Compaction of the myelin membranes is essential for it to function as insulator of axons. As the Po glycoprotein is the most abundant protein of peripheral nervous system (PNS) myelin and is a member of the immunoglobulin (Ig) gene superfamily, this molecule is believed to hold these membrane lamellae together via interactions of both its extracellular and its cytoplasmic domains. The long term goals of this study are to identify in molecular terms the role of Po in the formation and in the maintenance of compact myelin, in the hope of contributing to our understanding of the mechanisms of demyelinating diseases such as Guillain-Barre Syndrome (GBS). While it has been shown that the extracellular domains of Po interact homophilically it is not known what precise regions of the molecule are responsible for these interactions. Even less is known about the role of Po's cytoplasmic domain in myelination. Thus, the overall goals of this proposal are to determine what roles various regions of Po play in the synthesis and compaction of myelin. The adhesion assay we have developed will allow us to pursue the following aims. First, to determine by point-mutation, whether a 5-amino acid sequence, SDNGT, in Po's Ig domain, is crucial to adhesion. As Po is believed to be the closest relative to the ancestral gene for the whole Ig superfamily, and because of the conservation of this 5-amino acid sequence in a sub-set of Ig domains, SDNGT may be an important motif to all Ig adhesion molecules in which it is found. Also this sequence is also present in an influenza virus which elicitated Guillain-Barre syndrome in hundreds of people when administered as a vaccine. The connection between the SDNGT sequence and GBS will be addressed by screening sera from these particular patients for SDNGT antibodies and by determining if SDNGT peptide antibodies recognize only the strain of virus used in this vaccine and not others. If this amino acid sequence is found to be involved in demyelination then it is imperative that future vaccines are screened for its presence. Second, to establish if the cytoplasmic domain of Po is necessary for its extracellular adhesion, the adhesion of truncated Po will be assessed. In addition, by detergent extraction and affinity chromatography, the putative interactions of Po's cytoplasmic domain with cytoskeletal components will be identified and characterized; an interaction that may be crucial in the initial stages of compaction. Third, to elucidate the interactions of Po with myelin associated glycoprotein (MAG) , an interaction that may also be essential in the early stages of myelination, mixed adhesion assays will be conducted with Po-expressing and MAG-expressing cells. In addition, as MAG and Po are expressed in the same and opposing membranes simultaneously, adhesion as says with cells expressing both Po and MAG will be carried out. Accomplishment of these aims should substantially advance the long term understanding of the mechanism of myelin compaction, organization and the role of adhesion molecules in demyelinating diseases.
