Compaction of the myelin membrane into lamellae is essential for its function as an insulator in axons of the peripheral nervous system (PNS). P(O), the most abundant glycoprotein in the PNS, is believed to hold these lamellae together. The long term goals of this study are to clarify in molecular terms the role of P(O) (as well as other proteins) in myelination in the hope of contributing to our understanding of the mechanisms of demyelinating diseases such as Guillain-Barre syndrome (GBS) and in neuronal regeneration in the PNS. The extracellular domains of P(O) undergo hemophilic P(O)P(O) interactions in which the carbohydrate moieties play a major role. Heterophilic interactions of P(O) with neurons are believed to promote neurite outgrowth. The overall goals of this proposal are to determine what roles various regions of P(O) play in the synthesis and compaction of myelin. Our adhesion assay specifically developed to identify extracellular regions of P(O) participating in interactions will be used to pursue the following aims. First, to determine by mutation whether a 5-amino acid sequenced in immunoglobulin-like domain of P(O) is crucial to adhesion. This sequence is also found in a flu virus vaccine which elicited GBS, therefore the results may also bear on the mechanisms of demyelination in this disease. Second, to clarify the role of the intracellular domain of P(O) in the adhesive properties of its extracellular domain and in the sorting of P(O) to myelin. Sorting will be assessed by expressing shark P(O) in rat Schwann cells, allowing myelination to occur, and determining by means of species-specific antibodies whether truncated forms of P(O) reach myelin. Third, to elucidate the interactions of P(O) with myelin associated glycoprotein, possibly in the initial stages of myelination, by mixed adhesion assays and by determining the adhesive properties of cells co-expressing both proteins. Fourth, to map the regions of P(O) that may be involved in heterophilic interactions with neurons by comparing the ability of cells expressing unaltered and mutated P(O) to promote neurite outgrowth. Accomplishment of these aims should substantially advance the long term understanding of the mechanism of myelin organization.
