title: A glycosyltransferase-enriched reconstituted membrane system for the synthesis of branched O-linked glycans in vitro.
abstract: Mimicking the biochemical reactions that take place in cell organelles is becoming one of the most important challenges in biological chemistry. In particular, reproducing the Golgi glycosylation system in vitro would allow the synthesis of bioactive glycan polymers and glycoconjugates for many future applications including treatments of numerous pathologies. In the present study, we reconstituted a membrane system enriched in glycosyltransferases obtained by combining the properties of the wheat germ lectin with the dialysable detergent n-octylglucoside. When applied to cells engineered to express the O-glycan branching enzyme core2 beta (1,6)-N-acetylglucosaminyltransferase (C2GnT-I), this combination led to the reconstitution of lipid vesicles exhibiting an enzyme activity 11 times higher than that found in microsomal membranes. The enzyme also showed a slightly higher affinity than its soluble counterpart toward the acceptor substrate. Moreover, the use of either the detergent re-solubilization, glycoprotein substrates or N-glycanase digestion suggests that most of the reconstituted glycosyltransferases have their catalytic domains in an extravesicular orientation. Using the disaccharide substrate Galβ1-3GalNAc-O-p-nitrophenyl as a primer, we performed sequential glycosylation reactions and compared the recovered oligosaccharides to those synthesized by cultured parental cells. After three successive glycosylation reactions using a single batch of the reconstituted vesicles and without changing the buffer, the acceptor was transformed into an O-glycan with chromatographic properties similar to glycans produced by C2GnT-I-expressing cells. Therefore, this new and efficient approach would greatly improve the synthesis of bioactive carbohydrates and glycoconjugates in vitro and could be easily adapted for the study of other reactions naturally occurring in the Golgi apparatus such as N-glycosylation or sulfation.
authors.affiliation: INSERM UMR 911 Aix-Marseille Université, Centre de Recherche en Oncologie biologique et Oncopharmacologie, Marseille, France.
authors.forename: Sandrine
authors.initials: S
authors.lastname: Susini
authors.forename: Charlotte
authors.initials: C
authors.lastname: Jeanneau
authors.forename: Sylvie
authors.initials: S
authors.lastname: Mathieu
authors.forename: Sylvie
authors.initials: S
authors.lastname: Carmona
authors.forename: Assou
authors.initials: A
authors.lastname: El-Battari
mesh_terms: D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005960:Glucosides / Q000378:metabolism; D006001:Glycoconjugates / Q000096:biosynthesis*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism* / Q000648:ultrastructure; D008051:Lipid Bilayers / Q000378:metabolism; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism*; D009844:Oligosaccharides / Q000096:biosynthesis; D011134:Polysaccharides / Q000096:biosynthesis*; D014162:Transfection; D014909:Wheat Germ Agglutinins / Q000378:metabolism
chemical_list: D005960:Glucosides; D006001:Glycoconjugates; D008051:Lipid Bilayers; D009844:Oligosaccharides; D011134:Polysaccharides; D014909:Wheat Germ Agglutinins; D049452:Green Fluorescent Proteins; D017351:N-Acetylglucosaminyltransferases; C053024:beta-1,3-galactosyl-O-glycosyl-glycoprotein beta-1,6-acetylglucosaminyl transferase