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: 7723032
• 负责人: DANIEL A KIRSCHNER
• 金额: $ 2.72万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-06-01 至 2009-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7723032
• 关键词: Acylation; Adhesions; Affect; Behavior; Bos taurus; Carbohydrates; Cattle; Cell Adhesion; Cells; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Covalent Interaction; Custom; Deletion Mutation; Detergents; Development; Digestion; Electrospray Ionization; Endopeptidases; Equilibrium; Funding; Gel; Genes; Grant; Human; Hybrids; Inherited; Institution; Maintenance; Mannose; Manuscripts; Mass Spectrum Analysis; Membrane; Membrane Glycoproteins; Methods; Molecular; Mus; Myelin; Myelin P0 Protein; N-Glycosylation Site; Numbers; Oligosaccharides; Pattern; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Peripheral; Peripheral Nervous System Diseases; Phylogenetic Analysis; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Publications; Relative (related person); Reporting; Research; Research Personnel; Resources; Role; Series; Sodium Dodecyl Sulfate-PAGE; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stearic Acids; Structure; Temperature; Time; United States National Institutes of Health; Xenopus; Xenopus laevis; base; dimer; glycosylation; interest; mass spectrometer; monomer; nano

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. The Cys152 observed is acylated with stearic acid (C18:0) according to results from MALDI MS. It was observed that Xenopus P0 contains 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 that we have identified by MS differ from those reported for other species such as cattle. We propose that the unique acylation and glycosylation could 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, which might also contribute to the difference in their tendency toward aggregation. Our results from these studies will help us to investigate atypical adhesion in peripheral myelin and thus should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. A manuscript has been submitted for publication.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 髓磷脂零蛋白 (P0) 是形成和维持髓磷脂的主要蛋白质。它是一种完整的膜糖蛋白，含有单个 N-糖基化位点。 P0 基因的突变和缺失与遗传性周围神经病相关。 P0 的聚糖在细胞间粘附中发挥重要作用。 对牛、鼠和人 P0 的研究表明，P0 在膜中以四聚体形式存在。然而，爪蟾中 P0 的主要形式是二聚体。利用包括 SDS 聚丙烯酰胺凝胶电泳 (SDS-PAGE) 在内的质谱策略来确定 P0 二聚体的相互作用以及单体和寡聚体的翻译后修饰概况。这些结果应该有助于理解 P0 在髓磷脂中粘附作用的系统发育。 通过 SDS-PAGE 纯化非洲爪蟾 P0 寡聚物。切下感兴趣的条带并重新加载到迷你板凝胶中，以研究低聚物稳定性的相互作用。为了确定糖型谱并验证每个级分中存在的蛋白质的分配，使用 PNGase F 对单体和寡聚体进行凝胶内去糖基化，然后使用蛋白酶进行凝胶内消化。使用基质辅助激光解吸/电离飞行时间质谱仪 (MALDI-TOF MS)、纳米 ESI-TOF MS 和定制的 ESI-qQq-FTMS 对肽和寡糖进行表征。通过 MS/MS 对感兴趣的碳水化合物和肽进行测序。 在之前的研究中，使用了许多物理和化学方法来尝试破坏低聚物，包括温度、pH、去污剂和变性剂，但这些方法似乎只能增加相对于单体的低聚物的量。在这项研究中，当通过 SDS-PAGE 分离的二聚体条带重新加载到凝胶中时，观察到一半的二聚体分解成单体。此外，观察到，当使用“小型制备细胞”从混合物中纯化蛋白质时，几乎所有二聚体都解离成单体。基于这些观察，我们建议非洲爪蟾 PNS P0 二聚体通过非-共价相互作用，并且非共价二聚体和单体之间存在平衡，使用蛋白酶凝胶消化，然后进行 MALDI-MS 和 ESI-MS/MS，最初指定的二聚体和单体。基于大约 60% 的序列覆盖率，确认单体是 P0 的形式。 为了探索影响 Xenopus P0 折叠和聚集的因素，对 P0 单体和寡聚体进行了翻译后修饰 (PTM)。观察了特定的酰化和糖基化模式，并根据结果确定了观察到的Cys152被硬脂酸(C18:0)酰化。 MALDI MS.观察到非洲爪蟾P0含有一系列高甘露糖、杂合和复杂的聚糖。主要聚糖通过 ESI FTMS/MS (CAD) 进行高精度测定。此外，MALDI MS 和 ESI MS/MS 证实 Asn92 是单个完全占据的 N-糖基化位点。我们通过 MS 鉴定的非洲爪蟾 P0 中的 PTM 与报道的其他物种（例如牛）的 PTM 不同。我们认为，独特的酰化和糖基化可能是非洲爪蟾 P0 异常折叠和聚集行为的基础。此外，P0二聚体和单体的糖型和糖型分布略有不同，这也可能导致它们聚集倾向的差异。这些研究的结果将帮助我们研究外周髓磷脂的非典型粘附，从而有助于理解 P0 在髓磷脂中粘附作用的系统发育。手稿已提交出版。