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. Myelin protein zero (P0) is a major protein for the formation and maintenance of myelin. It is an integral membrane glycoprotein containing a single N-glycosylation site. The mutations and deletions in the P0 gene correlate with hereditary peripheral neuropathies. The glycans of P0 play an important role in cell-to-cell adhesion. Studies on bovine, murine and human P0 suggest that P0 exists as tetramers in the membrane. However, the predominant form of P0 in Xenopus is a dimer. A mass spectrometry strategy including SDS-polyacrylamide-gel-electrophoresis (SDS-PAGE) was utilized to determine interactions underlying the P0 dimer and the post-translational-modification profiles of the monomers and oligomers. These results should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. Xenopus P0 oligomers were purified by SDS-PAGE. Bands of interest were excised and reloaded in the mini-slab gel to study the interaction(s) underlying the stability of the oligomers. In order to determine the glycoform profiles and verify the assignments of the protein present in each fraction, the monomers and oligomers were in-gel deglycosylated with PNGase F, and then in-gel digested with proteases. The peptides and the oligosaccharides were characterized using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS), nano ESI-TOF MS and custom built ESI-qQq-FTMS. The carbohydrates and peptides of interest were sequenced by MS/MS. In previous studies, a number of physical and chemical methods were used in attempts to disrupt the oligomers, including temperature, pH, detergents, and denaturants, but these approaches seemed only to increase the amount of oligomers relative to the monomer. In this study, when the dimer bands separated by SDS-PAGE were reloaded in a gel, it was observed that half of the dimer decomposed into the monomer. Furthermore, it was observed that almost all of the dimer dissociated into the monomer when a Mini-prep-cell"""""""" was used to purify the proteins from the mixture. Based on these observations, we propose that the Xenopus PNS P0 dimer is stabilized by non-covalent interaction(s) and there is equilibrium between the non-covalent dimer and the monomer. Using protease in-gel digestion, followed by MALDI-MS and ESI-MS/MS, the initially assigned dimer and the monomer were confirmed to be forms of P0, based on ca. 60% sequence coverage. In order to explore the factors affecting the folding and aggregation of Xenopus P0, the post-translational-modifications (PTM) of the P0 monomers and oligomers were investigated. Specific acylation and glycosylation patterns were observed and the structures of the modifying groups were determined. MALDI-MS showed that the Cys152 was acylated with stearic acid (C18:0). Xenopus P0 was found to contain a series of high mannose, hybrid and complex glycans. The major glycans were determined by ESI FTMS/MS (CAD) with high accuracy. In addition, Asn92 was confirmed as the single fully occupied N-glycosylation site by MALDI MS and ESI MS/MS. The PTMs in Xenopus P0 differ from those reported for other species such as cattle. The unique acylation and glycosylation may underlie the unusual folding and aggregation behaviour of P0 from Xenopus laevis. In addition, P0 dimer and monomer have slightly different glycoforms, and slightly different glycoform distributions;this may contribute to the differences in their aggregation. Our results will aid investigation of atypical adhesion in peripheral myelin and thus should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. Two manuscripts based on these results were recently published, one appeared in the previous reporting year and the second during the current year.
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