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

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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.