C-Mannosylation of Thryptophan Residues - Characterization of Mammalian Enzymes and Functional and Structural Aspect of the Modification on Thrombospondin Type 1 Repeats
Final Report Abstract
C-mannosylation of tryptophan (W) residues of proteins is an animal specific glycosylation process that occurs in the endoplasmic reticulum. 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. The only recently identified C-mannosyltransferase 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. Most invertebrates have one C-mannosyltransferase gene, named dpy-19 after the dumpy phenotype of the worm C. elegans, but mammals have four homologous enzymes. The C. elegans enzyme uses only the first two tryptophans of the WxxWxxW motif. We have shown that mammals, however, have two enzymes, DPY19L1 and DPY19L3, to fully mannosylate all three tryptophans of the WxxWxxW motif. DPY19L1 has the same activity as the C. elegans homolog, but DPY19L3 is responsible for the modification of the third tryptophan. In thrombospondin repeat containing protein the full recognition motif for C-mannosylation is WxxWxxWxxC and we could show that the specificity of DPY19L3 depends on the presence of the cysteine on the +3 position after the third tryptophan. The dumpy phenotype of C. elegans dpy-19 increases with rising temperature and becomes lethal above 24°C. We could show that at least for one protein, the netrin receptor UNC-5, C-mannosylation is required for secretion from the endoplasmic reticulum at higher temperature (28°C) but can be omitted at 20°C. This led to the conclusion that C-mannosylation aids the folding and stability of proteins and provided an explanation for the temperature dependent phenotype of the worm. Furthermore, we could confirm the observed phenomenon in cells and worms by experiments with isolated protein. Non-mannosylated netrin receptor UNC-5 had an about 10° lower melting temperature than the mannosylated protein. Also the re-folding of the protein is aided by C-mannosylation after thermal and chemical denaturation of the protein.
Publications
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(2017) Distinct C-mannosylation of netrin receptor thrombospondin type 1 repeats by mammalian DPY19L1 and DPY19L3. Proc Natl Acad Sci USA. 114:2574-2579
Shcherbakova A, Tiemann B, Buettner FF, Bakker H
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(2018) Apicomplexan C-Mannosyltransferases Modify Thrombospondin Type I-Containing Adhesins of the TRAP Family. Glycobiology. 2018 Feb 8
Hoppe CM, Albuquerque A, Bandini G, Leon DR, Shcherbakova A, Buettner FFR, Izquierdo L, Costello CE, Bakker H, Routier FH
