CYTOSOLIC GLYCOSYLATION

描述：申请者对细胞周期调节蛋白的研究 在细胞黏液霉菌盘基网柄菌中的FP21导致了 一种新的羟基化前残基上连接的五糖的发现 在143号位置。这是一个体积庞大的取代基，位于一个高度 蛋白质的保守表面。这也是一种不同寻常的结构 一种细胞质/核蛋白，作为亲羟化和复合体 糖基化通常被认为局限于 牢房。申请者的长期目标是了解这一点 复杂的翻译后共价修饰，无法预测 从cdna序列数据，影响fp21和其他蛋白质的功能。 这种情况很可能会发生。本项目的重点将是如何 五糖是生物合成的，最初使用FP21作为底物 确定该途径中潜在的关键酶。这将首先涉及到 通过以下方法确定控制其生物合成的酶的特异性 完成HyPro五糖(AIM)的结构测定 #1)。随后将对两种早期酶活性进行纯化 途径、前羟基酶和GalNAcTransferase，并检测 它们的底物专一性和动力学常数(目标2)。这种方法是 预计将确定该途径的候选酶，建议细胞 各自工作的隔间及其基板 承认。克隆这两种酶和cFucTase的cDNA(目标3) 将导致每种酶蛋白的一级结构，这将 独立解决他们的区隔问题，提出一种进化性的 与分泌途径中相应的已知酶的关系，以及 为后面目标中的关键操作提供工具。足够的 每种酶多肽催化其所建议的途径步骤 将通过在异源系统中表达其cdna来建立 然后是酶分析(目标4)。最后，酶的必要性 在体内执行该途径步骤的多肽及其 与FP21的共同区划将通过基因破坏来解决 在分泌途径中的酶基因和FP21的表达， 分别在网柄网柄菌中(目标5)。除了坚定地建立起 HyPro途径最初针对的基本原则和 修改FP21，结果将提供一个重要的网关，通过它 我们可以在其他生物中检测到这一途径，无论FP21是否是 在被检查的特定细胞或组织类型中糖基化的。这些工具 已开发的将允许未来剖析复杂的糖基化作用 细胞质/核蛋白，可能基于FP21的表型 和糖基化突变体，对细胞增殖至关重要 和新陈代谢。

DESCRIPTION: The applicant's study of the cell cycle regulatory protein FP21 in the cellular slime mold Dictyostelium discoideum has led to the discovery of a novel pentasaccharide attached to a hydroxylated Pro-residue at position 143. This is a bulky substituent to be located on a highly conserved surface of a protein. It is also an unusual structure to find on a cytoplasmic/nuclear protein, as Pro hydroxylation and complex glycosylation are usually thought to be confined to the secretory pathway of the cell. The applicant's long term goals are to understand how this complex postranslational covalent modification, which cannot be predicted from cDNA sequence data, affects the function of FP21 and other proteins on which it presumably occurs. This project will focus on how the pentasaccharide is biosynthesized, initially using FP21 as a substrate to identify potentially key enzymes in the pathway. This will involve first establishing the specificity of the enzymes which govern its biosynthesis by completing the structure determination of the HyPro pentasaccharide (aim #1). This will be followed by purification of 2 early enzyme activities of the pathway, the ProHydroxylase and the GalNAcTransferase, and examining their substrate specificity and kinetic constants (aim#2). This approach is expected to identify candidate enzymes for the pathway, suggest the cellular compartment in which each operates, and the basis for its substrate recognition. Cloning cDNAs for these 2 enzymes and the cFucTase (aim#3) will lead to the primary structure of each enzyme protein, which will independently address their compartmentalization, suggest an evolutionary relationship with corresponding known enzymes in the secretory pathway, and provide tools for critical manipulations in the later aims. The sufficiency of each enzyme polypeptide for catalyzing its proposed step of the pathway will be established by expression of its cDNA in a heterologous system followed by enzymatic assay (aim #4). Finally, the necessity of the enzyme polypeptide for execution of the pathway step in vivo, and its co-compartmentalization with FP21, will be addressed by genetic disruption of the enzyme genes and expression of FP21 in the secretory pathway, respectively in Dictyostelium (aim#5). In addition to firmly establishing the basic principles by which the HyPro pathway initially targets and modifies FP21, the results will provide an important gateway through which we can detect this pathway in other organisms, regardless of whether FP21 is glycosylated in the particular cell or tissue type examined. The tools developed will permit future dissection of the role of complex glycosylation of cytoplasmic/nuclear proteins which may, based on the phenotype of FP21 and glycosylation mutants, be fundamentally important for cell proliferation and metabolism.