MYELIN PO PROTEIN: EXPRESSION/SORTING AND ADHESION

髓鞘质 PO 蛋白：表达/排序和粘附

• 批准号: 3477432
• 负责人: Marie T. Filbin
• 金额: $ 9.56万
• 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/3477432
• 关键词: 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; natural gene amplification; 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.

雪旺细胞髓磷脂的形成需要精确的 表达的时机和一些的精确定位 特定蛋白质。 这些蛋白质如何被输送到正确的位置 它们的位置和具体功能尚不清楚。 这 主要外周髓磷脂蛋白 Po，被认为与 本身（血友病结合）可能通过其碳水化合物残基 从而起到压缩髓磷脂的作用。 此外，改变 Po 糖基化的变化与 它的表达，尽管直接相关的证据是 缺乏。 该提案的总体目标是确定 Po寡糖链是否与血友病有关 相互作用以及糖基化的变化是否构成关键 雪旺 Po 表达和分选的调节机制 细胞。 由于这些研究需要大量的 Po， 近期目标是通过基因过度表达糖蛋白 使用二氢叶酸还原酶 (DHFR) 策略进行扩增 用Po的cDNA转染细胞。 对于粘附力研究， 没有内源性髓磷脂蛋白的细胞，中国仓鼠 将使用卵巢（CHO）细胞，这将有助于准确 数据的解释。 通过具体的操控 糖结构，评估其粘附力的贡献 被制作。 将进行Po的表达/分选研究 在施万细胞中。 然而，由于在雪旺细胞中 转染的 Po 必须与内源性的 Po 区分开来。 糖蛋白，一个由 7-10 个氨基酸组成的“报告”基团 连接至转染糖蛋白的C末端部分。 确保带有和不带有报告肽的 Po 是 以相同的方式表达/排序，两者都将是 在与 Schwann 进行研究之前，在 CHO 细胞中进行了表征 细胞。 最终Po-reporter的表达会被监测到 在雪旺细胞中与添加轴膜和/或 治疗前和治疗后培养物中的细胞外基质 具有特定的糖基化抑制剂。 最后是长期 本研究的目标是应用本研究中获得的专业知识 其他特定髓磷脂蛋白，因此获得 了解周围神经系统中的髓鞘形成。 这些信息对于理解基本机制至关重要 和任何涉及周围神经病变的病理学 脱髓鞘和髓鞘再生，例如腓骨肌萎缩症。