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: 7955933
• 负责人: DANIEL A KIRSCHNER
• 金额: $ 0.47万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-01 至 2010-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7955933
• 关键词: Acylation; Adhesions; Affect; Behavior; Biology; Carbohydrates; Cattle; Cell Adhesion; Cells; Chemicals; Complex; Computer Retrieval of Information on Scientific Projects Database; Covalent Interaction; Custom; Deletion Mutation; Detergents; Development; Digestion; Equilibrium; Funding; Gel; Genes; Grant; Human; Hybrids; Inherited; Institution; Investigation; Maintenance; Mannose; Manuscripts; Mass Spectrum Analysis; Medicine; Membrane; Membrane Glycoproteins; Methods; Molecular; Mus; Myelin; Myelin P0 Protein; N-Glycosylation Site; Oligosaccharides; Pattern; Peptide Hydrolases; Peptide N-glycohydrolase F; Peptides; Peripheral; Peripheral Nervous System Diseases; Phylogenetic Analysis; Play; Polysaccharides; Post-Translational Protein Processing; Proteins; Publishing; 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. MALDI-MS showed that the Cys152 was acylated with stearic acid (C18:0). Xenopus P0 was found to contain 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 differ from those reported for other species such as cattle. The unique acylation and glycosylation may 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; this may contribute to the differences in their aggregation. Our results will aid investigation of atypical adhesion in peripheral myelin and thus should contribute to understanding of the phylogenetic development of P0's adhesion role in myelin. Two manuscripts based on these results were recently published, one appeared in the previous reporting year and the second during the current year.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 髓鞘蛋白零(P0)是髓鞘形成和维持的主要蛋白质。它是一种含有单一N-糖基化位点的完整膜糖蛋白。P0基因的突变和缺失与遗传性周围神经病相关。P0的糖链在细胞与细胞间的黏附中起重要作用。对牛、鼠和人P0的研究表明，P0在膜上以四聚体的形式存在。然而，非洲爪哇P0的主要形式是二聚体。采用包括十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法(十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法)在内的质谱学方法来确定P0二聚体之间的相互作用以及单体和低聚物的翻译后修饰图谱。这些结果将有助于理解P0‘S在髓鞘中的黏附作用的系统发育。 利用SDS-PAGE对非洲爪哇P0寡聚体进行纯化。将感兴趣的条带切除并重新加载到迷你平板凝胶中，以研究低聚物稳定性背后的相互作用(S)。用PNGase F对单体和寡聚体进行凝胶内脱糖，然后用蛋白水解酶进行凝胶内消化，以确定糖型图谱和蛋白质在各组分中的归属。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对多肽和寡糖进行了表征。用MS/MS对目标碳水化合物和多肽进行了测序。 在以前的研究中，一些物理和化学方法被用来试图破坏低聚物，包括温度、pH、洗涤剂和变性剂，但这些方法似乎只增加了相对于单体的低聚物的数量。在本研究中，当用SDS-PAGE分离的二聚体条带重新加载到凝胶中时，观察到有一半的二聚体分解成单体。此外，还观察到，当使用Mini-Prep-cell“从混合物中纯化蛋白质时，几乎所有的二聚体都解离成单体。基于这些观察，我们认为非洲爪哇PNS P0二聚体是由非共价相互作用(S)稳定的，并且非共价二聚体和单体之间存在平衡。利用蛋白酶凝胶内消化，然后进行MALDI-MS和ESI-MS/MS，根据约60%的序列覆盖率，初步确定初始分配的二聚体和单体为P0形式。为了探索影响非洲爪哇P0折叠和聚集的因素，研究了P0单体和低聚物的翻译后修饰(PTM)。观察到特定的酰化和糖基化模式，并确定了修饰基团的结构。MALDI-MS表明，Cys152与硬脂酸(C18：0)发生了酰化反应。非洲爪哇P0含有一系列高甘露糖、杂交糖和复合糖。利用电喷雾飞行时间质谱仪(ESI FTMS/MS，CAD)对主要多糖进行了高精度测定。此外，MALDI MS和ESI MS/MS证实Asn92是唯一一个完全占据的N-糖基化位点。非洲爪哇P0中的PTMS与牛等其他物种的PTMS不同。这种独特的酰化和糖基化可能是非洲爪哇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略有不同，糖型分布也略有不同，这可能是导致它们聚集程度不同的原因之一。我们的结果将有助于研究外周髓鞘中的非典型黏附，从而有助于理解P0‘S在髓鞘中黏附作用的系统发育。最近出版了基于这些结果的两份手稿，一份出现在上一个报告年度，另一份出现在本年度。