MOLECULAR BASIS FOR THE STABILITY OF XENOPUS P0-GLYCOPROTEIN DIMER

该子项目是利用 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH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 中心，不一定是研究者的机构。 髓磷脂零蛋白（P0）是髓磷脂形成和维持的主要蛋白质。它是一种含有单个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的聚糖在细胞间粘附中起重要作用。 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。采用包括SDS-聚丙烯酰胺凝胶电泳（SDS-PAGE）的质谱分析策略来确定P0二聚体的相互作用以及单体和寡聚体的翻译后修饰特征。这些结果将有助于理解P0的系统发育髓鞘的粘附作用。 通过SDS-PAGE纯化爪蟾P0寡聚体。切下感兴趣的条带并重新加载到小平板凝胶中，以研究低聚物稳定性背后的相互作用。为了确定糖型谱并验证各组分中存在的蛋白质的归属，使用PNGase F对单体和低聚物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。 在以前的研究中，使用了许多物理和化学方法试图破坏低聚物，包括温度，pH值，洗涤剂和变性剂，但这些方法似乎只是增加相对于单体的低聚物的量。在本研究中，当将通过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Mini-prep-cell从混合物中纯化蛋白质时，几乎所有的二聚体解离成单体。基于这些观察结果，我们提出非洲爪蟾PNS P0二聚体是通过非共价相互作用稳定的，并且非共价二聚体和单体之间存在平衡。采用蛋白酶胶内消化，然后进行MALDI-MS和ESI-MS/MS，基于ca. 60%的序列覆盖率 为了探索影响爪蟾P0蛋白折叠和聚集的因素，对爪蟾P0蛋白单体和寡聚体的翻译后修饰进行了研究。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。根据MALDI MS的结果，观察到的Cys 152被硬脂酸（C18：0）酰化。观察到爪蟾P0含有一系列高甘露糖、杂合和复合聚糖。通过ESI FTMS/MS（CAD）以高准确度测定主要聚糖。此外，Asn 92被证实为单一的完全占据的N-糖基化位点，通过MALDI MS和ESI MS/MS。我们已经确定的非洲爪蟾P0的PTM不同于其他物种，如牛。我们建议，独特的酰化和糖基化可能是非洲爪蟾P0的不寻常的折叠和聚集行为的基础。此外，P0二聚体和单体具有略微不同的糖型和略微不同的糖型分布，这也可能导致其聚集趋势的差异。这些研究结果将有助于我们研究外周髓鞘的非典型粘附，从而有助于理解P0的系统发育。髓鞘的粘附作用。一份手稿已提交出版。

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 P0s 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 P0s adhesion role in myelin. A manuscript has been submitted for publication.