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

C-甘露糖基化在内质网蛋白质加工中的参与及其对特定靶蛋白的影响

• 批准号: 347511139
• 负责人: Dr. Hendrikus Hans Bakker
• 金额: --
• 依托单位: Institut für Klinische Biochemie
• 依托单位国家: 德国
• 项目类别: Research Units
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/347511139?language=en
• 关键词: Involvement; mannosylation; protein; processing; endoplasmic

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.

蛋白质色氨酸残基的C-甘露糖化是发生在内质网(ER)的动物特有的糖基化过程。它使用二羟甲基-磷酸-甘露糖作为糖供体，这也是N-糖基化和O-甘露糖化所必需的。虽然后一种途径已经得到了广泛的研究，但对于内质网中的C-甘露糖化过程和单个蛋白质的C-甘露糖化功能几乎一无所知。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-糖基化的复合体的催化亚单位(STT3)有关，这两个过程可能存在机制和功能上的相似之处。C-甘露糖存在于特定的靶序列(WxxW)上，通常以双基序(WxxWxxW)形式出现，其中所有色氨酸都可以C-甘露糖化。在这样一个基序上的两到三个C-甘露糖确实可以被视为相当于N-糖加工的阶段，暴露出类似的末端甘露糖。在2013年首次从线虫中克隆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置。DPY19L1使用前两个色氨酸，DPY19L3使用WxxWxxW基序的最后两个色氨酸。为了进一步研究C-甘露糖化的机制和功能，我们将遵循两个目标：第一，我们将结合其他糖基化途径研究内质网中的分子过程；第二，通过不同的C-甘露糖基转移酶，研究C-甘露糖化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，在细胞内和体外使用ER微体，并通过筛选相互作用伙伴来进行。此外，利用CRISPR/Cas技术产生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来研究不同哺乳动物酶的良好特异性。最后，现在我们有了一个可以表达C-甘露糖化和非甘露糖化蛋白的表达系统，我们想看看C-甘露糖化的功能后果，这些结果超出了内质网中的折叠过程。