Involvement of C-mannosylation in protein processing in the endoplasmic reticulum and implications for specific target proteins

蛋白质色氨酸残基的c -甘露糖基化是发生在内质网（ER）的动物特异性糖基化过程。它以磷酸多醇甘露糖为供糖体，n -糖基化和o -甘露糖基化也需要磷酸多醇甘露糖基化。尽管后一种途径已被广泛研究，但实际上对内质网中的c -甘露糖基化过程和单个蛋白质的c -甘露糖基化功能一无所知。分子、细胞和系统水平的研究一直受到阻碍，因为我们直到最近才能够识别编码c -甘露糖基转移酶的基因。该酶与负责n -糖基化的复合物的催化亚基（STT3）有关，这两个过程在机制和功能上可能存在相似性。c -甘露糖被发现在一个特定的靶序列（WxxW）上，通常作为双基序（WxxWxxW）出现，其中所有色氨酸都可以被c -甘露糖基化。这样一个基序上的两个或三个c -甘露糖确实可以看作是n -聚糖加工的等效阶段，暴露出类似的末端甘露糖。在2013年从秀丽隐杆线虫中克隆c -甘露糖基转移酶（DPY-19）后，我们已经能够证明哺乳动物有四种同源酶。对于其中两种酶，我们能够证明它们使用不同的受体位点。DPY19L1使用WxxWxxW基序的前两个色氨酸，DPY19L3使用WxxWxxW基序的最后一个色氨酸。使用c -甘露糖基化阴性细胞系，我们也可以证明c -甘露糖基化对蛋白质分泌和稳定性很重要。为了进一步研究c -甘露糖基化的机制和功能方面，我们将遵循两个目标：首先，我们将研究内质网中与其他糖基化途径相结合的分子过程；其次，我们将重点研究c -甘露糖基化通过不同的c -甘露糖基转移酶对不同靶蛋白的功能影响。第一部分将通过与其他糖基化过程的竞争实验进行，包括在细胞内和体外使用内质网微粒体，并筛选相互作用伙伴。此外，通过CRISPR/Cas技术生成几个不同的细胞突变体并分析其对糖基化的影响，将用于研究c -甘露糖与内质网中蛋白质折叠机制的相互作用。在第二部分中，将使用不同的受体蛋白研究不同哺乳动物酶的精细特异性。最后，现在我们有了一个表达系统，我们可以表达c -甘露糖基化和非甘露糖基化的蛋白质，我们想看看c -甘露糖基化的功能后果，它超出了内质网的折叠过程。

C-mannosylation of tryptophan residues of proteins is an animal specific glycosylation process that occurs in the endoplasmic reticulum (ER). It uses dolichol-phosphate-mannose as sugar donor, which is also required for N-glycosylation and O-mannosylation. Whereas the latter pathways have been extensively studied, virtually nothing is known about the C-mannosylation process in the ER and the function of C-mannosylation of individual proteins. Studies at the molecular, cellular and systemic levels have been hampered because we only recently have been able to identify the gene encoding the C-mannosyltransferase. The enzyme is related to the catalytic subunit (STT3) of the complex responsible for N-glycosylation and mechanistic and functional resemblances might exist between the two processes. C-mannose is found on a specific target sequence (WxxW), often occurring as a double motif (WxxWxxW) in which all tryptophans can be C-mannosylated. Two or three C-mannoses on such a motif can indeed be seen as an equivalent of stages of N-glycan processing, exposing similar terminal mannoses.After initially cloning the C-mannosyltransferase (DPY-19) from C. elegans in 2013, we have been able to show that mammals have four homologous enzymes. For two of the enzymes, we were able to show that they use different acceptor sites. DPY19L1 uses the first two and DPY19L3 the last tryptophans of a WxxWxxW motif. Using C-mannosylation negative cell lines, we could also show that C-mannosylation is important for protein secretion and stability.In order to further study mechanistic and functional aspects of C-mannosylation we will follow two objectives: Firstly, we will study the molecular processes in the ER in conjunction with other glycosylation pathways and secondly focus on functional consequences of C-mannosylation on different target proteins by the distinct C-mannosyltransferases. The first part will be carried out by competition experiments with other glycosylation processes, both in cells and in vitro using ER microsomes, and by screening for interaction partners. In addition, the generation of several distinct cellular mutants by CRISPR/Cas technology and the analyses of the consequences on glycosylation will be used to study the interaction of C-mannose with the protein folding machinery in the ER. In the second part, the fine specificity of the distinct mammalian enzymes will be studied using different acceptor proteins. Finally, now that we have an expression system in which we can express C-mannosylated and non-mannosylated proteins, we want to look at functional consequences of C-mannosylation that go beyond the folding process in the ER.