Analysis of a new Glycosyltransferase in LRP6/Wnt signalling

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Wnt/b-catenin signalling is transduced through Wnt receptor complexes on the cell membrane that contain a Frizzled receptor and the co-receptor LRP6. My previous research has helped demonstrate that LRP6 plays a crucial regulatory role in transducing Wnt signalling. We have recently identified several new modifiers of LRP6 using cell culture based expression screening approaches and this DFG grant proposal requests funding for the further characterisation of one of them, a novel glycosyltransferase of the B3GNT family. To date, no specific glycosyltransferase (GT) has been implicated in regulating Wnt signaling and it is known that LRP6, like most cell surface receptors, undergoes glycosylation for correct transport and functioning. Of the hundreds of diverse glycosyltransferases that operate in the protein glycosylation pathway, this B3GnT8 family member is predicted to function later in the pathway, likely in the trans-Golgi, to elongate polylactosamine chains on tetraantennary N-glycans. We have extensive preliminary data to show that B3GnT8 is a bona-fide regulator of LRP6 and Wnt signalling. Gain and loss of function experiments show it is both sufficient and required for Wnt signalling and epistasis experiments place its functioning at the level of LRP6. We also have initial evidence that it is involved in hexose induced Wnt signalling. We therefore propose to further characterise the role of B3GnT8 with respect to metabolic regulation of Wnt signalling. Our findings indicate that N-glycosylation of LRP6 plays an important regulatory role in Wnt signalling. Specific Aims 1. Identify LRP6 glycosylation sites (mutagenesis and MS analysis) 2. Characterise LRP6 glycan chain modification using a combination of MS analysis and azidosugar metabolic labelling. 3. Explore the link between glucose (hexose) metabolism and LRP6 signalling. 4. Show the in-vivo relevance of LRP6 glycosylation by the novel LRP6 GT using zebrafish embryos. 5. Understand the global role of the GT in cellular signalling by performing RNA seq experiments.