Dissecting and Reconstructing the Dolichol Pathway

描述(由申请人提供)：本项目的目标是研究组成二羟苯二酚途径的生物合成酶的综合作用。具体地说，研究的重点将是催化在内质网(ER)膜的胞液面上发生的前七个反应并导致焦磷酸二醇-GIcNAc2Man5的生物合成的酶。N-连接的糖基化是所有真核生物中必不可少的过程，而导致新生糖蛋白生物合成的步骤在整个真核生物王国中都是高度保守的。尽管这一途径在真核细胞中具有重要意义，但人们对组成酶的结构、机制和综合功能知之甚少。该计划的研究将集中在酿酒酵母中组成酒精醇途径的酶。了解酵母酶的进展将直接使鉴定哺乳动物的同源物成为可能，并为深入了解人类和其他哺乳动物的二羟苯基二醇途径提供帮助。 这项研究计划的具体目标如下： 1.鉴定、生化鉴定和开发催化Dolicol途径中7个连续转化序列的酶的同源或异源表达系统。将采用一种生化方法来鉴定序列中“缺失”的酶。 2.开发实验方法来研究底物是如何沿着多醇途径中的酶的“流水线”转移的。在体内和体外的方法，以评估途径和作用的二羟基苯酚结合的底物将介绍。 3.研究催化Dolicol途径早期步骤的酶的底物特异性。糖基供体和受体底物的特异性将被评估。具体地说，我们感兴趣的是评估Dolicol途径酶是否可以将糖直接转移到糖肽受体上。这一发现将有助于制备定制的糖蛋白产品。 4.对通路中的靶点进行结构分析。这一具体目标将与佐治亚大学的Prestegard教授进行核磁共振分析，以及波士顿大学医学院的Karen Allen教授进行X射线分析。

DESCRIPTION (provided by applicant): The goal of this program is to investigate the integrated action of the biosynthetic enzymes that comprise the dolichol pathway. Specifically, research will focus on the enzymes that catalyze the first seven reactions that take place on the cytosolic face of the endoplasmic reticulum (ER) membrane and lead to the biosynthesis of dolichol pyrophosphate-GIcNAc2Man5. N-linked glycosylation is an essential process in all eukaryotes, and the steps leading to the biosynthesis of nascent glycoproteins are highly conserved throughout the eukaryotic kingdom. Despite the significance of this pathway in eukaryotic cells, little is known about the structure, mechanism, and integrated function of the constituent enzymes. Studies in this program will focus on the enzymes comprising the dolichol pathway in the yeast S. cerevisiae. Progress in understanding the yeast enzymes will directly enable identification of mammalian homologs and provide insight into the dolichol pathway in humans and other mammals. The specific aims of this research program are as follows: 1.To identify, biochemically characterize, and develop homologous or heterologous expression systems for the enzymes that catalyze the sequence of seven contiguous transformations in the dolichol pathway. A biochemical approach will be adopted for the identification of the "missing" enzymes in the sequence. 2.To develop experimental approaches to investigate how substrates are transferred along the "assembly line" of enzymes in the dolichol pathway. Both in vivo and in vitro methods for evaluating the pathway and the role of the dolichol-bound substrates will be presented. 3.To investigate the substrate specificity of the enzymes catalyzing the early steps in the dolichol pathway. Specificity for both glycosyl donor and acceptor substrates will be evaluated. Specifically we are interested in evaluating whether the dolichol pathway enzymes can transfer saccharide directly to a glycopeptide acceptor. Such a finding would be of utility for the preparation of tailored glycoprotein products. 4.To carry out structural analysis of targets in the pathway. This specific aim will be carried out in collaboration with Prof. Prestegard at the University of Georgia for NMR analysis and Prof. Karen Allen in the at Boston University Medical School for X-ray analysis.