MOLECULAR BASIS FOR THE STABILITY OF XENOPUS P0-GLYCOPROTEIN DIMER

爪蟾 P0-糖蛋白二聚体稳定性的分子基础

• 批准号: 7369306
• 负责人: DANIEL A KIRSCHNER
• 金额: $ 2.85万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7369306
• 关键词: MOLECULAR; BASIS; STABILITY; XENOPUS; P0

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.

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子弹和调查员（PI）可能已经从其他NIH来源获得了主要资金，因此可以在其他清晰的条目中代表。列出的机构适用于该中心，这不一定是调查员的机构。糖蛋白髓磷脂蛋白零（P0）是免疫球蛋白超家族的蛋白质，是较高脊椎动物中周围神经系统髓鞘蛋白的主要蛋白质。 P0是通过细胞外和细胞内结构域的同粒细胞相互作用而形成和维持髓磷脂结构所必需的。 P0基因中的突变和缺失与遗传性周围神经病的严重程度不同。 P0包含单个N-糖基化位点，并具有异质性糖基化模式。 P0的聚糖部分通过均电相互作用在细胞到细胞粘附中起重要作用，因为非糖基化的P0没有显示均匀的粘附性。 对从牛髓磷脂分离的全长P0上的大鼠P0和小角度溶液的重组细胞外域的晶体学研究表明，P0在膜上作为四聚体存在；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，在与大鼠P0具有65％序列同一性的爪蟾中，P0的主要形式是二聚体。该二聚体似乎完全抵抗了用于减少二硫化物，脱酰酯和破坏疏水或离子相互作用的处理方法。因此，有人提出，将爪蟾P0单体共价键形成二聚体，并且聚糖的存在可能是在形成过程中的重要介体之一。 在这项研究中，进行了质谱法，以检验有关二聚体与爪蟾P0的单体形式的酶促消化的这一假设。发现二聚体所特有的糖基化和肽片段差异的发现可以支持该假设，允许阐明共价键，并在外围髓磷脂中表现出非典型的粘附。已经观察到了聚糖和肽的差异，并确定了相关的结构。这些二聚体和单体的表征将有助于我们理解P0在髓磷脂中粘合剂作用的系统发育。