Functional properties of Notch specific xylosyltransferases and effects of xylosylation on cellular Notch signaling: A comparative study of human and Drosophila enzymes

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It has been clearly established that O-fucosylation of EGF like repeats of the Notch receptor has a strong impact on the signaling function of the molecule. Extension of fucose by the glycosyltransferase Fringe influences the signaling strength of different ligands. In contrast, the importance of O-glucosylation, which provides the second type of abundant glycosylation on Notch, and the extension to the xylose-xylose-glucose-O-Ser glycotope is largely unknown because the genes encoding the enzymes involved in its biosynthesis were only recently cloned. We identified the human genes encoding xylosyltransferases involved in the building of the xylose-xylose-glucose trisaccharide. Two genes named GXYLT1 and GXYLT2 encode enzymes catalyzing the first xylose transfer, while a single gene (XXYLT1) is responsible for the addition of the second xylose residue. Pioneering experiments in Drosophila revealed that increased xylosylation attenuates Notch signaling strength and that the mammalian enzymes are more potent than the Drosophila enzymes. This study is directed at understanding the mode of action of the different Notch xylosyltransferases and their influence on Notch signaling at cellular level, with a particular focus on the difference between the human and Drosophila enzymes transferring the second xylose. This will be done by determination of the acceptor requirements of the enzymes, resolving subcellular localization and establishing of the minimal catalytic domain, which is directed at a collaborative effort to resolve the crystal structure. At cellular level, influence of xylosylation will be studied using reporter assays in Drosophila S2 cells.