interactions of MBP with the OLG cytoskeleton were investigated in OLGs transfected with fluorescently-tagged MBP, actin, tubulin, and zonula occludens 1 (ZO-1). MBP redistributes to distinct ‘membrane-ruffled’ regions of the plasma membrane where it had increased co-localization with actin and tubulin, and with the SH3-domain-containing proteins cortactin and ZO-1, when stimulated with PMA, a potent activator of the protein kinase C pathway. The results presented here advance our understanding regarding protein:protein interactions of MBP, and its multifunctionality in OLGs with regards to membrane formation and elaboration. / This work was supported by the Canadian Institutes of Health Research (MOP #86483, J.M.B. and G.H.), and Discovery Grants from the Natural Sciences and Engineering Research Council of Canada (NSERC, G.H., RG121541). G.S.T.S. was a recipient of a Doctoral Studentship from the Multiple Sclerosis Society of Canada

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Tetrabromobisphenol-A (TBBPA) is the most widely used flame retardant. Flame retardants are sprayed on furniture, mattress beds, children’s pajamas, car seats, upholstery, carpets, and rugs in the United States. Chemical immune reactivity may play a role in the epidemic of autoimmune disease. The goal of this research is to investigate whether any correlation exists between immunological reactivity to TBBPA, a key chemical used in most flame retardants, and neurological autoimmune target sites that are associated with neurological autoimmune diseases with a diverse and specific list of antibodies that include myelin basic protein, myelin-associated glycoprotein, alpha-synuclein, aquaporin receptors, and S100B antibodies with human serum samples. The outcomes of this research can be used to support the development of safety regulations and for identifying potential health concerns for current mandatory flame-retardant legislation. Additionally, this research may support the decisions made in respect of those suffering from neurological autoimmune diseases, as to whether removing flame retardant chemicals is a factor for consideration.

Downstream purification and analysis of a model biopharmaceutical protein (recombinant human myelin basic protein) is described. The recombinant protein was expressed in the milk of transgenic cows and was found exclusively associated with the casein micellar phase. Binding of milk calcium to the active sites of a cation exchanger resin was used beneficially in this study in order to gently disrupt the casein micelles and liberate the recombinant protein. This approach was found superior to the conventional micelle disruption procedures with respect to product recovery, resin fouling due to milk components and column hydrodynamic properties. Further purification was carried out using Ni2+ affinity chromatography and resulted in purity more than 90% and a total recovery of 78%. A capillary electrophoresis total protein assay employing large volume sample stacking and a microsphere-based, sandwich-type immunoassay were developed and validated. Both methods were successfully integrated with the downstream purification protocol in order to evaluate various quality attributes of the recombinant protein. A onestep capillary isoelectric focusing protocol was developed in order to monitor the recombinant protein in milk samples. The results showed extra protein bands in the transgenic milk that had isoelectric points significantly lower than the theoretically calculated one which indicated that the protein had been modified during expression. The association between the recombinant protein and bovine milk caseins was explored at the molecular level using the surface plasmon resonance technique. Results showed a calciummediated interaction between the recombinant protein and the phosphorylated caseins. This selective interaction was not noted between the human myelin basic protein and milk caseins which indicated mammary gland-related posttranslational modifications, most likely phosphorylation. The co-expression of the recombinant protein and caseins in the mammary gland, along with the ability of the recombinant protein to form calcium bridges with caseins explained its association with the casein micellar phase in the transgenic milk. Despite this and owing to the low expression levels of the recombinant protein in milk, light scattering investigations using diffusing wave spectroscopy showed no significant differences between the transgenic and the non-transgenic milk samples with respect to the average micelle size and the micelle surface charges.