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的糖基化类型，通常发生在凝血酶反应蛋白1型重复序列和I型细胞因子受体上。最近发现线虫DPY-19为C-甘露糖基转移酶，这表明C-甘露糖化和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转移酶(负责N-糖基化的酶)也是结构相关的蛋白质。然而，在线虫中只发现一种形式的酶，而在哺乳动物中有四种DPY-19的同源物(DPY19L1-4)。最值得注意的是，DPY19L2基因突变是球体精子症的主要原因，这是一种导致男性不育的罕见先天性疾病。目前的提议有两个目标。在提案的第一部分，重点放在C-甘露糖化的结构和功能后果上。在这些研究中，将使用已知的线虫目标蛋白。受线虫突变体dpy-19对温度敏感的启发，将针对C-甘露糖化对内质网温度依赖性折叠特性和蛋白质热稳定性的影响进行功能研究。另一个雄心壮志是建立纯化和分离甘露糖化和非甘露糖化凝血酶原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的四个哺乳动物同源物的活性。我们将克隆小鼠C-甘露糖基转移酶，并在C-甘露糖基转移酶阴性的果蝇S2细胞系中表达。酶活性将使用不同的合成肽，并通过共同表达假定的受体蛋白，代表不同类型的已知C-甘露糖化蛋白，以及一种可能被DPL19L2甘露糖化的精子特异性蛋白来确定。

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.