Pouilly, S., Bourgeaux, V., Piller, F. and Piller, V.
ACS Chem Biol. 7 (4) 753-760
publié le , mis à jour le
Changes in glycosylation are correlated to disease and associated with differentiation processes. Experimental tools are needed to investigate the physiological implications of these changes either by labeling of the modified glycans or by blocking their biosynthesis. N-acetylgalactosamine (GalNAc) is a monosaccharide widely encountered in glycolipids, proteoglycans and glycoproteins ; once taken up by cells it can be converted through a salvage pathway to UDP-GalNAc which is further used by glycosyltransferases to build glycans. In order to find new reporter molecules able to integrate into cellular glycans, synthetic analogs of GalNAc were prepared and tested as substrates of both enzymes acting sequentially in the GalNAc salvage pathway, the galactokinase 2 (GK2) and the uridylpyrophosphorylase AGX1. Detailed in vitro assays identified the GalNAc analogs which can be transformed into sugar nucleotides and revealed several bottlenecks in the pathway : a modification on C6 is not tolerated by GK2 ; AGX1 can use all products of GK2 although with various efficiencies ; all analogs transformed into UDP-GalNAc analogs except those with alterations on C4 are substrates for the polypeptide GalNAc transferase T1. Besides, all analogs which could be incorporated in vitro into O-glycans were also integrated into cellular O-glycans as attested by their detectetion on the cell surface of CHO-ldlD cells. Altogether our results show that GalNAc analogs can help to better define structural requirements of the donor substrates for the enzymes involved in GalNAc metabolism and those that are incorporated into cells will prove valuable for the development of novel diagnostic and therapeutic tools.