Analyse der funktionellen Eigenschaften der Notch spezifischen Xylosyltransferasen und Untersuchungen zur Beeinflussung der Notch Signalübertragung durch Xylosylierung: Eine vergleichende Studie in Mensch und Drosophila
Biochemie
Zusammenfassung der Projektergebnisse
The Notch receptor consists of 36 EGF repeats that are heavily glycosylated with unusual glycans. We study the O-glucose pathway, especially the modification of glucose by two xyloses by two different enzymes. In this project we have established the activity of the Drosophila enzyme responsible for the addition of the second xylose and could show it is, in contrast to the human enzyme, specific for certain EGF repeats. In the course of our analysis, we also discovered that the enzyme transferring the first xylose could alternatively use glucose as donor sugar. This leads to the intriguing idea that Notch glycosylation depend on the concentration of respective nucleotide sugars. To investigate the effect of expression of different enzymes on glycosylation and the consequences of glycosylation, we also generated mutant clones in the Drosophila Schneider (S2) cell line using CRISPR-Cas technology and generated stable cell lines overexpressing proteins. This provided a challenge as S2 cells do not survive alone, therefore excluding the usual method used in mammalian cells to generate clonal lines. We solved this by using cells not resistant to the antibiotic puromycin as feeder cells. This way, single mutated cells could survive till a colony was formed that could support itself. Using the generated mutant cells, we also set up a Notch signaling assay in S2 cells, that does not activate the endogenous notch pathway, but only detects the cleavage of the Notch intracellular domain. This has been done by adapting an existing mammalian cell assay in which the yeast Gal4 transcription factor is replacing the intracellular domain of the Notch receptor. We were not able to resolve the question how the xylosyltransferases are retained in the endoplasmic reticulum. A cysteine residue located in the membrane anchor of one of the xylosyltransferases was shown to be responsible for dimer formation of the enzyme, but mutation of the cysteine did not result in any mislocalization of the enzyme. The ability to generate clonal mutants in S2 cells will certainly bring this project further forward and will find its applications outside of this project as well.
Projektbezogene Publikationen (Auswahl)
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(2013) In vitro assays of orphan glycosyltransferases and their application to identify Notch xylosyltransferases. Methods Mol Biol. 1022:307-20
Sethi MK, Buettner FFR, Ashikov A, Bakker H
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(2013) Negative regulation of Notch signaling by xylose. PLoS.Genet. 9, e1003547
Lee TV, Sethi MK, Leonardi J, Rana NA, Buettner FFR, Haltiwanger RS, Bakker H, Jafar-Nejad H
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(2015) Notch-modifying xylosyltransferase structures support an SNilike retaining mechanism. Nat Chem Biol. 11:847-54
Yu H, Takeuchi M, LeBarron J, Kantharia J, London E, Bakker H, Haltiwanger RS, Li H, Takeuchi H
