MOLECULAR BASIS FOR THE STABILITY OF XENOPUS P0-GLYCOPROTEIN DIMER

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。糖蛋白髓鞘蛋白零(P0)是高等脊椎动物周围神经系统髓鞘的主要蛋白质，属于免疫球蛋白超家族。P0是形成和维持节间髓鞘结构所必需的，可能是通过胞外区和胞内区的亲水性相互作用实现的。P0基因的突变和缺失与不同严重程度的遗传性周围神经病相关。P0含有单一的N-糖基化位点，并具有异质性糖基化模式。由于未糖化的P0不表现出亲水性，因此P0的糖链在细胞间通过亲水性相互作用的黏附中起着重要的作用。对重组大鼠P0胞外区的结晶学研究和对从牛髓鞘分离的全长P0的小角度溶液散射研究表明，P0在膜上以四聚体形式存在；对哺乳动物髓鞘的SDS-PAGE分析表明，P0的主要形式是单体。相比之下，在与大鼠P0有65%序列同源性的非洲爪哇中，P0的主要形式是二聚体。该二聚体似乎完全抵抗用于减少二硫键、脱酰化和打破疏水或离子相互作用的处理的破坏。因此，有人认为非洲爪哇P0单体是共价键合形成二聚体的，而多糖的存在可能是形成过程中的重要介体之一。在这项研究中，对非洲爪哇P0的二聚体和单体形式的酶消化进行了质谱分析来验证这一假说。二聚体特有的糖基化和多肽片段的差异的发现可以支持这一假说，允许阐明共价键，并表明外周髓鞘中存在非典型的黏附。已经观察到了多糖和多肽的差异，相关结构正在确定中。这些对二聚体和单体的表征将有助于我们理解P0‘S在髓鞘中的黏附作用的系统发育。

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.