ACS Chemical Biology – Date de publication sur le web : 25 janvier 2012
Abstract:
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.
GalNAc is the initial sugar of mucin-type O-glycans, and is a component of several tumor antigens. The aim of this work was to determine whether synthetic GalNAc analogs could be taken up from the medium and incorporated into complex cellular O-glycans. The cell line employed was CHO ldlD, which can only use GalNAc and Gal present in the medium for the synthesis of its glycans. All GalNAc analogs with modified N-acyl groups (N-formyl, N-propionyl, N-glycolyl, N-azidoacetyl, N-bromoacetyl, and N-chloroacetyl) were incorporated into cellular O-glycans, although to different extents. The GalNAc analogs linked to Ser or Thr could be extended by the β3-galactosyltransferase glycoprotein-N-acetylgalactosamine 3β-galactosyl transferase 1 in vitro and in vivo and by α6-sialyltransferase α-N-acetylgalactosaminide-α-2,6-sialyltransferase 1. At the surface of CHO ldlD cells, all analogs were incorporated into sialylated O-glycan structures like those present on wild-type CHO cells, indicating that the GalNAc analogs do not change the overall structure of core-1 O-glycans. In addition, this study shows that the unnatural synthetic GalNAc analogs can be incorporated into human tumor cells, and that a tumor antigen modified by an analog can be readily detected by a specific antiserum. GalNAc analogs are therefore potential targets for tumor immunotherapy.
Dans le cadre du consortium « DNA-nanoparticles for skeletal muscle and airway epithelium in vivo gene therapy » soutenu par l’AFM, une étude comparée des voies d’endocytose de différents vecteurs chimiques a été réalisée dans l’équipe P. Midoux & C. Pichon. Pour cette étude, des outils cellulaires exprimant des compartiments intracellulaires fluorescents ont été préparés et utilisés afin de suivre par microscopie confocale de fluorescence l’entrée et le devenir de l’ADN vectorisé dans les myoblastes. Les résultats publiés dans Biomaterials montrent que l’efficacité de la transfection est plutôt corrélée à la nature de la voie endocytaire qu’à la quantité de matériel endocyté. Elle montre également l’importance de ces outils cellulaires pour suivre le devenir intracellulaire d’une nanoparticule par microscopie fluorescente en temps réel.
Billiet L, Gomez JP, Berchel M, Jaffrès PA, Le Gall T, Montier T, Bertrand E, Cheradame H, Guégan P, Mével M, Pitard B, Benvegnu T, Lehn P, Pichon C and Midoux P
De l’hydantoïne a été obtenue en laboratoire dans des conditions similaires à celles existant au sein des glaces interstellaires. C’est le résultat auquel viennent d’arriver des chercheurs de l’Institut d’Astrophysique Spatiale (CNRS, Université Paris-sud), du Centre de Biophysique Moléculaire (CNRS) et de la NASA. Issue de la condensation de l’urée et de l’acide glycolique présentes par ailleurs dans les météorites, l’hydantoïne, peut jouer le rôle de catalyseur pour la formation de chaines peptidiques. Cette formation de proto-protéines peut avoir lieu dans des conditions adéquates, telles celles qui existaient dans les océans de la Terre primitive, étape probablement essentielle à l’apparition de la vie.
Référence de l’article scientifique :
« Prebiotic Significance of Extraterrestrial Ice Photochemistry : Detection of Hydantoin in Organic Residues »
Pierre de Marcellus, Marylène Bertrand, Michel Nuevo, Frances Westall, and Louis Le Sergeant d’Hendecourt. L., Astrobiology, vol. 11, no. 9, p. 847-854, Novembre 2011.
