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 资助的中心拨款提供的资源的众多研究子项目之一。子项目和研究者 (PI) 可能已从另一个 NIH 来源获得主要资金，因此可以在其他 CRISP 条目中出现。列出的机构是中心的机构，不一定是研究者的机构。糖蛋白髓磷脂零蛋白（P0）是免疫球蛋白超家族的一种蛋白质，是高等脊椎动物周围神经系统髓磷脂的主要蛋白质。 P0 是节间髓磷脂结构形成和维持所必需的，可能是通过细胞外和细胞内域的同亲相互作用。 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 包含单个 N-糖基化位点并具有异质糖基化模式。 P0 的聚糖部分通过亲同性相互作用在细胞间粘附中发挥重要作用，因为非糖基化的 P0 不表现出亲同性粘附。 对大鼠 P0 重组胞外结构域的晶体学研究和从牛髓磷脂分离的全长 P0 上的小角溶液散射研究表明，P0 在膜中以四聚体形式存在；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，爪蟾与大鼠 P0 具有 65% 的序列同一性，P0 的主要形式是二聚体。该二聚体似乎完全能够抵抗用于减少二硫化物、脱酰基和破坏疏水性或离子相互作用的处理的破坏。因此，有人提出爪蟾P0单体通过共价键结合形成二聚体，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，采用质谱法来测试爪蟾 P0 的二聚体与单体形式的酶消化的这一假设。二聚体特有的糖基化和肽片段差异的发现可以支持这一假设，阐明共价键，并证明外周髓磷脂中的非典型粘附。已经观察到聚糖和肽的差异，并且正在确定相关结构。二聚体和单体的这些表征将有助于我们了解 P0 在髓磷脂中的粘附作用的系统发育发展。