C-Mannosylation of Thryptophan Residues - Characterization of Mammalian Enzymes and Functional and Structural Aspect of the Modification on Thrombospondin Type 1 Repeats

蛋白质中WXXW基序的C-甘露糖基化是一种独特类型的糖基化，通常发生在血小板反应蛋白1型重复序列和I型细胞因子受体上。根据最近对C.通过将线虫DPY-19作为C-甘露糖基转移酶，我们可以表明C-甘露糖基化和N-糖基化不仅在功能上相关，而且C-甘露糖基转移酶和寡糖基转移酶（负责N-糖基化的酶）也是结构上相关的蛋白质。而在C.哺乳动物中DPY-19有4个同源物（DPY 19 L1 - 4）。最值得注意的是，DPY 19 L2的突变是导致男性不育的罕见先天性疾病--球形精子症的主要原因。目前的建议有两个目标。在提案的第一部分，重点放在C-甘露糖基化的结构和功能后果上。对于这些研究，我们已经知道了C.将使用Elegans。受C.线虫突变体dpy-19是温度敏感的，将进行针对C-甘露糖基化对内质网中温度依赖性折叠性质和蛋白质热稳定性的影响的功能研究。另一个目标是建立纯化和分离甘露糖基化和非甘露糖基化血小板反应蛋白重复序列的方法，这是未来结构研究所需的。第二个目标是确定DPY-19的四种哺乳动物同系物的活性。将在C-甘露糖基转移酶阴性果蝇S2细胞系中克隆和表达推定的小鼠C-甘露糖基转移酶。将使用不同的合成肽并通过共表达推定的受体蛋白（代表不同类型的已知C-甘露糖基化蛋白）和可能被DPL 19 L2甘露糖基化的精子特异性蛋白来测定酶活性。

C-Mannosylation of the WXXW motif in proteins is a unique type of glycosylation typically occurring on thrombospondin type 1 repeats and type I cytokine receptors. By the recent identification of C. elegans DPY-19 as the C-mannosyltransferase, we could show that C-mannosylation and N-glycosylation are not only functionally related, but that the C-mannosyltransferase and the oligosaccharyltransferase (the enzyme responsible for N-glycosylation) are structurally related proteins as well. Whereas only one form of the enzyme is found in C. elegans, there are four homologs of DPY-19 in mammals (DPY19L1 - 4). Most notably, mutations in DPY19L2 are the main cause of globozoospermia, a rare congenital disorder causing male infertility. The current proposal has two objectives. In the first part of the proposal the focus lays on the structural and functional consequences of C-mannosylation. For these studies, the already known target proteins of C. elegans will be used. Inspired by the fact that the C. elegans mutant dpy-19 is temperature sensitive, functional studies will be carried out that are directed at the influence of C-mannosylation on temperature dependent folding properties in the endoplasmic reticulum and on thermostability of the proteins. Another ambition is to establish methods to purify and separate mannosylated and non-mannosylated thrombospondin repeats, required for future structural studies. A second goal is to establish the activity of the four mammalian homologs of DPY-19. The putative mouse C-mannosyltransferases will be cloned and expressed in the C-mannosyltransferase negative Drosophila S2 cell line. Enzymatic activity will be determined using different synthetic peptides and by co-expression of putative acceptor proteins, representing the different types of known C-mannosylated proteins, and a sperm specific protein that is potentially mannosylated by DPL19L2.