MYELIN PO PROTEIN--EXPRESSION/SORTING AND ADHESION

髓鞘质 PO 蛋白——表达/排序和粘附

• 批准号: 3477433
• 负责人: Marie T. Filbin
• 金额: $ 8.99万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-09-19 至 1993-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3477433
• 关键词: CHO cells; Schwann cells; cell adhesion; complementary DNA; extracellular matrix; gene expression; genetic manipulation; genetic markers; genetic regulation; glycosylation; laboratory rat; myelin; myelin glycoprotein; myelin proteolipid; myelination; polymerase chain reaction; protein engineering; protein structure; tissue /cell culture

The formation of myelin by Schwann cells requires the precise timing of expression and the exact positioning of a number of specific proteins. How these proteins are delivered to the correct location and what specific functions they have is unknown. The major peripheral myelin protein, Po, is thought to interact with itself (hemophilic binding) possibly via its carbohydrate residues and thus play a role in compaction of myelin. Moreover, changes in the glycosylation of Po have been associated with a changes in its expression, although evidence for a direct correlation is lacking. The overall goals of this proposal are to determine whether the oligosaccharide chain of Po is involved in hemophilic interaction and whether changes in glycosylation constitute the key mechanism for regulation Po expression and sorting in the Schwann cell. As an abundance of Po is required for these studies, the immediate goals are to over-express the glycoprotein by gene amplification using the dihydrofolate reductase (DHFR) strategy in cells transfected with the cDNA for Po. For the adhesion studies, cells that have no endogenous myelin proteins, Chinese hamster ovary (CHO) cells, will be used which will assist in the accurate interpretation of the data. Through specific manipulation of the sugar structure, assessment of its contribution of adhesion will be made. Studies on the expression/sorting of Po will be carried out in Schwann cells. However, since in Schwann cells the transfected Po must be distinguished from the endogenous glycoprotein, a "reporter" group of 7-10 amino acids will be attached to the C-terminal portion of the transfected glycoprotein. To ensure that Po with and without the reporter peptide is expressed/sorted in an identifical manner, both will be characterized in CHO cells, prior to the studies with Schwann cells. Eventually, the expression of Po-reporter will be monitored in Schwann cells in relation to the addition of axolemma and/or extracellular matrix to the culture, before and after treatment with specific inhibitors of glycosylation. Finally the long term goals of this study are to apply the expertise gained in this study ot other specific myelin proteins and hence to gain an understanding of myelin-ation in the peripheral nervous system. Such information is critical in understanding the basic mechanism and pathology of any peripheral neuropathy which involves demyelination and remyelination e.g., Charcot-Marie-Tooth disease.

雪旺细胞形成髓鞘需要精确的 表达的时机和一些准确的定位 特定的蛋白质。这些蛋白质是如何传递到正确的 目前尚不清楚它们的位置和具体功能。这个 主要的外周髓鞘蛋白Po被认为与 自身(血友病结合)可能通过其碳水化合物残基 从而在髓鞘的致密过程中发挥作用。此外，变化 在Po的糖基化中与一种 它的表达，尽管有直接关联的证据是 缺乏。这项提案的总体目标是确定 Po的寡糖链是否与血友病有关 相互作用和糖基化的变化是否构成关键 雪旺氏细胞中Po表达和分选的调控机制 手机。由于这些研究需要大量的PO，因此 目前的目标是通过基因过量表达糖蛋白。 利用二氢叶酸还原酶(DHFR)策略扩增 用该基因转染的细胞，可获得高表达的抗体。在粘附力研究中， 没有内源性髓鞘蛋白的细胞，中国仓鼠 卵巢(CHO)细胞，这将有助于准确 对数据的解释。通过特定的操作 糖的结构，评估其对粘附力的贡献 被创造出来。将对Po的表达/分类进行研究 在雪旺细胞里。然而，由于在雪旺细胞中 必须将转基因的Po与内源性的 糖蛋白，一个由7-10个氨基酸组成的报告基团 连接到转染糖蛋白的C-末端部分。 以确保有和没有报告肽的Po是 以标识的方式表示/排序，两者都将 在与Schwann的研究之前，在CHO细胞中具有特征 细胞。最终，博报记者的表达将受到监控 雪旺细胞中与轴膜和/或 治疗前后细胞外基质对培养物的影响 使用特定的糖基化抑制剂。最后，从长远来看 这项研究的目标是应用在这项研究中获得的专业知识 而不是其他特定的髓鞘蛋白，从而获得一个 对周围神经系统髓鞘形成的认识。 这些信息对于理解基本机制是至关重要的 以及任何外周神经病变的病理学，涉及 脱髓鞘和重新髓鞘形成，例如，夏科-玛丽-图斯病。