MOLECULAR BASIS FOR THE STABILITY OF XENOPUS P0-GLYCOPROTEIN DIMER

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。糖蛋白髓磷脂蛋白零（P0）是高等脊椎动物外周神经系统髓磷脂的主要蛋白，是免疫球蛋白超家族的一种蛋白。P0是在节间形成和维持髓鞘结构所必需的，可能是通过细胞外和细胞内区域的亲同性相互作用。P0基因的突变和缺失与不同程度的遗传性周围神经病变相关。P0含有一个单一的n -糖基化位点，具有异质糖基化模式。由于非糖基化的P0不表现出亲同源性粘附，因此P0的聚糖部分在细胞间通过亲同源性相互作用的粘附中起重要作用。对大鼠P0重组胞外结构域的晶体学研究和对牛髓鞘全长P0的小角溶液散射研究表明，P0以四聚体的形式存在于细胞膜中；哺乳动物髓磷脂的SDS-PAGE分析表明，P0的主要形式是单体。相比之下，非洲爪蟾与大鼠的P0序列同源性为65%，其P0的主要形式为二聚体。二聚体似乎完全不受用于减少二硫化物、脱烷基和破坏疏水或离子相互作用的处理的破坏。因此，我们认为爪蟾P0单体是共价键形成二聚体的，而聚糖的存在可能是形成过程中的重要介质之一。在本研究中，采用质谱法对二聚体和单聚体形式的爪蟾P0的酶促消化进行了测试。二聚体独特的糖基化和肽片段的差异的发现可以支持这一假设，允许共价键的阐明，并证明了外周髓鞘的非典型粘附。已观察到聚糖和多肽的差异，并正在确定相关结构。这些二聚体和单体的特征将有助于我们理解P0在髓鞘中的粘附作用的系统发育。

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.