This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Glycoprotein myelin protein zero (P0), a protein of the immunoglobulin superfamily, is the major protein of peripheral nervous system myelin in higher vertebrates. P0 is required for the formation and maintenance of myelin structure in the internode, likely through homophilic interactions at both the extracellular and intracellular domains. Mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies of varying severity. P0 contains a single N-glycosylation site and has a heterogeneous glycosylation pattern. The glycan moiety of P0 plays an important role in cell-to-cell adhesion via homophilic interactions, since non-glycosylated P0 does not show homophilic adhesion. Crystallographic studies on the recombinant extracellular domain of rat P0 and small-angle solution scattering on full-length P0 isolated from bovine myelin suggest that P0 exists as tetramers in the membrane; SDS-PAGE analysis of mammalian myelin shows that the predominant form of P0 is monomeric. By contrast, in Xenopus, which has 65% sequence identity with rat P0, the predominant form of P0 is a dimer. The dimer appears to be totally resistant to disruption by treatments used to reduce disulfides, to deacylate, and to break hydrophobic or ionic interactions. Therefore, it was proposed that Xenopus P0 monomers are covalently bonded to form the dimer, and the presence of the glycans may be one of the important mediators during the formation. In this study, mass spectrometry was undertaken to test this hypothesis on enzymatic digests of the dimeric versus the monomeric forms of Xenopus P0. The finding of differences in glycosylation and peptide fragments unique to the dimer could support the hypothesis, allow elucidation of the covalent bond, and demonstrate an atypical adhesion in peripheral myelin. Differences in glycans and peptides have been observed and the relevant structures are being determined. These characterization of the dimer and monomer will contribute to our understanding of the phylogenetic development of P0's adhesive role in myelin.
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